16. Advancing the double-seeded gill cell culture system for drug uptake studies in fish, Env. Tox. Che., 2025
Aya Takesono,Maciej Trznadel, Francesca Molinari, Chrisna Matthee, Anke Lange, Stewart F. Owen and Charles R. Tyler
https://doi.org/10.1093/etojnl/vgaf153
ABSTRACT
This study focuses on environmental risk assessment (ERA), a regulatory requirement for the approval of new drugs in Europe and the United States, which necessitates reliable methods for modeling how active pharmaceutical ingredients (APIs) are absorbed by fish. In this context, a model using rainbow trout (Oncorhynchus mykiss) gill cells cultured on double-seeded inserts (DSIs) has emerged as a potential tool for assessing API uptake in freshwater fish. However, variability in establishing and maintaining high transepithelial electrical resistance (TEER), which is crucial for replicating functional gill tissue, has posed challenges for standardizing this method.
The study aimed to optimize the DSI gill cell culture system and evaluate its utility for API transfer studies. Key factors affecting cell adherence and TEER development include the size and age of the donor fish, cell washing conditions, and membrane coating. Additionally, using serum-free L-15 medium instead of freshwater for apical exposure greatly improved TEER stability. These refinements enabled reproducible API transfer data with TEER values exceeding 1 kΩ·cm², allowing robust comparisons of transfer rates across various API types and doses. The authors conclude that optimizing the DSI gill cell culture system for apical freshwater application requires a deeper understanding of gill cell biology, especially regarding the role of specific cell types in gill epithelium development.
15. Critical insights into the potential risks of antipsychotic drugs to fish, including through effects on behaviour, Biol. Rev., 2025
Gabrielle Wasser-Bennett, A. Ross Brown, Samuel K. Maynard, Stewart F. Owen and Charles R. Tyler
https://doi.org/10.3389/ftox.2024.1406942
ABSTRACT
Antipsychotic drugs (APDs) are a diverse class of neuroactive pharmaceuticals increasingly detected in surface and ground waters globally. Some APDs are classified as posing a high environmental risk, due, in part, to their tendency to bioaccumulate in wildlife, including fish. Additional risk drivers for APDs relate to their behavioural effects, potentially impacting fitness outcomes. However, standard ecotoxicological tests used in environmental risk assessment (ERA) do not currently account for these mechanisms. In this review, we critically appraise the environmental risks of APDs to fish. We begin by reading-across from human and mammalian effects data to standard ecotoxicological effects endpoints in fish. We then explore the wide range of behaviours suitable for ecotoxicological assessment of APDs (and other neuroactive) pharmaceuticals, principally through laboratory studies with zebrafish, and assess the potential for using these behavioural phenotypes to predict adverse individual- and population-level outcomes in wild fish, taking into account phenotypic plasticity. Next, we illustrate the advantages and challenges of measuring and applying behavioural endpoints for fish, including within current regulatory risk assessments. In our final analysis, the implications of relying on apical endpoints for ERA of neuroactive drugs (including APDs) are assessed and recommendations provided for the development of a more refined and tailored mechanistic approach, which would enable more robust assessment of their environmental risk(s).
14. Read-Across of Biotransformation Potential between Activated Sludge and the Terrestrial Environment: Toward Making It Practical and Plausible, Env. Sci. Tech., 2025
Claudia Coll, Claudio Screpanti, Jasmin Hafner, Kunyang Zhang, Kathrin Fenner
https://doi.org/10.1021/acs.est.4c09306
ABSTRACT
Recent emphasis on the development of safe-and-sustainable-by-design chemicals highlights the need for methods facilitating the early assessment of persistence. Activated sludge experiments have been proposed as a time- and resource-efficient way to predict half-lives in simulation studies. Here, this persistence “read-across” approach was developed to be more broadly and robustly applicable. We evaluated 21 previously used reference plant protection products (PPPs) for their broader applicability in calibrating regression and classification models for predicting half-lives in soil (DT50OECD307) and water-sediment systems (DT50OECD308) based on their half-life in sludge and the organic carbon–water partition coefficient KOC as predictors. The calibrated regression models showed satisfactory predictions of DT50OECD307 for another 22 test PPPs. Performance was less satisfying for the prediction of DT50OECD308 for 46 active pharmaceutical ingredients (APIs), suggesting a need for expanding the set of calibration substances and more experimental KOC values. The classification models mostly correctly classified persistent and non-persistent test compounds for both PPPs and APIs, which is relevant for early-stage screening of persistence. Transformation products of the reference compounds in activated sludge samples were consistent with the reported degradation pathways in soil, particularly with respect to major aerobic, enzyme-catalyzed transformation reactions.
13. Defining the data gap: What do we know about environmental exposure, hazards and risks of pharmaceuticals in the European aquatic environment?, Wat. Res., 2024
F.D. Spilsbury, P.A. Inostroza, P. Svedberg, C. Cannata, A.M.J. Ragas, T. Backhaus
https://doi.org/10.1016/j.watres.2023.121002
ABSTRACT
Active pharmaceutical ingredients (APIs) and their transformation products inevitably enter waterways where they might cause adverse effects to aquatic organisms. Identifying the potential risks of APIs in the environment is therefore a goal and current strategic direction of environmental management described in the EU Strategic Approach to Pharmaceuticals in the Environment and the Green Deal. This is challenged by a paucity of monitoring and ecotoxicity data to adequately describe risks.
In this study we analyze measured environmental concentrations (MECs) of APIs from 5933 sites in 25 European countries as documented in the EMPODAT database or collected by the German Environment Agency for the time period between 1997 and 2020. These data were compared with empirical data on the ecotoxicity of APIs from the U.S. EPA ECOTOX database. Although 1763 uniquely identifiable APIs are registered with the European Medicines Agency (EMA) for sale in the European Economic Area (EEA), only 312 (17.7%) of these are included in publicly available monitoring data, 36 (1.8%) compounds have sufficient ecotoxicological data to derive a PNEC, and only 27 (1.5%) compounds meet both the hazard and exposure data requirements required to to perform an environmental risk assessment according to EMA guidelines. Four of these compounds (14.8%) had a median risk quotient (RQ) > 1. Endocrine disruptors had the highest median RQ, with 7.0 and 5.6 for 17α-ethinyl-estradiol and 17β-estradiol respectively.
12. A database on pharmaceuticals in the environment: What do stakeholders need?, Env. Sci. & Pol., 2024
Cristiana Cannata, Rodrigo Vidaurre, Ad M.J. Ragas, Caroline T.A. Moermond
https://doi.org/10.1016/j.envsci.2024.103946
ABSTRACT
Pharmaceutical pollution has raised concerns about how these contaminants affect ecosystems. Data to assess the environmental risk of pharmaceuticals exist but are dispersed and not always publicly accessible. To address this issue, the European Commission recently proposed to develop a data source for human medicinal products, involving relevant stakeholders, including healthcare and industry representatives. The aim of our study was to define the user requirements for such a database on pharmaceuticals in the environment (PiE). We reached out to over 100 professionals that work on PiE, asking what data should be incorporated in the database and what features it should have. The results show that most stakeholders are affected by data gaps, mainly related to ecotoxicity, monitoring, transformation products, metabolites and removal rates in wastewater treatment plants. Interest in the mechanism of action of active pharmaceutical ingredients was specifically expressed by the pharma sector, including stakeholders related to the authorisation, production and use of medicines. Researchers and stakeholders dealing with environmental and water quality have greater interest for mass spectrum data, modelled environmental concentrations and data on transformation products and metabolites. While showing that the key actors working on PiE strongly endorse the development of an accessible and transparent database, we provide recommendations for creating such a data repository on pharmaceuticals.
11. Prioritisation of data-poor pharmaceuticals for empirical testing and environmental risk assessment, Env. Int., 2024
Cristiana Cannata, Rhys Whomsley, Thomas Backhaus, Irene Bramke, Caroline T.A. Moermond, Maria Caraman, Anna Lombardo, Ad M.J. Ragas
https://doi.org/10.1016/j.envint.2023.108379
ABSTRACT
There are more than 3,500 active pharmaceutical ingredients (APIs) on the global market for human and veterinary use. Residues of these APIs eventually reach the aquatic environment. Although an environmental risk assessment (ERA) for marketing authorization applications of medicinal products is mandatory in the European Union since 2006, an ERA is lacking for most medicines approved prior to 2006 (legacy APIs). Since it is unfeasible to perform extensive ERA tests for all these legacy APIs, there is a need for prioritization of testing based on the limited data available. Prioritized APIs can then be further investigated to estimate their environmental risk in more detail. In this study, we prioritized more than 1,000 APIs used in Europe based on their predicted risk for aquatic freshwater ecosystems. We determined their risk by combining an exposure estimate (Measured or Predicted Environmental Concentration; MEC or PEC, respectively) with a Predicted No Effect Concentration (PNEC). We developed several procedures to combine the limited empirical data available with in silico data, resulting in multiple API rankings varying in data needs and level of conservativeness.
10. Many human pharmaceuticals are weak inhibitors of the cytochrome P450 system in rainbow trout (Oncorhynchus mykiss) liver S9 fractions, Front. Toxicol., 2024
Tea Pihlaja, Timo Oksanen, Netta Vinkvist, Tiina Sikanen
https://doi.org/10.3389/ftox.2024.1406942
ABSTRACT
Pharmaceutical residues are widely detected in aquatic environment and can be taken up by nontarget species such as fish. The cytochromes P450 (CYP) represent an important detoxification mechanism in fish, like in humans. In the present study, we assessed the correlation of the substrate selectivities of rainbow trout CYP1A and CYP3A homologues with those of human, through determination of the half-maximal inhibitory concentrations (IC50) of a total sixteen human pharmaceuticals toward CYP1A-like ethoxyresorufin O-deethylase (EROD) and CYP3A-like 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD) in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (RT-S9).
9. Design of greener drugs: aligning parameters in pharmaceutical R&D and drivers for environmental impact, Drug Discovery Today, 2024
Rodrigo Vidaurre, Irene Bramke, Neele Puhlmann, Stewart F. Owen, Daniela Angst, Caroline Moermond, Bastiaan Venhuis, Anna Lombardo, Klaus Kümmerer, Tiina Sikanen, Jim Ryan, Andreas Häner, Gemma Janer, Silvio Roggo, Alison Nimrod Perkins
https://doi.org/10.1016/j.drudis.2024.104022
ABSTRACT
Active pharmaceutical ingredients (APIs) in the environment, primarily resulting from patient excretion, are of concern because of potential risks to wildlife. This has led to more restrictive regulatory policies. Here, we discuss the ‘benign-by-design’ approach, which encourages the development of environmentally friendly APIs that are also safe and efficacious for patients. We explore the challenges and opportunities associated with identifying chemical properties that influence the environmental impact of APIs. Although a straightforward application of greener properties could hinder the development of new drugs, more nuanced approaches could lead to drugs that benefit both patients and the environment. We advocate for an enhanced dialogue between research and development (R&D) and environmental scientists and development of a toolbox to incorporate environmental sustainability in drug development.
8. The VERA software: Implementation of the acute fish toxicity endpoint and its application to pharmaceutical compounds , Chemosphere, 2024
Erika Colombo, Edoardo Luca Viganò, Giuseppa Raitano, Anna Lombardo, Alberto Manganaro, Alessio Sommovigo, Emilio Benfenati
https://doi.org/10.1016/j.ejps.2023.106614
ABSTRACT
The Virtual Extensive Read-Across software (VERA) is a new tool for read-across using a global similarity score, molecular groups, and structural alerts to find clusters of similar substances; these clusters are then used to identify suitable similar substances and make an assessment for the target substance. A beta version of VERA GUI is free and available at vegahub.eu; the source code of the VERA algorithm is available on GitHub. In the past we described its use to assess carcinogenicity, a classification endpoint. The aim here is to extend the automated read-across approach to assess continuous endpoints as well. We addressed acute fish toxicity. VERA evaluation on the acute fish toxicity endpoint was done on a dataset containing general substances (pesticides, industrial products, biocides, etc.), obtaining an overall R2 of 0.68. We employed the VERA algorithm also on active pharmaceutical ingredients (APIs). We included a portion of the APIs in the training dataset to predict APIs, successfully achieving an overall R2 of 0.63. VERA evaluates the assessment’s reliability, and we reached an R2 of 0.78 and Root Mean Square Error (RMSE) of 0.44 for predictions with high reliability.
7. Designing greener active pharmaceutical ingredients: Insights from pharmaceutical industry into drug discovery and development, Europ. Journal of Pharma. Sci., 2024
Neele Puhlmann, Rodrigo Vidaurre, Klaus Kümmerer
https://doi.org/10.1016/j.ejps.2023.106614
ABSTRACT
Pharmaceutical residues found in the environment may impact ecosystems negatively. Research and Development (R&D) of greener pharmaceuticals (greener in terms of reduced environmental occurrence and/or reduced effects on environmental organisms) could be a design approach to prevent or minimize potential environmental impacts by pharmaceuticals. One aim of PREMIER is to evaluate the feasibility of this design approach. To this purpose we interviewed drug designers working in major pharmaceutical companies to understand its opportunities and challenges. Drug designers saw manifold opportunities for incorporating environmental considerations along the common R&D process as long as there are design options. In general, they were open-minded and interested, and some already intrinsically motivated to contribute to this design approach. To bring environmental considerations into the drug design process drug designers will need suitable screening tools and related training for tool application. Collaboration across pharma companies, authorities, and academia is seen as highly promising to realize R&D of greener pharmaceuticals. Financial, social, and regulatory incentives would be supportive. As the entire structure of the healthcare sector influences the feasibility of R&D and use of greener pharmaceuticals, PREMIER will interview further stakeholders in the coming months, including general health insurances, practitioners, pharmacists, and others.
6. Combining predictive and analytical methods to elucidate pharmaceutical biotransformation in activated sludge, Environ. Science: Processes & Impacts, 2023
Jasmin Hafner, Kathrin Fenner and Andreas Scheidegger
https://doi.org/10.1039/D3EM00161J
ABSTRACT
While short biotransformation half-lives of pharmaceuticals in the environment are generally considered to reduce their potential for negative environmental impacts, the formation of stable transformation products (TPs) might question this view. Yet, very little information on TPs of pharmaceuticals is available, which makes it difficult to consider this aspect in their environmental fate and risk assessment. This is partially due to the analytical challenges when identifying all potential TPs from laboratory biotransformation testing. We therefore present an updated methodology that combines up-to-date biotransformation pathway prediction (envipath.org) with an experimental-analytical workflow. For the latter, we identified TPs formed during biotransformation of 42 pharmaceuticals, using biotransformation experiments in activated sludge and liquid chromatography coupled to high-resolution mass spectrometry. Using this workflow, we could identify 79 transformation products to different levels of identification confidence for 31 pharmaceutical compounds, thus significantly adding to the knowledge on typical biotransformation reactions and transformation products observed for pharmaceuticals. All pathways have been made available in electronic format in the activated sludge package of envipath.org.
5. Systematic Handling of Environmental Fate Data for Model Development─Illustrated for the Case of Biodegradation Half-Life Data, Environ. Sci. Technol. 2023
Jasmin Hafner, Kathrin Fenner and Andreas Scheidegger
https://doi.org/10.1021/acs.estlett.3c00526
ABSTRACT
The assessment of environmental hazard indicators such as persistence, mobility, toxicity, or bioaccumulation of chemicals often results in highly variable experimental outcomes. Persistence is particularly affected due to a multitude of influencing environmental factors, with biodegradation experiments resulting in half-lives spanning several orders of magnitude. Also, half-lives may lie beyond the limits of reliable half-life quantification, and the number of available data points per substance may vary considerably, requiring a statistically robust approach for the characterization of data. Here, we apply Bayesian inference to address these challenges and characterize the distributions of reported soil half-lives. Our model estimates the mean, standard deviation, and corresponding uncertainties from a set of reported half-lives experimentally obtained for a single substance. We apply our inference model to 893 pesticides and pesticide transformation products with experimental soil half-lives of varying data quantity and quality, and we infer the half-life distribution for each compound. By estimating average half-lives, their experimental variability, and the uncertainty of the estimations, we provide a reliable data source for building predictive models, which are urgently needed by regulatory authorities to manage existing chemicals and by industry to design benign, nonpersistent chemicals. Our approach can be readily adapted for other environmental hazard indicators.
4. Factors Determining the Susceptibility of Fish to Effects of Human Pharmaceuticals, Environ. Sci. Technol. 2023
Chrisna Matthee, Andrew Ross Brown, Anke Lange, and Charles R. Tyler
https://doi.org/10.1021/acs.est.2c09576
ABSTRACT
The increasing levels and frequencies at which active pharmaceutical ingredients (APIs) are being detected in the environment are of significant concern, especially considering the potential adverse effects they may have on nontarget species such as fish. With many pharmaceuticals lacking environmental risk assessments, there is a need to better define and understand the potential risks that APIs and their biotransformation products pose to fish, while still minimizing the use of experimental animals. There are both extrinsic (environment- and drug-related) and intrinsic (fish-related) factors that make fish potentially vulnerable to the effects of human drugs, but which are not necessarily captured in nonfish tests. This critical review explores these factors, particularly focusing on the distinctive physiological processes in fish that underlie drug absorption, distribution, metabolism, excretion and toxicity (ADMET). Focal points include the impact of fish life stage and species on drug absorption (A) via multiple routes; the potential implications of fish’s unique blood pH and plasma composition on the distribution (D) of drug molecules throughout the body; how fish’s endothermic nature and the varied expression and activity of drug-metabolizing enzymes in their tissues may affect drug metabolism (M); and how their distinctive physiologies may impact the relative contribution of different excretory organs to the excretion (E) of APIs and metabolites. These discussions give insight into where existing data on drug properties, pharmacokinetics and pharmacodynamics from mammalian and clinical studies may or may not help to inform on environmental risks of APIs in fish.
3. Minimizing Experimental Testing on Fish for Legacy Pharmaceuticals, Environ. Sci. Technol. 2023
Anja Coors, A. Ross Brown, Samuel K. Maynard, Alison Nimrod Perkins, Stewart Owen, Charles R. Tyler
https://doi.org/10.1021/acs.est.2c07222
ABSTRACT
There was no regulatory requirement for ecotoxicological testing of human pharmaceuticals authorized before 2006, and many of these have little or no data available to assess their environmental risk. Motivated by animal welfare considerations, we developed a decision tree to minimize in vivo fish testing for such legacy active pharmaceutical ingredients (APIs). The minimum no observed effect concentration (NOECmin, the lowest NOEC from chronic Daphnia and algal toxicity studies), the theoretical therapeutic water concentration (TWC, calculated using the fish plasma model), and the predicted environmental concentration (PEC) were used to derive API risk quotients (PEC/NOECmin and PEC/TWC). Based on a verification data set of 96 APIs, we show that by setting a threshold value of 0.001 for both risk quotients, the need for in vivo fish testing could potentially be reduced by around 35% without lowering the level of environmental protection. Hence, for most APIs, applying an assessment factor of 1000 (equivalent to the threshold of 0.001) to NOECmin substituted reliably for NOECfish, and TWC acted as an effective safety net for the others. In silico and in vitro data and mammalian toxicity data may further support the final decision on the need for fish testing.
2. GREENER Pharmaceuticals for More Sustainable Healthcare, Environ. Sci. Technol. Lett. 2022
Caroline T. A. Moermond, Neele Puhlmann, Stewart F. Owen, Jim Ryan, Jason Snape, Bastiaan J. Venhuis, Klaus Kümmerer
https://doi.org/10.1021/acs.estlett.2c00446
ABSTRACT
Medicines are essential to human health but can also impact the aquatic and terrestrial environment after use by patients and release via excreta into wastewater. We highlight the need for a GREENER approach to identify and meet important environmental criteria, which will help reduce the impact of medicinal residues on the environment. These criteria include effect reduction by avoiding nontarget effects or undesirable moieties, exposure reduction via lower emissions or environmental (bio)degradability, no PBT (persistent, bioaccumulative, and toxic) substances, and risk mitigation. With all of these criteria, however, patient health is of primary importance as medicines are required to be safe and efficacious for treating diseases. We discuss the feasibility of including these criteria for green by design active pharmaceutical ingredients in the process of drug discovery and development and which tools or assays are needed to accomplish this. The integrated GREENER approach can be used to accelerate discussions about future innovations in drug discovery and development.
1. A Generalized Physiologically Based Kinetic Model for Fish for Environmental Risk Assessment of Pharmaceuticals, Environ. Sci. Technol. 2022
Jiaqi Wang, Tom M. Nolte, Stewart F. Owen, Rémy Beaudouin, A. Jan Hendriks and Ad M.J. Ragas
https://doi.org/10.1021/acs.est.1c08068
ABSTRACT
An increasing number of pharmaceuticals found in the environment potentially impose adverse effects on organisms such as fish. Physiologically based kinetic (PBK) models are essential risk assessment tools, allowing a mechanistic approach to understanding chemical effects within organisms. However, fish PBK models have been restricted to a few species, limiting the overall applicability given the countless species. Moreover, many pharmaceuticals are ionizable, and fish PBK models accounting for ionization are rare. Here, we developed a generalized PBK model, estimating required parameters as functions of fish and chemical properties. We assessed the model performance for five pharmaceuticals (covering neutral and ionic structures). With biotransformation half-lives (HLs) from EPI Suite, 73 and 41% of the time-course estimations were within a 10-fold and a 3-fold difference from measurements, respectively. The performance improved using experimental biotransformation HLs (87 and 59%, respectively). Estimations for ionizable substances were more accurate than any of the existing species-specific PBK models. The present study is the first to develop a generalized fish PBK model focusing on mechanism-based parameterization and explicitly accounting for ionization. Our generalized model facilitates its application across chemicals and species, improving efficiency for environmental risk assessment and supporting an animal-free toxicity testing paradigm.
