Evaluating the Environmental Impacts of Chemicals for Sustainable Paper Recycling

Recycling paper resources is important for promoting resource circulation and reducing waste. However, during the collection and reprocessing of recovered paper, chemicals originally present in printed materials, thermal paper, and other paper products may migrate into wastewater and potentially affect aquatic ecosystems.

In particular, aromatic sensitizers and related compounds (SRCs), which are used to facilitate color-forming and color-developing reactions in paper products, have raised concerns because their aromatic structures resemble those of certain chemicals with endocrine-disrupting or dioxin-like activities.

However, little was known about how SRCs present in paper recycling effluent actually affect fish development.

Toxicity Assessment Combining Molecular Docking and Zebrafish Embryo Testing

Our laboratory conducted an integrated toxicity assessment of SRCs that may be detected in paper recycling effluent by combining molecular docking simulations with zebrafish (Danio rerio) embryo exposure experiments.

First, to predict whether SRCs could interact with toxicity-related receptors, we performed virtual screening by molecular docking against zebrafish Ahr2 and human AHR. The aryl hydrocarbon receptor (AHR) is a receptor critically involved in the toxic effects of aromatic chemicals, including dioxin-like compounds.

Subsequently, compounds identified as potentially concerning through molecular docking analysis were evaluated in zebrafish embryo exposure experiments conducted up to 96 hours post-fertilization. Morphological abnormalities and the expression of toxicity-related genes were assessed.

Developmental Toxicity and AHR Activation Induced by Benzyl 2-Naphthyl Ether (BNE)

Among the SRCs examined, benzyl 2-naphthyl ether (BNE) produced clear effects in zebrafish embryos at relatively low concentrations.

Exposure of zebrafish embryos to 1 µM BNE resulted in morphological abnormalities, including pericardial edema and reduced body length. In addition, expression of cyp1a and ahr2, marker genes associated with activation of the AHR pathway, was significantly increased.

These findings indicate that BNE may induce developmental toxicity in zebrafish embryos accompanied by activation of the AHR signaling pathway.

However, co-exposure to CH-223191, an AHR antagonist, only partially reduced the morphological abnormalities induced by BNE. In contrast, the effects of a dioxin-like compound used as a positive control were relatively well restored by CH-223191. These results suggest that BNE toxicity may involve not only AHR-mediated effects but also AHR-independent toxic mechanisms.

Potential Effects on Endocrine-Related Pathways

In addition, our study found that several SRCs, including BNE, showed predicted binding potential for the estrogen receptor in in silico analyses.

In zebrafish embryos, BNE exposure also increased the expression of cyp19a1b, which encodes brain aromatase. These findings suggest that SRCs present in paper recycling effluent may affect endocrine-related pathways in addition to the AHR signaling pathway.

Nevertheless, predicted receptor binding and changes in gene expression alone are insufficient to directly conclude that these compounds cause long-term ecological effects or reproductive toxicity. Further studies are required to evaluate longer-term exposure outcomes, including developmental and reproductive effects.

Significance of This Study: Toward Safer and More Sustainable Recycling

This study does not question the importance of paper recycling itself. Rather, to promote resource circulation in a sustainable manner, it is essential to scientifically evaluate the types and effects of chemicals that may be released into the environment during recycling processes and, where necessary, connect these findings to improved wastewater management and the development of safer alternative materials.

Our findings demonstrate that certain chemicals associated with paper recycling effluent may exert developmental toxicity in fish embryos. Furthermore, by integrating molecular docking simulations with zebrafish exposure experiments, we established a research framework for prioritizing environmental chemicals that require further evaluation and experimentally examining their biological effects.

Our laboratory investigates overlooked chemical risks in the environment by combining in silico prediction with in vivo and in vitro toxicological evaluation of environmental contaminants and consumer-use chemicals.

Original Research Article

The findings of this study were published in Environmental Toxicology and Chemistry.

Assessment of the Aryl Hydrocarbon Receptor–Mediated Effects of Aromatic Sensitizers in Paper Recycling Effluent Employing Zebrafish Embryos and In Silico Docking
Kazuki Takeda, Aoi Sarata, Masanori Terasaki, Akira Kubota, Keita Shimizu, Ryo Kamata
Environmental Toxicology and Chemistry, 43(10), 2176–2188, 2024.

DOI:
https://doi.org/10.1002/etc.5969

Environmental Toxicology and Chemistry is published on behalf of the Society of Environmental Toxicology and Chemistry (SETAC), an international scientific society in the fields of environmental toxicology and environmental chemistry.

Research Highlights

Student Poster Presentation Award at the Japanese Society of Toxicology Annual Meeting

Jiayan Sun, then a sixth-year undergraduate student in our laboratory, presented findings from this study at the 51st Annual Meeting of the Japanese Society of Toxicology, held in Fukuoka in July 2024, and received the Student Poster Presentation Award.

SETAC Award at the Joint Conference on Environmental Chemicals

Aoi Sarata, then a sixth-year undergraduate student in our laboratory, presented findings from this study at the 1st Joint Conference on Environmental Chemicals, held in Toyama in June 2022, and received the SETAC Award.