Front. Toxicol. Frontiers in Toxicology Front. Toxicol. 2673-3080 Frontiers Media S.A. 1272368 10.3389/ftox.2023.1272368 Toxicology Systematic Review A systematic review of the evaluation of endocrine-disrupting chemicals in the Japanese medaka (Oryzias latipes) fish Dasmahapatra et al. 10.3389/ftox.2023.1272368 Dasmahapatra Asok K. 1 2 Williams Charmonix B. 1 Myla Anitha 1 Tiwary Sanjay K. 1 Tchounwou Paul. B. 1 3 * 1 RCMI Center for Environmental Health, Jackson State University, Jackson, MS, United States 2 Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University, MS, United States 3 RCMI Center for Urban Health Disparities Research and Innovation, Morgan State University, Baltimore, MD, United States

Edited by: Rosaria Meccariello, University of Naples Parthenope, Italy

Reviewed by: Massimo Venditti, Second University of Naples, Italy

Azza Naija, Qatar University, Qatar

 *Correspondence: Paul. B. Tchounwou, paul.tchounwou@morgan.edu 27 11 2023 2023 5 1272368 04 08 2023 10 10 2023 Copyright © 2023 Dasmahapatra, Williams, Myla, Tiwary and Tchounwou. 2023 Dasmahapatra, Williams, Myla, Tiwary and Tchounwou

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Japanese medaka (Oryzias latipes) is an acceptable small laboratory fish model for the evaluation and assessment of endocrine-disrupting chemicals (EDCs) found in the environment. In this research, we used this fish as a potential tool for the identification of EDCs that have a significant impact on human health. We conducted an electronic search in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and Google Scholar (https://scholar.google.com/) using the search terms, Japanese medaka, Oryzias latipes, and endocrine disruptions, and sorted 205 articles consisting of 128 chemicals that showed potential effects on estrogen–androgen–thyroid–steroidogenesis (EATS) pathways of Japanese medaka. From these chemicals, 14 compounds, namely, 17β-estradiol (E2), ethinylestradiol (EE2), tamoxifen (TAM), 11-ketotestosterone (11-KT), 17β-trenbolone (TRB), flutamide (FLU), vinclozolin (VIN), triiodothyronine (T3), perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA), terephthalic acid (TPA), trifloxystrobin (TRF), ketoconazole (KTC), and prochloraz (PCZ), were selected as references and used for the identification of apical endpoints within the EATS modalities. Among these endpoints, during classification, priorities are given to sex reversal (masculinization of females and feminization of males), gonad histology (testis–ova or ovotestis), secondary sex characteristics (anal fin papillae of males), plasma and liver vitellogenin (VTG) contents in males, swim bladder inflation during larval development, hepatic vitellogenin (vtg) and choriogenin (chg) genes in the liver of males, and several genes, including estrogen–androgen–thyroid receptors in the hypothalamus–pituitary–gonad/thyroid axis (HPG/T). After reviewing 205 articles, we identified 108 (52.68%), 46 (22.43%), 19 (9.26%), 22 (17.18%), and 26 (12.68%) papers that represented studies on estrogen endocrine disruptors (EEDs), androgen endocrine disruptors (AEDs), thyroid endocrine disruptors (TEDs), and/or steroidogenesis modulators (MOS), respectively. Most importantly, among 128 EDCs, 32 (25%), 22 (17.18%), 15 (11.8%), and 14 (10.93%) chemicals were classified as EEDs, AEDs, TEDs, and MOS, respectively. We also identified 43 (33.59%) chemicals as high-priority candidates for tier 2 tests, and 13 chemicals (10.15%) show enough potential to be considered EDCs without any further tier-based studies. Although our literature search was unable to identify the EATS targets of 45 chemicals (35%) studied in 60 (29.26%) of the 205 articles, our approach has sufficient potential to further move the laboratory-based research data on Japanese medaka for applications in regulatory risk assessments in humans.

 Japanese medaka endocrine disruptors EATS pathways systematic review risk assessment National Institutes of Health10.13039/100000002 section-at-acceptance Developmental and Reproductive Toxicology 1 Introduction

Due to the increase in industrial and agricultural activities, endocrine-disrupting chemicals (EDCs), defined by the World Health Organization (WHO) as “Exogeneous substances that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism or its progeny, or (sub)populations,” are accumulated in the environment. A strategic approach to identify EDCs would be utilized by the existing knowledge to prioritize and focus on the screening and environmental monitoring efforts of these chemicals. The European Commission also set criteria for the identification of EDCs that require regulatory action. Currently, endocrine disruptors (EDs) are identified on a case-by-case basis using the available guidance provided in the OECD Guidance Document 150 (2018). The OECD Conceptual Framework for Testing and Assessment of EDs provided a tiered framework for the organization of study information to assess endocrine activity. This framework provides guidance for prioritizing relevant data streams and methods according to the type and level of information needed for a regulatory assessment. In the USA, EPA’s EDSP has developed the requirements for the prioritization, screening, and testing of environmental contaminants, including pesticides, commercial chemicals, and agricultural products, for their potential to impact the endocrine system, especially in relation to estrogen, androgen, and thyroid (EAT) hormones and their nuclear receptors (NIEHS, 2018). Moreover, the perturbation of the enzymes of steroidogenesis by EDCs has potential effects on EAT pathways. Therefore, a two-tier testing approach was designed by EDSP. Tier 1 assays detect the potential effects of a chemical by various modes of action (Tier 1: screening) on EATS pathways. The results of the Tier 1 assays are evaluated by using a “weight of evidence” approach to determine whether the potential of the chemical is to interact with EATS and whether a Tier 2 assay is necessary. The purpose of Tier 2 studies is to use in vivo testing to further characterize the EATS effects and establish a dose–response relationship for adverse effects produced by the chemicals. Tier 2 tests are much longer-term studies that include exposure during critical life stages and have a broad range of more tightly spaced treatment than Tier 1. Moreover, Tier 2 tests can encompass multiple generations, covering effects on fecundity and fertility, development, growth, and sexual maturity. The successful completion of Tier 2 testing provided information to establish exposure and effect relationships, and assessed relevant endpoints across most life stages.

In aquatic environments, fish are considered one of the primary risk organisms for EDCs, especially those interacting with reproductive hormones. Sex determination in fish is very labile and can be disrupted or functionally reversed by external agents at critical developmental stages (Francis, 1992). Fish populations are directly exposed to a wide variety of EDCs, originating from industrial, agricultural, or municipal effluents (Ternes et al., 1999; Chen et al., 2007; Kim et al., 2014a). Evidence shows that EDCs can have long-term effects on reproduction and subsequent population development in natural fish populations (Kidd et al., 2007). The effects of EDCs on nuclear receptors have been studied extensively in small fish models like zebrafish, Japanese medaka, stickleback, and roach (Rutilus rutilus) (Iguchi et al., 2006; Lange et al., 2009; Tohyama et al., 2015). Since endocrine disruptions are linked to the receptor level, to predict ED effects, the identification of appropriate biomarkers at molecular levels is necessary.

Japanese medaka (Oryzias latipes) fish are small, freshwater teleost fish that inhabit gently flowing rivers and waterways. Like zebrafish (Danio rerio) and fathead minnows (Pimephales promelas), it is one of the small fish models (vertebrate) used in EDC studies (OECD, 2018). The sex determination locus has been identified in this fish species, and external sex-specific markers (chromatophores, shape of the anal and dorsal fins, anal fin papillae) can be used to easily differentiate males from females both from phenotypic and genotypic standpoints (Scholz and Mayer, 2008). Several OECD test guidelines (OECD TG 229; OECD TG 240) were used during the evaluation of EDCs in Japanese medaka, following tier-based approaches (Tier 1 and Tier 2). Moreover, the effects of endocrine active chemicals on Japanese medaka were reviewed previously (Urushitani et al., 2007; Flynn et al., 2017; Onishi et al., 2021; Kawashima et al., 2022). Based on the available publications found in public databases, we hypothesized that a literature search can identify the number and sources of EDCs that disrupted the EATS-related pathways of Japanese medaka (O. latipes) and correlate the effects with specific receptors at the molecular level.

In this review, we summarized the data on EDCs available in public databases, highlighting the links between molecular, phenotypic, and physiological endpoints using Japanese medaka as a single fish species. Although majority of the data refer to interfering with reproductive and thyroid hormone signaling pathways (EATS), limited information about the disruption of other endocrine organs, like the endocrine pancreas and interrenal gland (fish homolog of the adrenal gland), is also available (Dasmahapatra and Tchounwou, 2022a; b, 2023a; b). We evaluated the selective effects of 128 EDCs reported in 205 articles. As a result, we believed that 43 of them (EDCs) show potential to proceed to Tier 2 tests, and 13 chemicals should be considered EDCs without any further tier-based studies.

 2 Materials and methods 2.1 Literature search strategy

The objectives of the literature search were to identify the relevant studies published in peer-reviewed journals that focused on the endocrine disruption of Japanese medaka (O. latipes) induced by various chemicals detected in the aquatic environments. The search was performed in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and Google Scholar (https://scholar.google.com/). PubMed was considered the main and reliable source of information; however, Google Scholar was used if the full text article was not available in PubMed. We initiated our search in PubMed using the search term Japanese medaka (Oryzias latipes), which provided 3,747 results (until 30 June 2023). We narrowed down the search by adding the term “endocrine” (Japanese medaka, Oryzias latipes, and endocrine), which reduced the number to 646, and finally, the addition of the term “endocrine disruption” reduced the results to 239 (Figure 1). We finally sorted 205 articles for review that focused on EATS pathways of Japanese medaka (Figure 1). We identified 128 chemicals that have potential ED effects on this fish (Japanese medaka, O. latipes) (Figure 2). After a literature search, we assembled ED-related information in Supplementary Table S1, which was also deposited in Figshare (doi/10.6084/mg.figshare. 22598068). For classification of these compounds as selective disruptors of EATS pathways, 14 chemicals from 128 searched chemicals were selected as reference chemicals (Figure 2; Table 1). For estrogen endocrine disruptors (EEDs), E2 and EE2 were used as reference chemicals for agonists, and TAM was used for antagonists. For androgen endocrine disruptors (AEDs), 11-KT and TRB were used for agonists, and FLU and VIN were used for antagonists. For thyroid endocrine disruptors, (TEDs), T3 was used for agonists, and PFOA and TBBPA were used for antagonists. For steroidogenesis, TPA and TRF were used for stimulators, and KTC and PCZ were used for inhibitors (Table 1). After critical evaluation of the ED effects of these reference chemicals, the criteria of evaluation of endocrine disruption induced by an EDC on Japanese medaka are determined (Table 1). The chemicals which were unable to fulfill the criteria were considered unclassified.

Strategies for the selection of literature reports from peer-reviewed articles published on Japanese medaka (Oryzias latipes).

Strategies for the selection of chemicals from peer-reviewed articles published on Japanese medaka (O. latipes).

The apical endpoints of the reference chemicals related to EATS pathways in Japanese medaka.

Endocrine targets Reference chemicals Agonists/Antagonists/Stimulator/Inhibitor Affinity Literature End points
Agonists Antagonists
EED E2 Agonist In vitro reporter gene assay (affinity) (Tohyama et al., 2015)i) esr1: EC50 = 1.31 × 10−10 Mii) esr1: EC50 = 1.31 × 10−10 Miii) esr2b: EC50 = 8.16 × 10−11 M Nimrod and Benson (1998), Patyna et al. (1999), Foran et al. (2000), Koger et al. (2000), Shioda and Wakabayashi (2000), Metcalfe et al. (2001), Tabata et al. (2001), Kang et al. (2002a), Kashiwada et al. (2002), Oshima et al. (2003), Balch et al. (2004b), Hall et al. (2005), Zeng et al. (2005), Balch and Metcalfe (2006), Hirai et al. (2006), Zhang et al. (2008d), Sun et al. (2009), Jin et al. (2011a), Kamata et al. (2011), Hirakawa et al. (2012), Lee et al. (2012), Flynn et al. (2013), Lei et al. (2013), Green et al. (2015), Tohyama et al. (2015), Inagaki et al. (2016), Flynn et al. (2017), Lee et al. (2017a), Lee Pow et al. (2017), Bertotto et al. (2019), Kang et al. (2019), Myosho et al. (2019), Ishibashi et al. (2020), Spirhanzlova et al. (2020), Onishi et al. (2021), Pandelides et al. (2021), Horie et al. (2022a). 1. Female biased sex ratio, testis-ova (male feminization)
2. Significant decrease in fecundity
3. Serum VTG level increased in males and females
4. Inhibition of swim bladder inflation
5. Intersex gonad
6. Increased HSI in males
7. Secondary sexual features reduced
8. gsdf expression in XY embryos remained unaltered
9. Vacuolization of hepatocytes in the liver
10. Hydropic degeneration in glomerulus of the kidney
11. Reproductive behavior suppressed with in both sexes
12. No effect on male sexual behavior
13. Histological structure of the kidney disrupted
14. Gene expression
A: Brain:
(i) Male:
1. Upregulation of gnrh1, cyp19b, esr1, fshβ, lhβ
2. Downregulation of gnrhR1, gnrhR2, arα
(ii) Female:
1. Upregulation of esr1 and esr2a
2. cyp19b remained unaltered
B. Liver:
(i) Male:
1. Upregulation of esr1, vtg 1, vtg 2, chgH, chgHm, chgL, cyp1c
2. No alteration in esr1, esr2a, and arα
(ii) Female:
1. vtg1 and vtg2 remained unaltered
In vitro reporter gene assay (Onishi et al., 2021): EC50 = 0.00098 µM (medaka esr1); IC50 = 2.0 µM (medaka arβ) 2. esr2a and arα remained unaltered
C. Gonad
i) Testis:
1. Aromatase expression increased
2. DNA methylation pattern reduced
3. esr1 transcripts decreased
4. Downregulation of cyp11a, cyp17
5. fshR reduced
(ii) Ovary:
1. Downregulation of cyp19a
EE2 Agonist In vitro reporter gene assay: EC50 = 0.00088 µM (Medaka esr1 agonist assay); IC50 = 0.14 µM (medaka arβ antagonist assay (Onishi et al., 2021) Scholz and Gutzeit (2000), Metcalfe et al. (2001), Foran et al. (2002), Islinger et al. (2002), Lee et al. (2002), Seki et al. (2002), Balch et al. (2004a), Nozaka et al. (2004), Chikae et al. (2004), Hano et al. (2005), Zeng et al. (2005), Orn et al. (2006), Zhang et al. (2008c), Hashimoto et al. (2009), Park et al. (2009), Sun et al. (2011a), Hirakawa et al. (2012), Liao et al. (2014), Schiller et al. (2014), Abdel-Moneim et al. (2015), Bhandari et al. (2015), Bhandari et al. (2020), Thayil et al. (2020), Onishi et al. (2021), Pandelides et al. (2021), Horie et al. (2022a), Myosho et al. (2022)
TAM Antagonist In vitro reporter gene assay: IC50 = 0.14 µM (medaka esr1 antagonist assay; (Onishi et al., 2021) Chikae et al. (2004), Sun et al. (2007a), Sun et al. (2011a), Flynn et al. (2017), Onishi et al. (2021). 1. Significant reduction in fecundity
2. Reduction in hatchability, hatching delay, developmental abnormalities
3. Liver VTG in male increased, reduced in females
4. Liver histology of male fish disrupted
5. HSI remained unaltered
6. Secondary sexual features reduced
A: Brain:
i) Male:
1. Upregulation of arα, esr1 and cyp19a
2. Downregulation of cyp19b
ii) Female:
1. No alteration in esr1 and esr2
2. Upregulation of arα and cyp19a
3. Downregulation of cyp19b
B: Liver:
i) Male:
1. Upregulation of vtg1, vtg2, esr1, arα
2. No alteration in esr2a
ii) Female:
1. Upregulation of esr2a
2. Downregulation of vtg1 and vtg2 and esr1
3. arα remained unaltered
C; Gonad
i) Testis:
1. Upregulation of StAR, cyp19b
2. Downregulation of esr1 and esr2a, cyp17a, cyp17b, cyp19a
3. cyp11a, cyp11b remained unaltered
ii) Ovary:
1. Upregulation of StAR, cyp11a
2. Downregulation of esr1, esr2a and arα
3. cyp11b remained unaltered
AED 11-KT Agonist In vitro reporter gene assay: IC50 = 0.0027 (medaka arβ agonist assay; Onishi et al., 2021) Asahina et al. (1989), Leon et al. (2007), Leon et al. (2008), Grillitsch et al. (2010), Onishi et al. (2021), Watanabe et al. (2023). 1. Increased anal fin papillary processes (masculinization in females)
2. Enhancement of growth is sex-specific (males are larger than females)
3. Hypertrophy in thyroid follicular cells induced in both sexes
4. Germ cell necrosis is induced in both sexes
5. Male biased sex-ratio
6. Upregulation of gsdf mRNA in XX embryos (sex reversal of the XX fish)
7. Ovo-testis
8. Decrease in VTG content in females
9. Gene Expression
A: Brain:
TRB Agonist In vitro reporter gene assay: EC50 = 0.0036 µM(medaka arβ agonist assay; Onishi et al., 2021) Orn et al. (2006), Seki et al. (2006), Zhang et al. (2008b), Park et al. (2009), Grillitsch et al. (2010), Flynn et al. (2013), Flynn et al. (2017), Abdel-Moneim et al. (2015), Mazukami-Murata et al. (2016), Kang et al. (2019), Onishi et al. (2021), Horie et al. (2022a), Myosho et al (2022). i) Female
1. Upregulation of gnrhR2, cyp19b
B: Liver:
1. down regulation of vtg1, vtg2, chgH, chgHm mRNAs in the both sexes
2. Down regulation of esr1 in males
3. Upregulation of cyp3A and annexin max2 in females
C; Gonad
(i) Testis:
1. Down regulation of StAR and cyp11b
(ii) Ovary:
1. Upregulation of cyp19a
2. Upregulation of a
FLU Antagonist In vitro reporter gene assay: IC50 = 12 µM (medaka arβ agonist assay; Onishi et al. (2021), Horie et al. (2022a) Chikae et al. (2004), Nozaka, (2004), Kang et al. (2006), Leon et al. (2007), Leon et al. (2008), Nakamura et al. (2014b), Schiller et al. (2014), Onishi et al. (2021). 1. Increased plasma VTG levels in females, not in males
2. Hepatic VTG unaltered in males, decreased in females
3. Fecundity and fertility were significantly decreased
4. Growth (length and weight) was inhibited in males, not in females (sex-specific)
5. No sex reversal
6. Formation of testis-ova; disruption of spermatogenesis and ovarian cell necrosis
VIN Antagonist In vitro reporter gene assay: IC50 = 5.1 µM(medaka arβ antagonist assay; Onishi et al., 2021). Kiparissis et al. (2003b), Nakamura et al. (2014b), Sun et al. (2016b), Flynn et al. (2017), Onishi et al. (2021) 7. Hypertrophy of thyroid follicular cells in males
8. Decrease in papillary process in the anal fin of male fish; females did not develop papillary process in the anal fin
9. Reduced expression of gnrhr2, cyp11b and 3βhsd
10. Repressed esr2a and cyp19a1b
11. No significant induction of gsdf expression in XY embryos
12. Gene expression:
A: Brain:
(i) Male:
1. Upregulation of esr2a, arα and cyp19a and cyp19b genes
B: Testis:
1. esr1 and cyp17b mRNAs were upregulated
2. cyp19a and cyp19b downregulated
TED T3 Agonist Godfrey et al. (2019), Horie et al. (2022d) 1. Decrease in the surface area of swim bladder in females
2. Upregulation of trα and trβ mRNAs
PFOA Antagonists Ji et al. (2008), Lee et al. (2017b), Kang et al. (2019), Godfrey et al. (2019) 1. Fecundity suppressed
2. Females displayed larger swim bladder
3. Thyroid follicles showed hyperplasia, hypertrophy, and colloidal depletion
4. No change in sex ratio
TBBPA Antagonists Horie et al. (2023a) 5. In liver, vtg1 in males, and vtg1 and vtg2 in females increased; VTG protein in males reduced
6. chgH and chgHm mRNA expression in liver of males
7. Increased tshβ, trβ and vtg in females
8. Upregulation of esr2a and vtg in males
9. No effect on expression of dio1 and dio2
MOS TPA Stimulator Jang and Ji (2015). 1. Upregulation of cyp19a, cyp19b, StAR, and cyp17 mRNAs in a concentration-dependent manner
2. erα, vtg1, vtg2, cyp11a, hsd3b. trα, dio2, ahr remained unaltered
TRF Stimulator Zhu et al., (2015)
KTC Inhibitor In vitro reporter gene assay: IC50 = 4.2 µM(medaka arβ antagonist assay) (Onishi et al., 2021) Zhang et al. (2008a), Onishi et al. (2021) 1. Reduced fecundity
2. Anal fin papillae increased in males not in females
3. Liver VTG decreased in both sexes
4. Gene expression
PCZ Inhibitor Zhang et al. (2008a), Schiller et al. (2014), Flynn et al. (2017), Kang et al. (2019), Onishi et al. (2021) A: Brain:
(i) Male:
1. Downregulation of cyp19b
(ii) Female:
1. Downregulation of gnrhR2 and gnrhR3
B: Liver:
(i) Male
1. Upregulation of esr1 and arα mRNAs
2. Downregulation of chgH and chgHm
(ii) Female
1. Upregulation of arα
2. Downregulation of esr1, vtgI, vtg2, cghL, chgH, chgHm
C: Gonad:
(i) Testis:
1. Upregulation of esr2a, lhr, cyp19a
(ii) Ovary:
1. Downregulation of esr2, arα, lhr
2. Downregulation of StAR, cyp11a, cyp11b
2.2 Genes sensitive to EDCs within the EATS modalities of Japanese medaka

Within the EATS modalities, most of the EDCs function via the hormone-responsive element of a target gene by binding to the ligands of nuclear receptors (NRs), including ESRs (esr1, esr2a, and esr2b), ARs (arα and arβ), or TRs (trα and trβ). The effects of EDCs on NRs have been studied in Japanese medaka (Myosho et al., 2022; Tohyama et al., 2015). The expression of estrogen-responsive genes is known to be induced or suppressed by estrogen via ESRs with the estrogen-responsive elements (EREs) of responsive genes. Specifically, VTGs and CHGs encode complex precursor proteins in the egg yolk and eggshell, respectively, and are synthesized in the liver. EDCs with estrogenic potential induced the expression of VTG and CHG in juvenile and mature male fish, respectively, in which the expression levels of vtg and chg are typically low. Therefore, to evaluate the estrogenic potential of EDCs in Japanese medaka, vtgs (vtg1 and vtg2) and chg (chgL, chgH, and chgHm) can be used as markers.

The androgenic effects of EDCs are mediated via direct binding to ARs (arα and arβ) with distinctive binding properties or transactivation activity (Onishi et al., 2021; Kawashima et al., 2022). Molecular effects of AEDs could be identified from secondary sex characteristics (anal fin papillae of males) or indirectly by analysis of the induction/suppression of VTG (vtg1 and vtg2), LH, FSH, aromatase, ESRs, or T-hormone levels (Scholz and Mayers, 2008). The formation of papillary processes in the anal fin of Japanese medaka (males) is augmented by the bone morphogenic protein (bmp7) and lymphoid enhancer-binding factor (lef1), along with arα and arβ which can be used as markers for AEDs during evaluation (Ogino et al., 2014).

EDCs having TH-disrupting potential inhibit or accelerate TH-dependent processes, either directly or indirectly, including TH-dependent gene expression. The HPT axis is highly conserved among vertebrates, and the TH and receptors (trα and trβ) play crucial roles in the regulation of development, growth, and energy metabolism. A number of high-profile environmental pollutants adversely affect the TH system of Japanese medaka, including development, visual performance, malformation of the swim bladder, and TH-dependent gene (tshβ, trα, trβ, dio1, and dio2) expression (Godfrey et al., 2019; Dang et al., 2021; Horie et al., 2022c). Therefore, to evaluate the thyroid-disrupting potential of EDCs, in Japanese medaka, these genes (tshβ, trα, trβ, dio1, and dio2) can be used as markers for TEDs.

Moreover, within the estrogen–androgen–steroidogenesis (EAS) modalities, the steroid hormones, estrogen (E2) and androgen (A), are derived from cholesterol and secreted from the gonads (testis or ovary). The production, conversion, and breakdown of E2 and A in the endocrine glands and target tissues are carefully controlled by a range of steroidogenic enzymes (steroidogenesis), many of which belong to the cytochrome P450 family (CYP11, CYP17, and CYP19). Many EDCs have the abilities to disrupt the synthesis and function of steroidogenic enzymes, resulting in inappropriate concentrations of E2 or A, which impacts the reproduction, development, growth, and metabolism of fish (Japanese medaka). The enzyme aromatase (CYP19) converts testosterone/androgen (A) into estradiol/estrogen (E) and controls the fine balance between these two potent sex steroids. Therefore, the genes that show potential to regulate steroidogenesis in Japanese medaka are used as markers during EDC evaluation.

 3 Results

Depending on the ED effects, we sorted 205 articles (Table 2) consisting of 128 chemicals (1.6 articles/chemicals or the approximate ratio is 8 articles:5 chemicals) that showed potential effects on Japanese medaka. Furthermore, based on the apical endpoints selected from 14 reference chemicals (Table 1) and after reviewing 165 articles, we identified 83 chemicals that target EATS pathways of Japanese medaka (Tables 3–10), and due to the lack of sufficient information, 45 chemicals reviewed from 60 articles remained unclassified (Table 11). Moreover, among the 83 chemicals that target EATS pathways, 43 chemicals were recommended for Tier 2 tests, and 13 chemicals show enough potential to be considered EDCs without any further tier-based studies (Tables 3–10). The rest of the EATS chemicals need further studies on Tier 1 screening. Moreover, with regard to the apical endpoints, the EATS chemicals were further classified as agonists and antagonists of EEDs, AEDs, and TEDs, and stimulators or inhibitors of steroidogenesis (Figure 2; Tables 3–10).

Literature reports sorted for the evaluation of the effects of EDCs on Japanese medaka (Oryzias latipes).

Literature author EED AED TED MOS Unclassified
1 Abdel-Moneim et al. (2015)
2 Asahina et al. (1989)
3 Asala et al. (2021)
4 Asala et al. (2022)
5 Balch et al. (2004a)
6 Balch et al. (2004b)
7 Balch and Metcalfe (2006)
8 Beltran et al. (2022)
9 Bertotto et al. (2019)
10 Bhandari et al. (2015)
11 Bhandari et al. (2020)
12 Cheek et al. (2001)
13 Chen et al. (2022)
14 Chikae et al. (2004)
15 Chu et al. (2016)
16 Coronado et al. (2008)
17 Dasmahapatra et al. (2020a)
18 Dasmahapatra et al. (2020b)
19 Dasmahapatra and Tchounwou (2022a)
20 Dasmahapatra and Tchounwou (2022b)
21 Dasmahapatra and Tchounwou (2023a)
22 Dasmahapatra and Tchounwou (2023b)
23 Devoy et al. (2023)
24 Edmunds et al. (2000)
25 Flippin et al. (2007)
26 Flynn et al. (2013)
27 Flynn et al. (2017)
28 Flynn et al. (2018)
29 Foran et al. (2000)
30 Foran et al. (2002)
31 Foran et al. (2004)
32 Fujita et al. (2022)
33 Godfrey et al. (2019)
34 Gonzalez-Doncel et al. (2016)
35 Gonzalez-Doncel et al. (2014a)
36 Gonzalez-Doncel et al. (2014b)
37 Gonzalez-Doncel et al. (2017)
38 Gray and Metcalfe (1997)
39 Gray et al. (1999a)
40 Gray et al. (1999b)
41 Green et al. (2015)
42 Grillitsch et al. (2010)
43 Gronen et al. (1999)
44 Hall et al. (2005)
45 Hall et al. (2007)
46 Hamm and Hilton, (2000)
47 Han et al. (2010)
48 Hano et al. (2005)
49 Hano et al. (2007)
50 Hashimoto et al. (2009)
51 Hirai et al. (2006)
52 Hirakawa et al. (2012)
53 Hirako et al. (2017)
54 Hishida and Kawamoto. (1970)
55 Hong et al. (2007)
56 Horie et al. (2017)
57 Horie et al. (2018)
58 Horie et al. (2019)
59 Horie et al. (2021)
60 Horie et al. (2022a)
61 Horie et al. (2022b)
62 Horie et al. (2022c)
63 Horie et al. (2022d)
64 Horie et al. (2023a)
65 Horie et al. (2023b)
66 Horng et al. (2010)
67 Inagaki et al. (2016)
68 Inui et al. (2003)
69 Ishibashi et al. (2004)
70 Ishibashi et al. (2006)
71 Ishibashi et al. (2020)
72 Islinger et al. (2002)
73 Jang and Ji (2015)
74 Ji et al. (2008)
75 Ji et al. (2010)
76 Ji et al. (2012)
77 Jin et al. (2011a)
78 Jin et al. (2011b)
79 Jin et al. (2020)
80 Kamata et al. (2011)
81 Kang et al. (2002a)
82 Kang et al. (2002b)
83 Kang et al. (2003)
84 Kang et al. (2006)
85 Kang et al. (2008)
86 Kang et al. (2019)
87 Kashiwada et al. (2002)
88 Kawashima et al. (2022)
89 Kim et al. (2007)
90 Kim et al. (2012)
91 Kim et al. (2014)
92 Kim et al. (2017)
93 Kim et al. (2020)
94 Kiparissis et al. (2003a)
95 Kiparissis et al. (2003b)
96 Knorr and Braunbeck (2002)
97 Kobayashi et al. (2005)
98 Koger et al. (2000)
99 Kuhl and Brouwer., 2006
100 Kwak et al. (2018)
101 LaLone et al. (2013)
102 Lee et al. (2002)
103 Lee et al. (2011)
104 Lee et al. (2012)
105 Lee et al. (2013)
106 Lee et al. (2014)
107 Lee et al. (2017a)
108 Lee et al. (2017b)
109 Lee et al. (2019a)
110 Lee et al. (2019b)
111 Lee Pow et al. (2017)
112 Lei et al. (2013)
113 Lei et al. (2016)
114 Leon et al. (2007)
115 Leon et al. (2008)
116 Li et al. (2016)
117 Li et al. (2017)
118 Li et al. (2019)
119 Liang et al. (2020)
120 Liao et al. (2014)
121 Lin et al. (2014)
122 Liu et al. (2018)
123 Matten et al. (2023)
124 Mazukami-Murata et al. (2016)
125 Metecalfe et al. (2001)
126 Mihaich et al. (2019)
127 Miyagawa et al. (2014)
128 Myla et al. (2021a)
129 Myla et al. (2021b)
130 Myosho et al. (2019)
131 Myosho et al. (2022)
132 Nair et al. (2017)
133 Nakamura et al. (2014a)
134 Nakamura et al. (2014b)
135 Nakayama et al. (2011)
136 Nimrod and Benson (1998)
137 Nozaka et al. (2004)
138 Ogino et al. (2014)
139 Onishi et al. (2021)
140 Orn et al. (2006)
141 Oshima et al. (2003)
142 Pandelides et al. (2021)
143 Papoulias et al. (2000)
144 Park et al. (2008)
145 Park et al. (2009)
146 Patyna et al. (1999)
147 Paul et al. (2021)
148 Powe et al. (2018)
149 Richter et al. (2016)
150 Robinson et al. (2017)
151 Saunders et al. (2015)
152 Schiller et al. (2013)
153 Schiller et al. (2014)
154 Scholz and Gutzeit (2000)
155 Seki et al. (2002)
156 Seki et al. (2003a)
157 Seki et al. (2003b)
158 Seki et al. (2004)
159 Seki et al. (2006)
160 Shioda and Wakabayashi (2000)
161 Smith et al. (2019)
162 Song et al. (2020)
163 Spirhanzlova et al. (2017)
164 Spirhanzlova et al. (2020)
165 Su et al. (2023)
166 Sun et al. (2007a)
167 Sun et al. (2007b)
168 Sun et al. (2009)
169 Sun et al. (2011a)
170 Sun et al. (2014)
171 Sun et al. (2016a)
172 Sun et al. (2016b)
173 Sun et al. (2016c)
174 Suzuki et al. (2004)
175 Tabata et al. (2001)
176 Tabata et al. (2004)
177 Tagawa and Hirano (1991)
178 Teather et al. (2005)
179 Thayil et al. (2020)
180 Thresher et al. (2011)
181 Tilton et al. (2003)
182 Tohyama et al. (2015)
183 Uchida et al. (2010)
184 Wagner et al. (2017)
185 Wang et al. (2016)
186 Watanabe et al. (2017)
187 Watanabe et al. (2023)
188 Yamamoto et al. (2007)
189 Yamamoto et al. (2011)
190 Yan et al. (2020)
191 Yokota et al. (2000)
192 Yokota et al. (2001)
193 Yokota et al. (2005)
194 Yokota et al. (2017)
195 Yokota et al. (2018)
196 Yum et al. (2010)
197 Zeng et al. (2005)
198 Zha et al. (2006)
199 Zhang and Hu (2008)
200 Zhang et al. (2008a)
201 Zhang et al. (2008b)
202 Zhang et al. (2008c)
203 Zhang et al. (2008d)
204 Zhang et al. (2008e)
205 Zhu et al. (2015)

Only the names of the authors are listed in the first column of the table. The cells filled in colors (red = EED; blue = AED; green = TED; yellow = MOS; black = unidentified pathways) represent the specific endocrine pathways/organs disrupted by EDCs. EDCs, endocrine-disrupting chemicals; EED, estrogen endocrine disruptor; AED, androgen endocrine disruptor; TED, thyroid endocrine disruptor; MOS, modulators of steroidogenesis.

Potential EED agonist chemicals identified from the literature search.

Serial number Name of the chemical Nature (source) Significant endpoints Reference Recommendation
1 AR-1260 Polychlorinated biphenyl (persistent organic pollutant)  1. Induced vtg, chgL, and chgHm mRNAs in the liver of males Yum et al. (2010) Tier 2 (thyroid-dependent mechanisms)
2 AZM Organophosphate pesticide [agricultural]  1. Female-biased sex ratio Teather et al. (2005) Tier 1 (thyroid-dependent mechanisms)
3 BZP Antimicrobial agent [personal care product]  1. Serum VTG enhanced in males Yamamoto et al. (2007) Tier 2 (thyroid-dependent mechanisms)
 2. vtg1, vrg2, chgL, chgH, chgHm, esr1, and cyp1a genes upregulated in the liver of males
4 BMT Synthetic glucocorticoid (pharmaceuticals)  1. Both vtg1 and vtg2 mRNAs were induced in the liver of male fish Su et al. (2023) Tier 2 (thyroid-dependent mechanisms)
 2. Ova found in the testis
 3. Serum T reduced, while E2 induced
5 BPA Raw material for polycarbonate plastic [industrial]  1. Anal fin papillae in males disappeared Shioda and Wakabayashi (2000) (Thyroid-dependent mechanisms)
 2. Testis–ova Yakota et al. (2000)
 3. VTG mRNAs (vtg1 and vtg2) and protein in the liver of males increased Metcalfe et al. (2001)
 4. Upregulation of chgL and chgH in the liver of male fish Tabata et al. (2001)
 5. Expression of esr1, esr2a, and esr2b genes remained unchanged Kang et al. (2002b) Kashiwada et al. (2002) Lee et al. (2002) Zeng et al. (2005) Kamata et al. (2011) Lee et al. (2012) Schiller et al. (2014) Bhandari et al. (2015, 2020) Tohyama et al. (2015) Inagaki et al. (2016) Li et al. (2016); Li et al. (2017) Horie et al. (2019) Ishibashi et al. (2020) Thayil et al. (2020) Kawashima et al. (2022)
6 CFR Antibiotic (pharmaceutical)   1. Plasma E2 level increased in females Kim et al. (2017) Tier 2 (thyroid-dependent mechanisms)
  2. Sex-specific alteration in the gene expression pattern of the HPG axis
7 CLT Organochlorine pesticide (fungicide) [agricultural]  1. Female-biased sex ratio Teather et al. (2005) Tier 1 (gene analysis on EATS pathways}
8 CTC Antimicrobial agent [pharmaceutical]  1. Enhancement of serum E2 and liver VTG content in male fish Kim et al. (2007) Ji et al. (2010) Tier 2 (thyroid-dependent mechanisms)
9 p,p′-DDE DDT metabolite [agricultural]  1. Increased HSI Zhang and Hu (2008) Tier 2 (thyroid-dependent mechanisms)
 2. Development of intersex Horie et al. (2022a)
 3. Upregulation of vtg1, vtg2, chgH, chgL, and esr1 genes in the liver of male fish
10 o,p′-DDT Organochlorine pesticide [agricultural]  1. Female-biased sex ratio in fish Edmunds et al. (2000) (Thyroid-dependent mechanisms)
 2. Ova–testis Cheek et al. (2001)
 3. chgH, chgL, chgHm, vtg1, vtg2, and esr1 mRNAs induced in the liver of male Kuhl and Brouwer (2006) Uchida et al. (2010)
11 DES Nonsteroidal estrogen [pharmaceutical]  1. vtg1 mRNA was upregulated in males Zeng et al. (2005) Tier 2 (thyroid-dependent mechanisms)
 2. Sex-reversed males laid eggs Lei et al. (2016)
12 EDS Organochlorine pesticide [agricultural]  1. Female-biased sex-ratio Teather et al. (2005); Lee et al. (2013) Tier 2 (thyroid-dependent mechanisms)
 2. Serum VTG induced in male fish
13 EQ Metabolite of the soy isoflavone daidzein [natural product]  1. Testis–ova formation in males Kiparissis et al. (2003a) Tier 2 (thyroid-dependent mechanisms)
 2. Intersex Wang et al. (2016)
 3.11-KT in the plasma reduced
14 E3 Natural estrogen [pharmaceutical]  1. Testis–ova Metcalfe et al. (2001) Tier 2 (thyroid-dependent mechanisms)
 2. Induced vtg1 mRNA in males Zeng et al. (2005)
15 E1 Natural estrogen [pharmaceutical]  1. Female-biased sex ratio Metcalfe et al. (2001) (Thyroid-dependent mechanisms)
 2. Liver VTG induced in both sexes Nakamura et al. (2014a) Kawashima et al. (2022)
16 4-MBC Camphor derivative [personal care product]  1. Enhancement in the serum VTG in both sexes Inui et al. (2003) Tier 2 (thyroid-dependent mechanisms)
 2. Upregulation of vtg1, vtg2, chgL, and chgH, and esr1 mRNAs in the liver of males Liang et al. (2020)
 3. Decrease in 11-KT in the plasma of males and enhancement of E2 in females
 4. Inhibition of spermatogenesis in the testis
17 MPB Antimicrobial agent [personal care product]  1. Plasma VTG content increased in males Yamomoto et al. (2011) Tier 2 (thyroid-dependent mechanisms)
 2. Upregulation of vtg2, chgL, chgH, chgHm, and esr1 in the liver of males Kawashima et al. (2022)
18 4-NP Alkylphenol [industrial]  1. Males developed testis–ova with the sex ratio skewed toward female Gray and Metcalfe, (1997); Nimrod and Benson, (1998); Shioda and Wakabayashi, (2000); Tabata et al. (2001); Yokota et al. (2001); Islinger et al. (2002); Kashiwada et al. (2002); Lee et al. (2002); Kang et al. (2003); Seki et al. (2003a); Nozaka et al. (2004); Kobayashi et al. (2005); Zeng et al. (2005), Balch and Metcalfe, (2006); Ishibashi et al. (2006); Jin et al. (2011a); Lee et al. (2012); Miyagawa et al. (2014); Tohyama et al. (2015); Watanabe et al. (2017); Ishibashi et al. (2020); Horie et al. (2021); Kawashima et al. (2022) (Thyroid-dependent mechanisms)
 2. HSI in adult males increased
 3. Serum VTG in males and hepatic VTG in both sexes increased
 4. Female-like anal fins in some males
19 OMC Organic UV-B filter (PCP) [personal care product]  1. Enhancement of plasma VTG in males Inui et al. (2003) Tier 2 (thyroid-dependent mechanisms)
 2. Upregulation of vtg1, vtg2, chgL, chgH, and esr1 mRNAs in males
20 4-OP De-ethoxylated alkylphenol [industrial]  1. Female-biased sex ratio Knorr and Braunbeck (2002) Tier 1 (thyroid-dependent mechanisms)
 2. Some F1 males developed testis–ova
21 PCPL Insecticide [agricultural]  1. Nonlinear enhancement in the plasma VTG levels in males and a concentration-dependent decrease in plasma VTG levels in females Zha et al. (2006) Tier 1 (thyroid-dependent mechanisms)
 2. Testis–ova formation in males and a degenerative ovary in females
22 PPB Personal care product [personal care product]  1. Enhancement in the plasma VTG content in males Inui et al. (2003) Tier 2 (interruption in swim bladder inflation needs further studies in thyroid-dependent mechanisms)
 2. Upregulation of vtg1, vtg2, chgL, chgH, and esr1 in the liver of male fish Gonzalez-Doncel et al. (2014a) Kawashima et al. (2022)
23 4t-OP Alkylphenol [industrial]  1. Sex ratio skewed toward females Gray et al. (1999a) Effect on swim bladder inflation needs further study on thyroid-dependent mechanisms
 2. Testis–ova observed in male fish Gray et al. (1999b)
 3. Liver VTG increased in both sexes Gronen et al. (1999)
 4. Inhibition of spermatogenesis Seki et al. (2003a)
 5. HSI in adult males increased Nozaka et al. (2004)
 6. Basophilia in the male liver Flynn et al. (2017) Horie et al. (2022a) Kawashima et al. (2022)
24 4t-PP Alkylphenol [industrial]  1. The appearance of secondary sexual features was reduced in males Seki et al. (2003b) Tier 2 (thyroid-dependent mechanisms)
 2. Testis–ova in the gonad of males Yokota et al. (2005)
 3. Hepatic VTG enhanced in both sexes Onishi et al., 2021
 4. HSI increased in males Kawashima et al. (2022)
25 TBCO Brominated flame retardant [industrial]  1. Upregulation of chgHm in the liver of males Saunders et al. (2015) Tier 2 (thyroid-dependent mechanisms)
 2. Upregulation of chgH, vtg2, and esr1 in the liver of females Sun et al. (2016c)
 3. Downregulation of esr1, esr2a, and arα in both the testis and ovary Devoy et al. (2023)

Potential EED antagonist chemicals identified by the literature search.

Serial number Name of the chemical Nature (source) Significant endpoint Reference Recommendation
1 ATZ Herbicide [agricultural]  1. cyp19a mRNA upregulated in the brain Zhang et al. (2008d) Tier 2 (thyroid-dependent mechanisms)
 2. Downregulation of esr1 mRNA in the testis Richter et al. (2016)
 3. VTG in the liver of females reduced
2 MET Drug (pharmaceutical)  1. Intersex observed in females Lee et al. (2019a) Tier 2 (studies related to thyroid-related gene expression are necessary)
 2. vtg1 declined in males
 3. Thyroid histology remained unchanged
3 PFOA Fluorinated organic compounds (wastewater effluent)  1. Reduced fecundity Lee et al. (2017a) Tier 2 (studies related to thyroid-related gene expression are necessary)
 2. Increase in the serum VTG content in F2 males
 3. Male-biased sex ratio with no change in intersex either in F1 or F2
4 TPhP Flame retardant/plasticizer [industrial]  4. Larval exposure reduced ovarian development in females Li et al. (2019) Tier 2 (thyroid-dependent mechanisms)
 5. Plasma T enhanced in females Kawashima et al. (2022)
 6. Hepatic VTG in females reduced

Potential androgen endocrine-disrupting agonist chemicals identified from the literature search.

Serial number Name of the chemical Nature (source) Significant endpoint Reference Recommendation
1 11-OA Glucocorticoid metabolite [pharmaceutical]  1. Male-biased sex ratio Grillitsch et al. (2010) Tier 1 (gene expression analysis and thyroid-dependent mechanisms)
2 BF Pyrethroid insecticide [agricultural]  1. Induced masculinization in the anal fin papillae Bertotto et al. (2019) Tier 2 (gene expression analysis related to EATS pathways and thyroid-dependent mechanisms)
 2. Male-biased sex ratio
3 CFD Antibiotic (pharmaceutical)  1. Plasma E2 was decreased in males and enhanced in females Kim et al. (2017) Tier 2 (thyroid-dependent mechanisms)
 2. Downregulation of cyp19a in the testis and upregulation of cyp19a in the ovary
 3. Sex-specific alteration in the gene expression of the HPG axis
4 DHT Metabolite of testosterone [pharmaceutical]  1. Anal fin papillae increased in both sexes Spirhanzlova et al. (2020); Onishi et al. (2021) Tier 1 (gene expression analysis related to EATS pathways and thyroid-dependent mechanisms)
 2. Sex ratio skewed toward males
5 GEN Isoflavone [natural Product]  1. Masculinization features in the secondary sex characteristics of XX females Hishida and Kawamoto (1970); Kiparissis et al. (2003a) Schiller et al. (2013, 2014) Tier 2 (downregulation of dio2 indicated more studies on thyroid-dependent mechanisms are necessary)
6 LNG Second-generation progestin (pharmaceutical)  1. Liver VTG downregulated in females Onishi et al. (2021); Pandelides et al. (2021); Watanabe et al. (2023) Tier 2 (effects on the swim bladder suggest more studies on thyroid-dependent mechanisms are necessary)
 2. Masculinization of the anal fin papillae in females
 3. Ovotestis in females
7 MT Synthetic androgen [pharmaceutical]  1. Sex reversal of XX females Papoulias et al. (2000); Chikae et al. (2004); Nozaka et al. (2004); Seki et al. (2004); Kang et al. (2008); Ogino et al. (2014); Myosho et al. (2019); Onishi et al. (2021) Thyroid-dependent mechanisms
 2. Serum VTG decreased in females
 3. Upregulation of gsdf mRNA in XX fish
8 P4 Female hormone (steroid) [pharmaceutical]  1. Females developed papillae on the anal fin rays Onishi et al. (2021) Tier 2 (thyroid-dependent mechanisms)
9 SPR Synthetic aldosterone receptor agonist [pharmaceutical]  1. Anal fin papillae increased in both sexes LaLone et al. (2013) Tier 2 (thyroid-dependent mechanisms)
 2. Hepatic vtg reduced in female fish
10 T Male hormone (steroid) [pharmaceutical]  1. Intersex gonad Koger et al. (2000) Tier 1 (thyroid-dependent mechanisms)

Potential AED antagonist chemicals identified by the literature search.

Serial number Name of the chemical Nature (source) Significant endpoint Reference Recommendation
1 CPA Male contraceptive [pharmaceutical]  1. Testis–ova observed in male fish Kiparissis et al. (2003b) Tier 2 (gene expression analysis of EATS pathways and thyroid-related mechanisms)
 2. No difference in the phenotypic sex ratio
 3. Inhibition of spermatogenesis
2 DZ Organophosphate insecticide [agricultural]  1. Number of anal fin papillae in F1 male fish reduced Hamm and Hinton, (2000); Flynn et al. (2018); Kawashima et al. (2022) Tier 2 (effects on the swim bladder suggests more studies on thyroid-dependent mechanisms are necessary)
3 2-EHHB Antimicrobial agent (personal care product)  1. Hepatic vtg1 upregulated in F1 males and downregulated in F2 males Matten et al. (2023) Tier 2 (gene expression analysis of EATS pathways and thyroid-related mechanisms)
 2. Anal fin papillae in F2 males reduced
 3. Delay in reproductive tract development in F1 males
 4. Eosinophilia observed in renal ducts (kidney) of females
4 FNT Organophosphate pesticide [agricultural]  1. Number of papillary processes decreased in XY medaka Horie et al. (2017; 2022a) Tier 1 (thyroid-related mechanisms)
5 KC-400 Polychlorinated biphenyl (industrial)  1. Downregulation of chgL, chgHm, and arα, in both males and females Nakayama et al. (2011) Tier 1 (thyroid-related mechanisms)
 2. Downregulation of vtg1 in males and upregulation in females
6 LD-BP Structural analog of bisphenol A [industrial]  1. Liver VTG in males and females increased Li et al. (2016, 2017) Tier 1 (thyroid-related mechanisms)
 2. Aggregation and hyperplasia of interstitial cells occurred in the testis, while atretic follicles, with interstitial cell fibrosis, occurred in the ovary
7 PCB 126 Coplanar PCB (persistent organic pollutants)  1. Downregulation of chgL, chgHm, and arα, in both males and females Nakayama et al. (2011) Tier 1 (thyroid-related mechanisms)
 2. Downregulation of vtg1 in males and upregulation of vtg1 in females
8 TCrP Organophosphate flame retardant [industrial]  3. Suppression of 11-KT and T levels and enhanced E2 level in the plasma of male fish Chen et al. (2022) Tier 1 (thyroid-related mechanisms)
 4. Dilated the efferent duct of the testis
 5. Intersex development

Potential TED agonist chemicals identified from the literature search.

Serial number Name of the chemical Nature (source) Significant endpoint Reference Recommendation
1 MTC Herbicide [agricultural] 1. Upregulation of the expression of trα, trβ, and dio2 mRNAs in females Jin et al. (2011b) Tier 1 (thyroid histopathology and EATS-dependent mechanisms)

Potential TED antagonist chemicals identified from the literature search.

Serial number Name of the chemical Nature (source) Significant endpoint Reference Recommendation
1 ATBC Non-phthalate plasticizer [industrial]  1. Disruption of swim bladder inflation Horie et al. (2022b); Horie et al. (2023b) Tier 1 (downregulation of vtg1 and vtg2 mRNAs in the liver of XX fish indicated more studies needed on EAS pathways are required)
 2. Downregulation of trα, trβ, and dio2
2 DEHS Plasticizer [industrial]  1. Downregulation of dio2 Horie et al. (2022c) Tier 1 (more studies needed on EATS pathways)
3 DIC NSAID [pharmaceutical]  1. Swim bladder inflation inhibition in larvae Hong et al. (2007); Lee et al. (2011): Yokata et al. (2017), Yokata et al. (2018); Pandelides et al. (2021) Tier 2 (more studies needed on EATS pathways)
4 EHMC Organic ultraviolet UV-B filter [personal care products]  1. T3 and T4 concentrations decreased Lee et al. (2019b) Tier 2 (studies other than those based on EATS pathways are necessary)
 2. Downregulation of dio2
 3. Upregulation of trh
5 PFBA Halogenated chemical [industrial]  1. No swim bladder inflation Godfrey et al. (2019); Horie et al. (2022d) Tier 1 (more studies needed on EAS pathways)
6 (PFOS/PFOSA) Halogenated compound [industrial]  1. Hyperplasia, hypertrophy, and colloidal depletion in thyroid follicles Ji et al. (2008) Tier 1 (more studies needed on EATS pathways)
Kang et al. (2019)
7 PTU Anti-thyroid medicine [pharmaceutical]  1. Modulation of swim bladder inflation Horie et al. (2023a) Tier 1 (studies related to EAS pathways)
8 SPC Anti-thyroid chemical [industrial]  1. Downregulation of trα and trβ genes Lee et al. (2014) Tier 1 (studies related to EAS pathways)
 2. Upregulation of dio2
 3. Decrease in T4 levels but T3 remained unaltered
 4. Fecundity decreased with the increase in temperature
9 TU Anti-thyroid chemical [industrial]  5. Decreased thyroid hormone levels in adult fish and fertilized eggs Tagawa and Hirano (1991) Tier 1 (although anti-thyroid effects were established, EAS-mediated pathways need to be investigated)
 6. No effect on the length and weight of the larvae
10 RND Herbicide (commercial formulation of glyphosate) [agricultural] 1. Uninflated swim bladder Smith et al. (2019) Tier 1 (studies related to thyroid histophysiology and thyroid-dependent gene expression)
11 TDCPP Halogen-containing organophosphorus compound [industrial]  1. Females failed to inflate the swim bladder Godfrey et al. (2019) Tier 1 (upregulation of vtg1 and vrg2 mRNAs indicates further studies on EAS mechanisms are necessary)
Horie et al. (2022a); Horie et al. (2022d)

Potential steroidogenesis stimulating EDCs identified from the literature search.

Serial number Name of the chemical Nature (source) Significant endpoint Reference Recommendation
1 OCL Organic UV filter (PCP) [personal care product]  1. Upregulation of fshβ, lhβ, fshr, lhr, ar, esr1, esr2a, StAR, hsd3β, cyp17α, and cyp19β mRNAs in the HPG axis Yan et al. (2020) Tier 2 (thyroid-dependent mechanisms)
 2. E2 and 11-KT increased in plasma
 3. Upregulation of vtg in the liver of males and females
2 FPN Phenylpyrazole insecticide [agricultural]  1. Upregulation of StAR, cyp17a, and cyp19b in males Sun et al. (2014) Tier 2 (thyroid-dependent studies)
 2. Upregulation of both vtg1 and vtg2 mRNAs in both sexes Wagner et al. (2017)
 3. No alteration occurred in esr1, esr2a, and arα in both sexes
3 RCT β-adrenergic agonist drug [pharmaceutical]  4. Upregulation of cyp19a and cyp19b mRNAs in females Sun et al. (2016a) Tier 2 (thyroid-dependent mechanisms)
 5. Upregulation of vtg1, vtg2, esr1, and esr2 mRNAs in females
4 TRI Pharmaceuticals [pharmaceutical]  1. Upregulation of StAR, 3β-hsd, 20β-hsd, cyp11a, cyp11b, cyp17a, cyp17b, and cyp19a in males Sun et al. (2014) Tier 2 (thyroid-dependent mechanisms)
 2. Upregulation of vtg1 and vtg2 in males and downregulation of vtg1 and vtg2 in females
 3. Upregulation of esr1 and arα in males

Potential steroidogenesis inhibitory EDCs identified by the literature search.

Serial number Name of the chemical Nature (source) Significant endpoint Reference Recommendation
1 BP UV filters used in cosmetics [personal care product]  1. Liver VTG in both male and females increased by BP2 Coronado et al. (2008) Tier 2 (thyroid-dependent mechanisms)
 2. Enhanced T concentration in the serum of male fish by BP3 Kim et al. (2014); Kawashima et al. (2022)
 3. Upregulation of vtg1 and vtg2 mRNAs, and the VTG protein in the liver of male fish by BP3
 4. Downregulation of gonadal StAR, cyp17, hsd3b, hsd17b3, and cyp19a by BP3
2 FAD Nonsteroidal aromatase inhibitor [pharmaceutical]  1. Aromatase enzyme activity reduced Suzuki et al. (2004) Tier 2 (thyroid-dependent mechanisms)
 2. Upregulation of cyp19a in the ovary Kuhl and Brouwer, (2006)
 3. Downregulation of esr1 and chgL in the liver of females Thresher et al. (2011) Park et al. (2008) Zhang et al. (2008b)
3 LET Nonsteroidal triazole [pharmaceutical]  1. Male-biased sex ratio Sun et al. (2007b, 2009, 2011a) Tier 2 (thyroid-dependent mechanisms)
 2. Downregulation of esr1, vtg1, and vtg2 in the liver of males Liao et al. (2014)
 3. Serum VTG levels remained unaltered in males and decreased in females
 4. Upregulation of StAR, cyp11a, cyp11b, cyp17a, cyp17b, and esr2 and downregulation of cyp19 b and arα in the ovary
 5. Upregulation of cyp11a and cyp11b and no alteration in cyp17a, cyp17b, cyp19a, and cyp19b mRNAs in the testis
4 LNR Herbicide [agricultural]  1. Downregulation of 3β-hsd and cyp11b Schiller et al. (2014) Tier 1 (gene expression analysis related to EATS pathways and thyroid-dependent mechanisms)
 2. E2 or T-induced expression of chgH was downregulated Spirhanzlova et al. (2017)
5 PRN Herbicide [agricultural]  1. Downregulation of cyp11b, 3β-hsd, gnrhr2, and cyp19a1b Schiller et al. (2014) Tier 1 (gene expression analysis related to EATS pathways and thyroid-dependent mechanisms)
6 TPT-Cl Organotin compound [industrial]  1. Downregulation of 17β-hsd1 and cyp19a in the ovary Zhang et al. (2008e) Tier 1 (studies on EATS pathways and thyroid-dependent mechanisms)
 2. Upregulation of cyp1a and cyp2a1 Horie et al. (2022a)
 3. ugt2a3 and 17β-hsd1 in the liver of both sexes
 4. No change in gsdf mRNA expression in both XX and XY embryos

Potential EDCs with unidentified EATS pathways.

Serial number Name of the chemical Nature (source) Reference Reason
1 ACT NSAID (pharmaceutical) Kim et al. (2012) Limited data (nonlinear induction of hepatic VTG in males was due to stress)
2 AMT Herbicide [agricultural] Horie et al. (2022a) Insufficient data
3 BZT-UV UV stabilizer; persistent organic pollutants (POPs) [personal care product] Fujita et al. (2022) Due to stress
4 BKC Quaternary ammonium compound [personal care product] Kim et al. (2020) Insufficient data (enhancement of vtg1 in the whole body was probably due to stress)
5 i-BP Antimicrobial [personal care product) Yamamoto et al. (2007) Insufficient data (estrogenic potential)
6 n-BP Antimicrobial [personal care product) Yamamoto et al. (2007) Insufficient data (estrogenic potential)
7 Cd Metal [inorganic] Tilton et al. (2003), Hirako et al. (2017) Insufficient data (anti-androgenic effects were probably mediated through stress)
8 ClxBPA Chlorinated product of BPA Tabata et al. (2004) Limited data (the compound showed estrogenic potential with regard to serum VTG in male fish)
9 CMP Biocide [personal care product] Flynn et al. (2017); Onishi et al. (2021) Inconsistent alteration of liver VTG in both sexes indicate the estrogenic potential of the compound
10 CYN Herbicide [agricultural] Kawashima et al. (2022) Effects are not mediated through EATS pathways
11 CHDM Plasticizer [industrial] Jang and Ji, (2015) Effects are not mediated through EATS pathways
12 DBP Plasticizer [industrial] Nozaka et al. (2004) VTG in male fish remained unchanged
13 DEHP Plasticizer [industrial] Metcalfe et al. (2001) Effects are not mediated through EATS pathways
14 DIBP Plasticizer [industrial] Kawashima et al. (2022) Limited AED features (hepatic VTG reduced in females)
15 END Organochlorine pesticide [agricultural] Horie et al. (2022a) Limited information (not related to EATS-mediated pathways)
16 FNC Insecticide [agricultural] Spirhanzlova et al. (2017) Effects not related to EATS pathways
17 FV Pyrethroid insecticide [agricultural] Kawashima et al. (2022) No effects on estrogen-dependent mechanisms
18 FLX Antidepressant [pharmaceutical] Foran et al. (2004) Mostly due to toxicity and not mediated through EATS pathways
19 FLR Herbicide (Agricultural) Jin et al. (2020) Effects are mediated through oxidative stress
20 GLP Herbicide [agricultural] Smith et al. (2019) Effects are mediated through oxidative stress
21 GO Nanocarbon [inorganic] Dasmahapatra et al. (2020a, b) Effects are not mediated through EATS pathways
Myla et al. (2021a); Myla et al. (2021b)
Asala et al. (2021); Asala et al. (2022)
Dasmahapatra and Tchounwou (2022a); Dasmahapatra and Tchounwou (2022b); Dasmahapatra and Tchounwou (2023a); Dasmahapatra and Tchounwou (2023b)
22 IBP Nonsteroidal anti-inflammatory drug [pharmaceutical] Flippin et al. (2007); Han et al. (2010) VTG induction in male fish serum is probably due to stress
23 LIN Antibiotic (pharmaceutical) Kim et al. (2012) Insufficient data (insignificant increase in hepatic VTG in male fish)
24 MTZ Goitrogen [pharmaceutical] Godfrey et al. (2019) Insufficient data (vtg gene expression upregulated in males)
25 MXC Organochlorine pesticide [agricultural] Nimrod and Benson (1998) Insufficient data
Zeng et al. (2005)
26 MCB Fungicide [agricultural] Lin et al. (2014) Induced cyp3a enzyme activities
27 1NT Insecticide [agricultural] Kawashima et al. (2022) Limited data (hepatic VTG enhanced in females)
28 NPX NSAID [pharmaceutical] Kwak et al. (2018) Although transcription of vtg1, erβ2, and cyp17 genes significantly increased, data are still limited for consideration as EEDs
29 NDEA Carcinogen [industrial] Nair et al. (2017) Limited data (sex-specific reduction in germ cells occurred only in the ovary)
30 OYZ Herbicide [agricultural] Hall et al. (2005, 2007) Insufficient data (induction of choriogenin in liver and abnormal gonad histology)
31 OXF Herbicide [agricultural] Powe et al. (2018) Toxicological effects
32 OTC Antibiotic [pharmaceutical] Ji et al. (2010, 2012) Insufficient data
33 PDM Herbicide [agricultural] Kawashima et al. (2022) Insufficient data (hepatic VTG enhanced in males)
34 PHN Aromatic hydrocarbon [burning of fuels] Horng et al. (2010) No significant EATS-mediated effects
35 PHT Epileptic drug [pharmaceutical] Kawashima et al. (2022) Insufficient data
36 RLX SERM [pharmaceutical] Onishi et al. (2021) Insufficient data (liver VTG enhanced in males and reduced in females)
37 SFT Veterinary pharmaceutical [pharmaceutical] Ji et al. (2010) Limited data (enhancement of the serum E2 level in male fish)
38 SRF Herbicide [agricultural] Hall et al. (2005) Limited data (only chg in males enhanced)
39 BDE-47 Flame retardants [industrial] Gonzalez-Doncel et al. (2014b); Gonzalez-Doncel et al. (2016) Lack of ED effects related to EATS pathways
Gonzalez-Doncel et al. (2017)
Beltran et al. (2022)
40 TRA Metabolite of TRB (agricultural) Robinson et al. (2017) Lack of adverse effects on fecundity
41 TRF Fungicide (agricultural) Lin et al. (2014) Induced cyp1a and cyp3a activities in the liver
42 TRD Fungicide [agricultural] Lin et al. (2014); Chu et al. (2016); Liu et al. (2018) Limited information (upregulation of vtg2 and cyp3a40 and downregulation of cyp3a38, vtg1, esr1, and cyp1a in the liver of females)
43 TBT Biocide [agricultural] Nozaka et al. (2004) Limited data (inhibition of brain aromatase)
Kuhl and Brouwer, (2006)
Hano et al. (2007)
Zhang et al. (2008d)
Horie et al. (2018, 2022a)
44 TCS Antimicrobial [industrial] Foran et al. (2000) Inconsistent data (hepatic VTG increased in males)
Ishibashi et al. (2004)
Mihaich et al. (2019)
Song et al. (2020)
Kawashima et al. (2022)
45 Nano zinc oxide (nZnO)/zinc sulfate (ZnSO4) Metal [inorganic] Paul et al. (2021) Toxic effects (reduced follicular growth and maturation in the ovary)
3.1 EEDs

For the identification and classification of EEDs from the searched chemicals, we considered three chemicals as references, E2 and EE2 as agonists, and TAM as antagonists (Table 1). Based on these reference chemicals, several endpoints, such as the female-biased sex ratio, induction of serum VTG (protein) in male fish, alteration of the secondary sex characteristics (anal fin papillae in the male fish), and up- or downregulation of vtg and chg genes/mRNAs in the liver of male fish, as well as the estrogen receptors (ERs) of the HPG axis in both sexes, were considered (Table 1). Using these strategies, we reviewed 108 articles, which is 52.68% of the searched articles, consisting of 25 chemicals as agonists and 4 chemicals as antagonists (Tables 3, 4). Adding three reference chemicals to the list, the number of EED agonists increased to 27 (21.09% of 128 chemicals) and antagonists to 5 (3.9% of 128 chemicals), altogether 32, which is 25% of the total (128 chemicals) chemicals searched by the literature survey. Alternatively, it appears that for every 100 EDCs, ∼21 of them are identified as EED agonists and ∼4 of them are identified as EED antagonists. Moreover, considering the 108 articles that studied EEDs, every EED chemical was studied in 3.375 articles (27 articles: 8 EEDs). Moreover, among EED agonists, other than two reference chemicals (E2 was reviewed in 37 articles and EE2 in 27 articles), 4-nonylphenol (4-NP; 23 articles), bisphenol A (BPA; 21 articles), and 4-tert-octylphenol (4-t-OP; 8 articles) are the most studied EED agonist chemicals in Japanese medaka (Table 3). Among others, o,p′-DDT (4 articles), 4t-PP (4 articles), E1 (3 articles), PPB (3 articles), and TBCO (3 articles) have drawn significant interest among investigators. The remaining 17 estrogen agonists were studied either twice (8 chemicals) or once (9 chemicals). For EED antagonists, the reference chemical TAM was studied in five articles, whereas ATZ was studied twice (Ritcher et al., 2016), MET once (Lee et al., 2019), and TPhP in two articles (Li et al., 2019; Kawashima et al., 2022). Moreover, 16 of the EEDs as agonists and 4 as antagonists were recommended for Tier 2 tests. Therefore, based on the literature search, we recommend that eight chemicals (E1, E2, EE2, BPA, o,p′-DDT, 4-NP, 4-t-OP, and TAM) showed enough potential to be considered EEDs in Japanese medaka and did not require any further Tier 2 tests for estrogen signaling mechanisms. Furthermore, except PPB (Gonzalez-Doncel et al., 2014a), 4t-OP (Gray et al., 1999b), and MET (Lee et al., 2019), in most of the EED chemicals, whether agonists or antagonists, the thyroid-related endpoints remained uninvestigated, even though the reference agonists (E2 and EE2) have the potential to inhibit swim bladder inflation (a thyroid-related endpoint) in a concentration-dependent manner in larvae if the embryos were exposed either to E2 or EE2 during development (Pandelides et al., 2021).

 3.2 AEDs

For AEDs, four chemicals, 11-KT and TRB as agonists and FLU and TRB as antagonists, were considered references (Table 1). Based on these reference chemicals, the apical endpoints, such as masculinization of females (development of anal fin papillae), male-biased sex ratio, upregulation of gsdf mRNA in XX embryos, ovotestis, and downregulation of vtg1, vtg2, chgH, and chgHm gene transcripts in the liver of both male and female fish (Table 1), were mostly considered during the evaluation of AEDs. With these efforts, from 46 articles, which is 22.43% of the sorted articles (Table 2), we identified 10 chemicals as agonists (Table 5) and 8 chemicals as antagonists (Table 6). With the addition of four reference chemicals, the number of AEDs increased to 22 (∼9% agonists and ∼8% antagonists), which is 17.18% of the 128 chemicals screened through the literature search. Alternatively, for every 100 EDCs, ∼9 chemicals are identified as AED agonists and ∼8 chemicals are identified as AED antagonists. Moreover, with regard to 46 articles that studied 22 AEDs, it appears that one AED chemical was studied in 2.09 articles (approximately 2 articles:1 AED). Moreover, among the reference chemicals, effects of TRB were observed in 14 articles, FLU was in 8 articles, and 11-KT and VIN were included in 5 articles (Table 1). Other than the references, the ED effects of three compounds, DHT, LNG, and P4, were evaluated together (Onishi et al., 2021). Furthermore, among the androgen agonists, the AED effects of MT were peer-reviewed in eight articles, followed by GEN (four articles) (Table 4). Among the other agonists, LNG was reviewed in three articles, and the remaining seven chemicals were studied only once (Table 5). Among the apical endpoints, masculinization was induced by BF, GEN, LNG, P4, and SPR, while downregulation of hepatic vtg in females was observed in MT and SPR (Table 5). Among the eight chemicals identified as potential antagonists, the most studied chemical was DZ, which was studied in three articles (Hamm and Hinton, 2000; Flynn et al., 2018; Kawashima et al., 2022), followed by LD-BP and FNT, which were studied in two articles each (Li et al., 2016; 2017; Horie et al., 2017; 2022a). Other than these chemicals, the remaining five chemicals were studied once (one article/chemical). Moreover, based on the targeted apical endpoints related to AED and the literature review, we recommend that nine chemicals showed enough potential to proceed to Tier 2 tests, and five chemicals (FLU, 11-KT, MT, TRB, and VIN) did not require Tier 2 tests for the evaluation of androgen signaling mechanisms. In addition, similar to EEDs, the thyroid-related apical endpoints, such as hypertrophy of thyroid follicular cells, were induced by 11-KT (reference agonist) and FLU (reference antagonist) in Japanese medaka (Leon et al., 2007). Other than the references, LNG (agonist) and DZ (antagonist) showed the potential to modulate swim bladder inflation in Japanese medaka larvae during development (Hamm and Hinton, 2000; Pandelides et al., 2021). Furthermore, GEN (agonist) shows potential to regulate the expression of dio2 mRNAs in larvae if the embryos were exposed to GEN during development (Schiller et al., 2013; 2014). Therefore, during the classification of EDCs as AED, the thyroid-related apical endpoints should not be ignored.

 3.3 TEDs

For TEDs, three chemicals, T3 as the agonist and PFOA and TBBPA as antagonists, were considered references (Table 1). The apical endpoints, such as swim bladder inflation in larvae, disruption of thyroid histopathology, and up- or downregulation of TH receptor genes (trα and trβ) and deiodinases (dio1 and dio2), were considered during TED evaluations. Our literature search found only 19 articles, which is 9.26% of the total articles (205 articles) sorted are focused on TED. From these articles, 12 chemicals, one as agonist (Table 7), and 11 chemicals as antagonists, were identified as TEDs (Table 8). Considering three references, 15 chemicals, 2 as agonists (1.56% of 128 EDCs) and 13 as antagonists (10.16% of 128 EDCs), which is only 11.72% of the screened chemicals (128 chemicals), showed TED effects on Japanese medaka. Alternatively, for 100 EDCs, 1.56 chemicals are identified as TED agonists, and ∼10 chemicals are identified as TED antagonists. Moreover, 19 articles identified 15 chemicals, which indicated that one TED was reviewed in 1.266 articles (approximately 5 articles:4 chemicals). Moreover, 5 chemicals, including three references and two antagonists (DIC and EHMC), were recommended to proceed to Tier 2 tests. The reference agonist T3 was studied in two articles, and the reference antagonists PFOA and TBBPA were included in four articles and 1 article, respectively (Table 1). Other than the references, the most studied chemical as a TED antagonist in Japanese medaka was DIC, which was peer-reviewed in five articles (Hong et al., 2007; Lee et al., 2011; Yokota et al., 2017; 2018; Pandelides et al., 2021). Other chemicals, such as ATBC, PFBA, PFOS/PFOSA, and TDCPP, were studied in two articles each. The remaining four antagonists were studied only once (Table 8). Although ATBC and TDCPP were evaluated as TED antagonists, the downregulation of liver vtg1 and vtg2 genes in XX fish by ATBC (Horie et al., 2023b) and upregulation of vtg mRNA in both male and female larvae by TDCPP (Godfrey et al., 2019) indicated that TED chemicals have the potential to regulate EAS pathways, which need further verifications.

 3.4 MOS

For identification of the MOS chemicals in Japanese medaka, four chemicals, TPA and TRF as stimulators and KTC and PCZ as inhibitors, were used as reference chemicals (Table 1). The apical endpoints selected for steroidogenesis are either the up- or downregulation of cyp19 genes that show potential to regulate the aromatase enzyme activity and lead to an increase or decrease in the circulating estrogen level in Japanese medaka. Our literature search selected 26 articles, which is 12.68% of the sorted articles, for the evaluation of steroidogenesis in Japanese medaka (Table 2). After reviewing these literature reports, four chemicals were considered stimulators of steroidogenesis and six chemicals were considered inhibitors (Table 9). Including the references, the total number of chemicals that interrupt steroidogenesis is 14, 6 stimulators (∼5%), and 8 inhibitors (∼6%), which is 10.93% of the identified chemicals that showed potential ED activities in Japanese medaka. Alternatively, for every 100 EDCs, 5 chemicals show potential to stimulate steroidogenesis and 8 chemicals inhibit steroidogenesis. Moreover, 14 MOS were identified after reviewing 26 articles, which indicated that for the identification of a chemical as MOS, 1.857 articles/MOS are reviewed (approximately 9 articles: 5 chemicals). Moreover, although the thyroid-related endpoints were not considered in these chemicals, including two references (TPA and TRF as agonists), nine chemicals (six as agonists and three as antagonists) were recommended for Tier 2 tests (Tables 9, 10). Among the stimulators, the ED activities of FPN, an insecticide, and TRI, a pharmaceutical product, were studied together (Sun et al., 2014). However, FPN was included separately in two articles (Sun et al., 2014; Wagner et al., 2017); the remaining three chemicals, OCL, RCT, and TRI, were investigated once (Sun et al., 2014; 2016a; Yan et al., 2020) (Table 9). Among inhibitors, the most studied chemical is FAD, a nonsteroidal aromatase inhibitor, which was studied in five articles (Suzuki et al., 2004; Kuhl and Brower, 2006; Zhang et al., 2008b; Park et al., 2008; Thresher et al., 2011). Moreover, LET, a nonsteroidal triazole, was included in four articles (Sun et al., 2007b; 2009; 2011a; Liao et al., 2014). BP, a UV filter used in cosmetics, was evaluated in three articles (Coronado et al., 2008; Kim et al., 2014; Kawashima et al., 2022), while the herbicide LNR and the organotin compound TPT-Cl were studied in two articles each (Table 10), and PRN was studied only once (Schiller et al., 2014). Although the apical endpoints of MOS are mainly concentrated on aromatase enzyme genes and enzyme activities, the ED effects of these compounds on Japanese medaka either as an EED or AED can also be observed in TRI (Yan et al., 2020), RCT (Sun et al., 2016a), BP (Coronado et al., 2008; Kawashima et al., 2022), FAD (Zhang et al., 2008b), and LET (Sun et al., 2007b).

 3.5 Unclassified

Due to limitations in the selection of apical endpoints, we were unable to identify the targeted EATS pathways of 45 chemicals (35.15% of the EDCs) identified from 60 (29.26% of the articles sorted) articles (Table 11). Alternatively, among 100 EDCs, 35 chemicals remained unclassified within the EATS modalities due to the lack of sufficient information (Table 11). Moreover, 45 unidentified EDCs in 60 sorted articles indicated that one chemical remained unidentified in 1.33 articles reviewed (4 articles:3 chemicals). Among these chemicals, the ED potential of GO was described in the maximum number of articles (10 articles) targeting the gonads, thyroid, interrenal glands, and endocrine pancreas of Japanese medaka (Dasmahapatra et al., 2020a; Dasmahapatra et al., 2020b; Myla et al., 2021; Asala et al., 2021; Myla et al., 2021; Dasmahapatra and Tchounwou, 2022a; Asala et al., 2022; Dasmahapatra and Tchounwou, 2022b; Dasmahapatra and Tchounwou, 2023a; Dasmahapatra and Tchounwou, 2023b). Moreover, TBT, a biocide used in agriculture, has been studied in six articles and showed the potential to inhibit brain aromatase in Japanese medaka (Nozaka et al., 2004; Khul and Brouwer, 2006; Hano et al., 2007; Zhang et al., 2008; Horie et al., 2018; Horie et al., 2022a). Furthermore, TCS, an antimicrobial product, was peer-reviewed in five articles that showed potential to enhance hepatic VTG in male fish (Foran et al., 2000; Ishibashi et al., 2004; Mihaich et al., 2019; Song et al., 2020; Kawashima et al., 2022). In addition, the flame retardant 2,2′,4,4′-BDE47 was peer-reviewed in four articles, although it was unable to target any of the EATS-related pathways in Japanese medaka (Gonzalez-Doncel et al., 2014b; 2016; 2017; Beltran et al., 2022). Among others, ACT, BKC, ClxBPA, CMP, IBP, LIN, MET, PDM, RLX, SFT, and TCS, although studied in a limited number of articles (except TCS, in most cases one or two articles), showed estrogenic potential by inducing the serum or liver VTG content in male fish (Foran et al., 2000; Ishibashi et al., 2004; Kim et al., 2012; Flynn et al., 2017; Godfrey et al., 2019; Mihaich et al., 2019; Kim et al., 2020; Song et al., 2020; Onishi et al., 2021; Kawashima et al., 2022). Furthermore, NPX, a NSAID, showed estrogenic potential by upregulating the expression of vtg1, erβ, and cyp17 genes in Japanese medaka (Kwak et al., 2018). Moreover, the potential ED effects produced by the rest of the chemicals (Table 11) are either due to induction of stress or mediated through pathways other than EATS.

 4 Discussion

Japanese medaka (Oryzias latipes) is one of the small laboratory fish models used for the evaluation of EDCs found in the environment (OECD, 2018). Like all other vertebrates, EATS pathways and their associated hypothalamus pituitary-releasing and -stimulating hormones are targeted by EDCs and disrupt the normal development and reproductive processes of this fish. For the identification of EDCs that specifically affect the endocrine systems of Japanese medaka (O. latipes), we searched the research articles in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and Google Scholar (https://scholar.google.com/) databases with the search terms, Japanese medaka, O. latipes, and endocrine disruptions. We hypothesized that literature search and evaluation can identify the number and sources of EDCs that disrupted the EATS-related pathways of Japanese medaka (Oryzias latipes) and provide additional evidence for the selection of a chemical as to whether to proceed to Tier 2 tests or not.

We sorted 205 articles that involved 128 chemicals for review (Figures 1, 2; Tables 1–11). Due to wide variations in experimental protocols and methodologies described in the research articles (n = 205), especially in non-TG studies, interpretation of the data from the literature survey became more complex. Moreover, the use of different life stages (embryos/larvae/adults), diversity in the modes of exposure (injection, immersion, and feeding), or in the duration of exposure (restricted either only in one generation or continued through multiple generations) made the problem even more complex. Therefore, to maintain consistency in the apical endpoints associated with ED effects, among the 128 identified chemicals, we selected 14 chemicals as the reference (Table 1). These chemicals (references) are either evaluated in this model (Japanese medaka) as reference chemicals by other investigators or screened through Tier 2 tests, following OECD guidelines (Flynn et al., 2017; Onishi et al., 2021; Kawashima et al., 2022; Myosho et al., 2022). Among these chemicals, E2 and EE2 (estrogen agonists), TAM (estrogen antagonist), 11-KT and TRB (androgen agonists), FLU and VIN (androgen antagonists), and KTC and PCZ (steroidogenesis inhibitors) were verified as agonists or antagonists for esr1 (for estrogen) and arβ (androgen) genes of Japanese medaka in vitro by RGA (Onishi et al., 2021; Kawashima et al., 2022). Additionally, the potential of E2, TAM, TRB, VIN, KTC, and PCZ as an EDC was evaluated in medaka through Tier 2 tests, following the MEOGRT protocol (Flynn et al., 2017). For stimulators of steroidogenesis, we considered TPA and TRF as reference chemicals (Jang and Ji, 2015; Zhu et al., 2015). For the thyroid, T3 as the agonist and PFOA and TBBPA as antagonists were considered, which were recently referenced by Godfrey et al. (2019) and Horie et al. (2023a) in Japanese medaka. Therefore, we think that the selection of reference chemicals for the identification of EATS-related apical endpoints and to set up guidelines is very reasonable and acceptable. Our approach identified 69 chemicals that show potential to target the EATS pathways of Japanese medaka, and 45 chemicals remained unclassified due to limited information, even though these unclassified chemicals induced ED-like effects in Japanese medaka (Table 11). Taken together, considering 14 references, 83 (69 identified +14 references = 83) chemicals are identified as EDCs (∼65%) that disrupt EATS pathways of Japanese medaka (O. latipes), and 45 EDCs (∼35%) remain unclassified due to the lack of sufficient information.

We further classified the EATS chemicals as agonists/stimulators and antagonists/inhibitors of EEDs, AEDs, and TEDs, and MOS. The apical endpoints selected for agonists should be in contrast with antagonists, and in many cases, these borderlines cannot be maintained. For example, one of the significant apical endpoints of an EED as an agonist is the upregulation of VTG in the liver of male (XY) medaka (Flynn et al., 2017); however, TAM, which was used as a reference chemical of the EED antagonist, increased the liver VTG content in male fish (Flynn et al., 2017). To avoid complicacy, during analysis, we ignored the classification of EATS chemicals as agonists and antagonists, and simply included all the agonists and antagonists together and expressed them as EEDs, AEDs, TEDs, and MOS where applicable (Table 2).

As mentioned previously, 128 EDCs were identified after reviewing 205 individual articles, which indicates that for the identification of a chemical as an EDC in Japanese medaka, more than one article was reviewed (1.60 articles/chemical, or the approximate ratio is 8 chemicals: 13 articles). Our studies also showed that after reviewing 165 articles, 83 EDCs were identified that targeted EATS pathways (Tables 3–10), and 45 chemicals remained unidentified after reviewing 60 articles (Table 11). Accordingly, approximately 65% of the EDCs were identified with their specific EATS targets after reviewing 80% of the searched articles and 35% of the EDCs remained unclassified after reviewing 20% of the searched articles (Table 2). Therefore, it appears that the databases consist of more articles as classified EDCs (related to EATS) than unclassified EDCs (Table 2). Moreover, as the EATS pathways are interdependent on each other through the common hypothalamus–pituitary axis (HP axis), it is very difficult to classify the EDCs on the basis of apical endpoints specific to the EATS pathways. However, our studies showed that more than 65% of the articles identified EDCs as EED, 28% of the articles identified EDCs as AED, 12% of the articles identified EDCs as TED, and 16% of the articles identified EDCs as MOS (Table 2), which can be arranged in the order of TED < MOS < AED < EED. Furthermore, among 83 EDCs that targeted EATS pathways, 39% of them are identified as EEDs, 27% are AEDs, 18% are TEDs, and 17% are MOS (Tables 3–10), and the order of arrangement appears to be MOS < TED < AED < EED. Therefore, the potential of literature searching to identify EATS-targeted chemicals in Japanese medaka partially supports the concept that the more the number of articles in the databases, the more the number of EDCs should be identified.

As recommended by USEPA, the effects of an EDC should be evaluated using a tier-based approach. In Tier-1 studies, the endpoints are focused mainly on lethal concentrations (LC/LD/IC50, NOEC, and LOEC), reproductive activity (fecundity, fertility, breeding behavior, and hatching of the embryos), sex reversal, secondary sexual features (the number of papillae in the anal fin rays which are present in juvenile/adult males and absent in female Japanese medaka), VTG (the egg yolk precursor protein), and choriogenins (the eggshell protein), which are absent in the liver of male fish, and histopathology of the gonad, liver, and kidney. The Tier 2 approach is multigenerational, consisting mostly of the same features evaluated in Tier 1 (fecundity, fertility, hatching, VTG content of the male fish liver, secondary sexual features, sex reversal, survivability of embryos, larvae, and adults, and histopathology of the gonad, liver, and kidney). Even though the Tier 2 tests are time-consuming, expensive, and need proper validation of the chemicals as an EDC through Tier 1 screening, for proper classification of the EDCs and their respective target endocrine organs or hormones in fish (Japanese medaka), multigenerational studies (Tier 2) are necessary (Kawashima et al., 2022). Accordingly, among 83 EATS (69 classified and 14 references), we recommend that six of the references (11-KT, T3, PFOA, TBBPA, TPA, and TRF), due to the limited number of articles (studies in Japanese medaka), should be considered high-priority candidate substances for Tier 2 testing. The eight other references (E2, EE2, TAM, TRB, FLU, VIN, KTC, and PCZ) were already verified either as reference chemicals during the evaluation of other EDCs or through multigenerational MEOGRT tests (OECD TG 240) (Flynn et al., 2017; Onishi et al., 2021; Kawashima et al., 2022). Therefore, these eight reference chemicals did not need any further Tier 2 tests for potential EAS-related effects; however, evaluation of thyroid-dependent mechanisms of these chemicals may require investigation (Myosho et al., 2019, 2021; Pandelides et al., 2021).

During screening of EEDs, among the identified chemicals, we recommend 16 (AR-1260, BZP, BMT, CFR, CTC, p,p′-DDE, DES, EDS, EQ, E3, 4-MBC, MPB, OMC, PPB, 4t-PP, and TBCO) as agonists, and 4 chemicals (ATZ, MET, PFAA, and TPhP) as antagonists were high-priority chemicals for Tier 2 tests. Among the rest, EED potentials of o,p′-DDT and 4-t-OP were evaluated by multigenerational MEOGRT tests (Flynn et al., 2017) and probably did not require any further Tier 2 tests as well (Flynn et al., 2017). In addition, BPA, E1, 4-NP, and 4-t-OP were recommended for Tier 2 tests after successful evaluation through the OECD TG 229 protocol (Kawashima et al., 2022). Moreover, our literature search found that BPA was reviewed in 21 articles, 4-NP in 23 articles, 4-t-OP in 8 articles, and E1 in 3 articles (Table 3). Therefore, we believe that these EEDs (E1, BPA, o,p′-DDT, 4-NP, and 4-t-OP) showed enough potential to be considered EED agonists without performing any further Tier 2 tests. In AEDs, six chemicals (BF, CFD, GEN, LNG, P4, and SPR) as agonists and three chemicals (CPA, DZ, and 2-EHHB) as antagonists were recommended for Tier 2 tests (Tables 5, 6). Moreover, our literature search showed that MT was studied in eight articles and probably did not require Tier 2 tests anymore. However, P4 and LNG, as progestins, induced secondary sexual features in female Japanese medaka (XX) (Onishi et al., 2021), and further evaluation by Tier 2 tests is necessary. Among TEDs, two antagonists (DIC and EHMC) were recommended for Tier 2 tests and for MOS, four chemicals (OCL, FPN, RCT, and TRI), as stimulators, and three chemicals (BP, FAD and LET), as inhibitors, were recommended for Tier 2 tests. Taken together, among the 83 EDCs that targeted EATS pathways, 43 chemicals were recommended for Tier 2 tests, and 13 chemicals can be considered potential EDCs without any further Tier 2 tests in Japanese medaka.

Our literature search did not classify the EATS pathways of 45 chemicals (35%), even though several of them induced specific EATS-related apical endpoints (Table 11). Generally, in in vivo studies, probably due to the HPG and HPT axes, the overlapping effects of the chemicals within the EATS pathways cannot be ruled out; therefore, many of these unclassified chemicals demonstrated effects on endocrine-related apical endpoints, such as alteration in the liver VTG content (upregulated by CMP, CHDM, IBP, MTZ, NPX, 1NT, PDM, and RLX, and downregulated by DIBP), upregulation of chg in the liver of male fish (OXY and SRF), impaired reproductive activity and gonad histology (Cd, LD-BP, and nZnO), histopathological changes in the thyroid (BDE-47), inhibition of aromatase (TBT), and regulation of the E2 concentration in the blood of fish (Cd and SFT) (Table 11). In addition, several of the unclassified EDCs have potential as ESR agonists (CMP, DIBP, and FU) or antagonists (CYN, PHT, and RLX), and the ESR agonist and ARβ agonist (INT) and ESR agonist and ARβ antagonist (CMP) were observed in in vitro RGA with medaka esr1 and arβ genes (Onishi et al., 2021; Kawashima et al., 2022). Moreover, the nanocarbon, GO, was evaluated in 10 articles targeting the gonads, thyroid, interrenal glands, and pancreas in adults; and gonads, thyroids, and interrenal glands in larvae (Dasmahapatra et al., 2020a; Dasmahapatra et al., 2020b; Dasmahapatra and Tchounwou, 2022a; Dasmahapatra and Tchounwou, 2022b; Dasmahapatra and Tchounwou, 2023a; Dasmahapatra and Tchounwou, 2023b; Asala et al., 2021; 2022; Myla et al., 2021a; b). Despite the histopathological alterations and cellular disruptions induced in the gonads, liver, kidneys, thyroid, interrenal glands, and pancreas of the adults and larvae of Japanese medaka by GO, due to the lack of specific Tier 1 and Tier 2 tests, GO remained unclassified without identifying any EATS-specific pathways (Dasmahapatra et al., 2020a; Dasmahapatra et al., 2020b; Dasmahapatra and Tchounwou, 2022a; Dasmahapatra and Tchounwou, 2022b; Dasmahapatra and Tchounwou, 2023a; Dasmahapatra and Tchounwou, 2023b; Asala et al., 2021; 2022; Myla et al., 2021a; b). Therefore, we think that, before excluding the potential of these unclassified chemicals as an ED, further validations using tier-based approaches are necessary. Alternatively, the effects should be considered nonspecific, mediated through oxidative stress, or not related to EATS-specific mechanisms.

Although the effects of 128 EDCs in Japanese medaka are classified based on EATS modalities, the disruptions of non-EATS pathways by these chemicals need to be investigated carefully (Martyniuk et al., 2022). Moreover, compared to EATS, less attention has been given to other endocrine organs, including the endocrine pancreas and the interrenal gland (adrenal gland), which should belong to non-EATS pathways of Japanese medaka. Due to the lack of validated in vivo or in vitro methods and the availability of the appropriate literature in the public databases, the evaluation of EDCs targeting non-EATS modalities of Japanese medaka is not properly focused on this review. Our literature search on the effects of EDCs on the endocrine pancreas and interrenal glands of Japanese medaka found only four articles, two for pancreas (Dasmahapatra and Tchounwou, 2023a; Dasmahapatra and Tchounwou, 2023b), and two for interrenal glands (Dasmahapatra and Tchounwou, 2022a; Dasmahapatra and Tchounwou, 2022b) (Tables 2, 11) in PubMed (www.ncbi.gov). Therefore, despite the significant importance of non-EATS modalities in Japanese medaka, due to the lack of sufficient literature and standard methods, the evaluation of EDCs mediated through non-EATS pathways is not appropriately described in this review article.

In conclusion, our strategies on the literature survey sorted 205 articles on Japanese medaka (O. latipes) that focused on 128 chemicals as EDCs. We found that 83 chemicals (∼65%) show potential as EDs targeting the EATS pathways. Although the overlapping of the endocrine-related apical endpoints cannot be ruled out, from the literature search, we classified 32 chemicals from 108 articles as EEDs, 22 chemicals from 46 articles as AEDs, 15 chemicals from 19 articles as TEDs, and 14 chemicals from 26 articles as MOS, and 45 EDCs from 60 articles remained unclassified. The number of EATS chemicals arranged in order (MOS < TED < AED < EED) fits well with the numbers identified by the literature search (TED < MOS < AED < EED). Moreover, 43 EDCs belonging to EATS are recommended for Tier 2 tests (∼34%), and 13 chemicals showed enough potential to be considered EDCs without any further tier-based studies (∼10%). Our evaluation of EDCs in Japanese medaka shows significant potential to further apply the laboratory-based research data for applications in regulatory risk assessments in humans.

 Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

 Author contributions

AD: conceptualization, resources, data curation, formal analysis, investigation, methodology, and writing–original draft and review and editing. CW: formal analysis, investigation, resources, writing–original draft, and review and editing, validation, and visualization. AM: formal analysis, validation, and writing–original draft. ST: validation and writing–original draft. PT: conceptualization, funding acquisition, resources, supervision, and writing–review and editing.

 Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The research was supported by the NIH/NIMHD grant #G12MD07581 (RCMI Center for Environmental Health), NIH/NIMHD grant #1U54MD015929 (RCMI Center for Health Disparities Research) and NSF grant #HRD 1547754 (CREST Center for Nanotoxicity Studies) at Jackson State University, Jackson, Mississippi, United States, and NIH/NIMHD grant #U54MD013376 (RCMI Center for Urban Health Disparities Research and Innovation) at Morgan State University, Baltimore, Maryland, United States. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH or NSF.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ftox.2023.1272368/full#supplementary-material

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ACT Acetaminophen
ATBC Acetyl tributyl citrate
APEO Alkylphenol polyethoxylate surfactants
AMT Amitrole
AChE Acetylcholinesterase enzyme
11-OA 5α-Androstan-3,11,17-trione
AED Androgen endocrine disruptors
AR Androgen receptor
AR-1260 Aroclor 1260
ATZ Atrazine
AZM Azinphos-methyl
BP2 Benzophenone 2
BP3 Benzophenone 3
BF Bifenthrin
DEHS Bis(2-ethylhexyl) sebacate
BPA Bisphenol A
BZT-UV Benzotriazole ultraviolet stabilizer
BKC Benzalkonium chloride
i-BP i-Butylparaben
n-BP n-Butylparaben
Cd Cadmium
CFD Cefadroxil
CFR Cefradine
ClxBPA Chlorinated BPA
CMP 4-Chloro-3-methylphenol
CLT Chlorothalonil
CTC Chlortetracycline
CHG Choriogenin
CYN Cyanazine
CHDM 1,4-Cyclohexanedimethanol
CPA Cyproterone acetate
dpf Day post-fertilization
dph Day post-hatch
DZ Diazinon
DBP Dibutyl phthalate
DIC Diclofenac
p,p′-DDE Dichlorodiphenyldichloroethylene
o,p′-DDT Dichlorodiphenyltrichloroethane
DEHP Diethylhexyl phthalate
DES Diethylstilbestrol
DHT 5α-Dihydrotestosterone
DIBP Diisobutyl phthalate
EDs Endocrine disruptors
EDSP Endocrine Disruptor Screening Program
EDS Endosulfan
END Endrin
E2 17-β-Estradiol
E3 Estriol
ESR Estrogen receptor
E1 Estrone
EQ Equol
EAS Estrogen–androgen–steroidogenesis
EATS Estrogen–androgen–thyroid–steroidogenesis
EHMC 2-Ethylhexyl-4-methoxycinnamate
EFSA European Food Safety Authority
EMA European Medicine Agency
EE2 17α-Ethinylestradiol
EU European Union
FAD Fadrozole
FNT Fenitrothion
FNC Fenoxycarb
FV Fenvalerate
FLFII Female leukophore-free strain
FAT Fish acute toxicity test
FELS Fish, early life stage toxicity test
FET Fish embryo toxicity test
FSTRA Fish short-term reproduction assay
FPN Fipronil
FLX Fluoxetine
FLR Fluridone
FLU Flutamide
GEN Genistein
GLP Glyphosate
gsdf Gonadal soma-derived factor
GSI Gonadosomatic index
GO Graphene oxide
HSI Hepatosomatic index
HBCD Hexabromocyclododecane
hpf Hour post fertilization
HPA Hypothalamus–pituitary–adrenal axis
HPG Hypothalamus–pituitary–gonadal axis
HPT Hypothalamus–pituitary–thyroid axis
IBP Ibuprofen
KC-400 Kanecholor 400
KTC Ketoconazole
11-KT 11-Ketotestosterone
LC Lethal concentration
LET Letrozole
LNG Levonorgestrel
LD-BP Lignin-derived bisphenol
LIN Lincomycin
LNR Linuron
LOEC Lowest observed effect concentration
LOEL Lowest observed effect level
MEOGRT Medaka extended one-generation reproduction test
MELA Medaka embryo-larval development assay
MDA Malondialdehyde
MET Metformin
MTZ Methimazole
MXC Methoxychlor
4-MBC 3-(4-Methylbenzylidene) camphor
MPB Methylparaben
MT Methyltestosterone
MTC Metolachlor
MOE Ministry of Environment
mph Month post-hatch
MOS Modulator of steroidogenesis
MCB Myclobutanil
NPX Naproxen
1NT 1-Naphthol
NDEA N-Nitrosodiethylamine
NSAID Nonsteroidal anti-inflammatory drug
4-NP 4-Nonylphenol
NP1EC; NP2EC Nonylphenol ethoxycarboxylate
NP1EO; NP2EO; NP9EO Nonylphenol ethoxylate
NPEO Nonylphenol polyethoxylates
NOEC No-observed-effect concentration
OCL Octocrylene
OMC Octyl methoxycinnamate
4-OP 4-Octylphenol
OECD Organization of Economic Cooperation and Development
OYZ Oryzalin
OXF Oxyfluorfen
OTC Oxytetracycline
PDM Pendimethalin
PCPL Pentachlorophenol
4t-PP 4-Tert-pentylphenol
PFAA Perfluoroalkyl acid
PFBA Perfluorobutyric acid
PFOA Perfluorooctanoic acid
PFOS Perfluorooctane sulfonate
PFOSA Perfluorooctane sulfonic acid
PFAA Perfluoroalkyl acid
PFBS Perfluorobutane sulfonate
PFNA Perfluorononanoic acid
PPAR Peroxisome proliferator-activated receptor
PHT Phenytoin
PCB 126 Polychlorinated biphenyl 126
PCZ Procloraz
P4 Progesterone
PRN Propanil
PPB Propylparaben
PTU 6-Propyl-2-thiouracil
RCT Ractopamine
RLX Raloxifene
REP Relative estrogenic potency
RGA Reporter gene assay
RND Roundup
SERM Selective estrogen receptor modulator
SDS Sodium dodecyl sulfate
SPC Sodium perchlorate
SPR Spironolactone
SFT Sulfathiazole
SRF Surflan
TAM Tamoxifen
TPA Terephthalic acid
TG Test guidelines
4t-OP 4-Tert-octylphenol
T Testosterone
TBBPA Tetrabromobisphenol A
TBCO 1.2,5,6-Tetrabromocyclooctane
BDE-47 2,2′,4,4′Tetrabromodiphenyl ether
TU Thiourea
TED Thyroid endocrine disruptors
TH Thyroid hormone
TR Thyroid hormone receptor
TSH Thyroid-stimulating hormone
T4 Thyroxine
TRA 17α-Trenbolone
TRB 17β-Trenbolone
TRD Triadimenol
TRF Triadimefon
TBT Tributyltin
TCS Triclosan
TCrP Tricresyl phosphate
TRF Trifloxystrobin
T3 Triiodothyronine
TRI Trilostane
TPhP Triphenyl phosphate
TPT-Cl Triphenyltin chloride
TDCPP Tris (1,3-dichloro-2-propyl) phosphate
USEPA; EPA United States Environmental Protection Agency
USFDA United States Food and Drug Administration
VIN Vinclozolin
VTG Vitellogenin
wph Week post-hatch
nZnO Nano zinc oxide
ZnSO4 Zinc sulfate