**2. Methodology**

A literature search related to zebrafish used as a model in ecotoxicity assessments was conducted in databases such as Web of Science, Science Direct, Scopus, and PubMed. The following keywords were used in the search process: "*Zebrafish*" and "*Ecotoxicity*", "*Danio rerio*" and "*Acute toxicity*", "*Zebrafish development*" and "*Fish assay*", and "*Zebrafish embryo test*" and "*Exposure*", etc. A total of 225 articles were reviewed in all databases. Studies that did not comply with the study's purpose were not included in the evaluation. Of the 225 articles published until January 2023, 92 were thoroughly reviewed. Each article used in the study was compiled according to the following parameters: (i) publication year; (ii) the type of pollutant; (iii) exposure factors; (iv) toxicity; (v) stage of development; (vi) types of tests; and (vii) ecotoxic effects (Figure 1) [1,11,12].

**Figure 1.** Systematic review methodology.

#### **3. Advantages and Disadvantages of Zebrafish in Ecotoxicity Tests**

Zebrafish is a successful monitoring model applied in both medicine (drug production, disease diagnosis, treatment, etc.) and environmental pollution (determination of the effect level of pollutants) [13]. Zebrafish has been widely used in ecotoxicity studies in recent years due to its advantageous features, such as reproduction process and developmental stage [14]. The most important feature that distinguishes zebrafish from other model creatures is that it is used in both sexes, which has brought zebrafish to the forefront in ecotoxicity tests [1]. One of the main advantages of this model is that experimental studies

can be carried out in the embryo process [13]. Additionally, embryos (laying from egg: 48–72 h (hpf); organ formation: 120 hpf; adulthood: 3 months) grow rapidly [15,16]. Rapid development is an excellent feature in toxicity studies with animal models as it allows instant monitoring of toxic exposure [17]. All genetic features of zebrafish are stored in a database (www.zfin.org, accessed on 1 March 2023). In addition, 70% of zebrafish genes are similar to human genes [8]. In recent years, zebrafish has been evaluated as an alternative ecotoxicity model to replace mice and other fish species. It has proven to be a bio-indicator that complies with the 3R (Reduce, Reuse, and Recycle) concept, as tests with zebrafish are economical, provide fast results, and the presence of a large number of embryos reduces the chemical requirements [18]. Among the disadvantages of zebrafish, it can be said that different reactions occur according to the sex type, and the embryo development pools are large.

#### **4. Development and Distribution of Zebrafish**

The zebrafish was described in 1882 by Francis Hamilton, who found it near the Ganges River in India [19]. After George Streisinger first used the zebrafish to study vertebrate development in the 1980s, it became one of the most important laboratory animals with unprecedented speed [8]. The zebrafish is native to most of the Indian subcontinent, from Pakistan in the west through India, Nepal, and Bangladesh to Myanmar in the east. Zebrafish can be found in a wide variety of habitats. The adult zebrafish is 2 to 4 cm long, and its body is characterized by zebra-like stripes (Figure 2).

**Figure 2.** Adult zebrafish illustration.

In toxicological studies, especially in order to determine the toxic levels of environmental pollutants, parameters close to the natural environment of zebrafish should be provided in the laboratory environment. In this context, water quality parameters come to the fore (Table 1) [20–23]. The zebrafish is found in rivers, streams, canals, and rice fields in India, Pakistan, Bangladesh, Nepal, Myanmar, and Bhutan [22]. Wild zebrafish distribution includes Brazil, Colombia, Malaysia, Sri Lanka, Thailand, and the United States [21,23]. Native zebrafish is found in more than 3000 institutions in more than 100 countries and is a popular aquarium fish [22,24]. In research studies with zebrafish in the literature, China (26.7%), Brazil (14.8%), France (12.5%), Germany (8.5%), India (6.3%), and Italy (5.1%) take the lead [1]. The University of Oregon in the United States is home to the Zebrafish International Resource Center (ZIRC), the world's first and largest zebrafish resource center located on the Zebrafish Information Network (ZFIN). According to data from the Federation of European Aquaculture Producers (FEAP), this type of fish is produced at a rate of 1.8% (https://feap.info/, accessed on 1 March 2023).


**Table 1.** Physico-chemical properties of water for lab-raised zebrafish.

### **5. Zebrafish-Based Experimental Applications**

In addition to fish species, experiment design is an important factor in ecotoxicity studies. The type of exposure (in vivo or in vitro), exposure route, exposure times, and stage of development are considered experimental parameters in data generation [13]. The majority of tests are performed in vivo (90.3%) compared to in vitro tests (9.7%) (Figure 3). Embryos (7.3%) and larvae (32.1%) are used less frequently than adults (55.8%) in ecotoxicity evaluation according to the development process (Figure 3). In terms of exposure route and duration, water (94.9%) and hour (51.1%) are evaluated at the highest rates, respectively (Figure 3). In Table 2, toxicity studies for which zebrafish are the models are given.

**Figure 3.** (**a**) *Danio rerio* development; (**b**) type of studies in vivo and in vitro, zebrafish exposure route, exposure time, and developmental stages (Adapted from Refs. [1,11]).


**Table 2.** Different toxicity studies in the literature.

### **6. Results**

The zebrafish's small size, resistance to biotic and abiotic conditions, rapid development process, short reproduction period, compatibility with laboratory conditions, easy supply, economy, high fertility, ability to manipulate embryos, and genetic similarity with humans increase the importance of this species for toxicity studies. Thanks to these advantages, zebrafish can be used instead of other living models in scientific research and can support the 3R rule. As a result of studies researched in international databases, it is thought that different uses of this species will be widespread, and it will be a key model organism in the future.

**Author Contributions:** Conceptualization, methodology, formal analysis, investigation, resources, writing—original draft preparation, writing—review and editing, visualization, H.Ç., T.B., ˙I. ¸S. and S.T. All authors have read and agreed to the published version of the manuscript. ¸

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This study was carried out in Aksaray University Central Library and Engineering Faculty Environmental Engineering Department.

**Conflicts of Interest:** The authors declare no conflict of interest.
