Application of Fish Embryo Assay Using Zebrafish and Oryzias latipes for Toxicity Testing and Deriving Water Quality Criteria

Abstract

To protect aquatic organisms in ecosystems, each country and continental union has established guidelines for deriving the water quality standards (WQS) of specific substances. These guidelines mandate the use of acute and chronic toxicity data for fish, which are high-trophic-level organisms. However, due to increasing concerns about animal welfare and experimental ethics, there is a growing need for alternative methods to determine substance toxicity in fish. Fish toxicity tests using early life stages, such as embryos or larvae, have been utilized as alternative methods for adult fish toxicity assays. This review of the WQS guidelines and relevant test protocols confirmed the classification of acute and chronic toxicity in fish assays using different developmental stages. Fish toxicity data derived from exposure periods longer than one week using embryonic- or larval-stage organisms can be considered as indicative of chronic toxicity. There is a high correlation between fish embryo toxicity and adult effects, suggesting that fish embryo toxicity tests with appropriate exposure durations could replace adult fish toxicity tests, addressing experimental animal ethics concerns.

1. Introduction

Fish are high-trophic-level organisms among various aquatic eco-receptors. As key receptors in aquatic systems, it is necessary to protect them from chemical or contaminant pollution. Water quality standards (WQS) are derived by considering both the level of contaminant pollution in the environment and its toxicity to aquatic eco-receptors [1,2,3,4,5]. The United States, Canada, the European Commission, and the governments of Australia and New Zealand derive the WQS for contaminants to protect aquatic eco-receptors, including fish [1,2,3,4]. Moreover, the WQS in Japan primarily aims to protect fish, leading to a high demand for chemical toxicity data on fish [5]. These demands have resulted in conducting animal tests on fish to evaluate the toxicity of specific chemicals.

From the perspective of animal care and use ethics, researchers must strive to maintain the welfare of experimental animals, including vertebrates such as fish and amphibians in aquatic environments, under the 3R principles (reduction, refinement, and replacement) [6,7]. Direct pain or unnecessary harm to test organisms are not permitted, and this has driven the development of alternative testing methods; one such alternative tool is the fish embryo assay, which uses organisms that have not yet developed a nervous system. Since fish embryos have primitive nervous systems and do not experience discomfort or pain, they are considered replacements for organisms used in traditional toxicity testing [6,7,8]. According to European Directive 2010/63/EU, fish embryos and larvae, which are not capable of independent feeding, are excluded from the group of protected animals [8].

In accordance with the fish embryo test guidelines [9,10,11,12,13,14,15,16], various fish species are used for assays, including Catostomus commersonii, Cyprinodon variegatus, Cyprinus carpio, Danio rerio, Esox lucius, Ictalurus punctatus, Lepomis macrochirus, Misgurnus anguillicaudatus, Oncorhynchus kisutch, Oncorhynchus mykiss, Oncorhynchus tshawytscha, Opsanus beta, Oryzias latipes, Oryzias sinensis, Pimephales promelas, Salmo salar, Salmo trutta, Salvelinus fontinalis, and Salvelinus namaycush. By screening previous studies for “each species name”, “fish” AND “toxicity”, or “fish embryo” AND “toxicity” in Google Scholar, the research ratios of “fish embryo” in “fish” toxicity research were shown to be 8.30% for zebrafish and 7.48% for O. latipes, respectively (Figure 1). These two fish species have high proportions of fish embryo studies. Furthermore, these two main fish species are included in the test guidelines of individual countries and international organizations to assess the toxicity of chemicals and contaminants for deriving water quality criteria, which helps to protect aquatic ecosystems.

Focused on both fish species (zebrafish and O. latipes), this study examined the overall development of each species from birth to adulthood in laboratory conditions. We also reviewed the test guidelines related to fish embryos for specific nations and international organizations, and analyzed previous research on both acute and embryonic experiments to understand how toxicity levels change. The ultimate purpose of this study is to provide suggestions for suitable exposure duration to determine acute and chronic toxicity using fish embryo assays. Ultimately, we aim to promote the fish embryo assay as an alternative tool for adult fish tests.

2. Development of Zebrafish and O. latipes

2.1. Fertilization of Embryo from Adult Fish

For obtaining the fish embryo, mating behavior is induced between male and female fish. In zebrafish, two males and one female are put into a single spawning unit, and mating behavior occurs. After the mating behavior among the adult zebrafish, the sudden light transition causes female adults to spawn fertilized embryos by injecting a single sperm into a zebrafish egg; the average spawning rates of female zebrafish were estimated as 200–300 eggs per week [17], and these embryos have a fragile chorion without villi.

Meanwhile, the recommended female-to-male ratio for breeding O. latipes embryos is recommended is 2.5:1 [18]. After fertilization, female O. latipes spawns a bundle of embryos, which are connected using long attaching filaments. Fertilized embryos can be separated from the attaching filaments, and non-attached filaments, called “chorion villi,” are observed via stereoscopic microscope. On average, adult O. latipes spawn 20–30 embryos per individual birth [18]. The collected embryos of zebrafish and O. latipes were cultured in embryonic rearing solutions (ERS; NaCl, 1 g/L; MgSO₄, 0.163 g/L; CaCl₂·2H₂O, 0.04 g/L; KCl, 0.03 g/L) and in an E3 medium (NaCl, 0.29 g/L; MgSO₄, 0.04 g/L; CaCl₂, 0.04 g/L; KCl, 0.01 g/L), respectively, until the hatching stage [17,18]. In this study, hatched organisms were moved into dechlorinated tap water in a 15 L aquarium under continuous aeration, and developmental changes were observed.

2.2. Developmental Stage from Embryo to Juvenile

The developmental changes of fish embryos can be shown through the chorion- and species-dependent difference in developmental speed observed from the hatching time (Figure 2). Fish embryos start hatching from 48 h post fertilization (hpf) in zebrafish and 8 days post-fertilization (dpf) in O. latipes. Hatched organisms are called “larvae,” and these still have yolk for nourishment supply without independent feeding activity; fin development commences at this stage. Subsequently, sac-fry-stage fish have yolk that is almost adsorbed and an air bladder that is inflated through cardiac impulse, and bloodstreams are observed. With a month of growth, the sac-fry reaches the juvenile stage under the exponential growth rate [18,19,20]. Zebrafish have a rapid growth rate compared to O. latipes; the body sizes of 2-month-old juvenile zebrafish were clearly distinguished from those of O. latipes (Figure 2D,H). Juveniles undergo sexual maturation and achieve full reproduction ability as females or males by genetically, and the life cycles of fish restart with the embryo production of young adults.

3. Derivation of Water Quality Criteria and Relevant Fish Assay

3.1. US EPA

The United States Environmental Protect Agency (US EPA) suggested the guideline for deriving WQS to protect aquatic organisms in 1985, and the revised version was re-released in 2010 [1]. This guideline proposed to protect aquatic eco-receptors, including fish, algae, and invertebrates, in freshwater and marine ecosystems. The toxicity data of fish are required, including acute and chronic data, which are essential for deriving WQS by calculating the acute–chronic ratio (ACR) for at least three other family groups of aquatic organisms.

The ACR requires the acute and chronic toxicity values of fish [1]; the revised guideline suggested that short-term impacts, including mortality, and sublethal symptoms, such as abnormality or growth inhibition, should be used to estimate the effective concentrations (EC_x) rather than lethal concentrations (LC_x), which only reflect the mortality over 96 h of exposure (OPPTS 850.1085; OCSPP 850.1075) [20,21]. Chronic toxicity can be assessed by the life cycle toxicity test. In US EPA, there are five test methods using embryos and larval fish. These test methods are conducted for at least 4 weeks [8,9,22,23,24,25,26]; Zebrafish and O. latipes are exposed to substances for 28 days post-hatching (dph), with the observation of survival, hatching, growth, abnormalities, and behaviors performed. Using O. latipes, the extended one-generation reproduction test can be conducted measuring the sex-maturation-related endpoints, including gonad histology, fecundity, and reproduction rate [23]. The test period for chronic toxicity assessment can be applied differently depending on the life span of the test species [9,24]. The fish species with short lifespans of less than a year are exposed for at least 30 days from the larval stages, and the species have a longer lifespan, 90 days of exposure are applied [24]. Additionally, at least one life cycle of substance exposure or short-term exposure of reproduction toxicity for 3 weeks can be applied [8,25] (Table 1).

3.2. Canada

The Canadian Council of Ministers of the Environment (CCME) also has a national protocol for the derivation of WQS to protect aquatic life. This protocol requires data on algae, invertebrates, and vertebrates, particularly fish and amphibians [2,27]; fish and amphibians are considered to be top-trophic-level organisms. In short-term exposures to determine acute toxicity in fish, two test species (O. mykiss and Gasterosteus aculeatus) were exposed to chemicals for 96 h and LC₅₀ was used as the acute toxicity value [28,29]. Considering the effective level of long-term exposure, test durations are determined in accordance with the developmental stage of the test species; the Canadian guideline specifies that exposures of longer than 7 days for early-life-stage embryos and larval fish or 21 days for juvenile and adult fish can be considered for chronic toxicity assays [11,30].

Environment Canada recommended two procedures for toxicity tests for early-life-stage fish using salmonid (O. mykiss) and Pimephales promelas [11,30]. A revised biological test method using O. mykiss included three options for developmental stages, namely embryos, alevins, and swim-up fry; embryo tests are conducted for 7 days after fertilization, the embryo/alevin test for 7 days, measuring the effect of each developmental stage after hatching, and the embryo to swim-up fry for 30 days. All these options start from the embryonic development and survival of early-life-stage organisms; since these early-life-stage organisms are at a sensitive stage in the life cycle, the assays are considered to be meaningful and powerful. Environment Canada also revealed the revised method of performing the toxicity test for 7 d to measure the survival of larval P. promelas [30]; seven days is not a long duration compared to a fish’s life-span, but the larval stage is a vulnerable stage in the context of the entire life cycle [31]. Accordingly, the larval fish assay for 7 days’ duration cannot replace chronic toxicity tests, but it can be used to predict the chronic effects of the conventional lethality test using juvenile fish (Table 2) [30,32].

3.3. European Commission

The European Commission has developed a strategy for the Water Framework Directive (WFD) [33]; the WFD requires the identification of priority substances with significant risks to aquatic environments. Since 2001, the European Commission has conducted chemical assessments of waterbodies alongside tests of the ecological status, and the priority substance lists have been selected. For deriving the environmental quality standard (EQS), a risk assessment paradigm has been developed based on worst-case scenarios, and the expert group on the EQS initiated a guideline for EQS derivation in water field policy in 2007.

The EQS derivation requires ecological toxicity data for targeted substances for use in the species sensitivity distribution approach [34,35,36]. Fish are among the ecological receptors for which it is required to conduct acute or chronic toxicity tests. In terms of EQS data, both acute and chronic toxicity data are required; chronic toxicity is regarded by considering the toxicant exposure over at least a complete lifespan or in sensitive developmental stages. The EQS data can be secured by using the no observed effect concentration (NOEC) values from these chronic toxicity tests [37,38,39]. The technical guidance for deriving the EQS suggested the Organisation for Economic Co-operation and Development (OECD) test guidelines (TG) as reference methods for fish (Table 3) [3]; acute toxicity can be considered if the mortality is determined in the 96 h exposure test [40]. The acceptable chronic toxicity tests for fish are early-life-stage tests using embryo or larval organisms and observing hatching, growth inhibition, and abnormalities [13]; the reproduction test is also regarded as a chronic test [41].

There are three fish embryo assays, OECD TG 236, with a zebrafish embryo for 96 h, and OECD TG 210 and TG 212, with an early-life stage toxicity test, starting from embryonic stages, can be used as acute and chronic test methods [12,13,14]. The apical endpoints are observed in OECD TG 236, including coagulated embryos, deformation of the somite, and lack of development with low heartbeat; the LC₅₀ for 96 h is used as acute toxicity data for EQS derivation [12]. The OECD TG 212 [13,14] is an alternative method for OECD TG 210, conducted for relatively long exposures, and covering the vulnerable life stages from the embryo, considered a chronic test [14]. Both OECD TG 210 and 212 start exposure to substances from the embryonic stage and use endpoints to derive the EQS by observing survival at all developmental stages, the hatching rate, and growth inhibition with malformations or behaviors [3,13,14].

Juvenile or adult fish assays are also mentioned; representative methods are the acute test for adult fish with 96 h of exposure (OECD TG 203) and chronic tests on juvenile fish with at least 4 weeks of exposure (OECD TG 215) [15,42,43]. For sexual development or reproduction, both the 60-day TG 234 sexual development test and the one-generation reproduction test (OECD TG 240) using O. latipes are considered chronic toxicity tests [15,43]; these two tests are mainly focused on endpoints such as gonadal histology, vitellogenin concentration, or sex hormone levels. The short-term tests, lasting less than 4 weeks, can be used to support the toxicity data for deriving the EQS in the European Commission, but are deferred from the priority lists of chronic toxicity tests (OECD TG 229 and TG 230) [3,41,44].

3.4. Australia and New Zealand

Australia and New Zealand published the freshwater and marine water quality guideline in 1992 and updated the content in 2000, and the current version has made some amendments regarding the technical rationale for deriving toxicity values in 2018 [4,45,46]. The WQS derivation for water quality in Australia and New Zealand covers the various taxonomies associated with trophic levels and nutrient cycling. Fish are a typical families of a realistic biological community, which consume some living organisms as a feeding activity. Furthermore, the ethical problem of animal experimentation has been an issue for fish assays, so relevant matters have been reviewed. Accordingly, the Commonwealth Scientific and Industrial Research Organisation (CRISO) modified the technical rationale for some detailed explanations for deriving the WQS value for toxicants in Australia and New Zealand [4,46].

Firstly, the definitions of acute and chronic toxicity were re-defined. In 2000, acute toxicity was defined as the rapid effects caused by substances in living organisms (for instance, death); continuous or prolonged exposures of test organisms for weeks to years on test organisms were considered as chronic toxicity [45]. Previously, chronic toxicity only included some biological responses of long duration with slow processes affecting one life-stage of living organisms [45]. The revised definition covered several common tests, including larval development tests, which had been classified as acute or chronic assays. Re-defined acute toxicity means lethal or sub-lethal effects occurring after a short exposure to chemicals; chronic toxicity has also been defined as lethal or sub-lethal effects for substantial portion of the lifetime of an organism (greater than 10% of the life span). Adverse effects on sensitive early-life-stage organisms have also been considered as chronic toxicity [4].

Especially for fish toxicity, the classification of acute and chronic toxicity is determined by the initial stage of development at the time of exposure and test duration with various endpoints. In juvenile or adult fish, the effects of substances for durations of less than 3 weeks are considered as acute toxicity; impacts from a longer exposure period are considered to be chronic toxicity for fish. Accordingly, the 7-d toxicity test for juvenile or adult fish is regarded as an acute toxicity assessment. For early-stage fish, including embryos and larvae, the classification of the exposure duration is one week, with a test duration of less than 7-d being an acute effect; if the exposure duration is longer than 7 days, the effects are considered to be chronic toxicity.

Considerable endpoints have been expanded in the revised guideline [46]; both traditional endpoints (survival growth and reproduction) and non-traditional endpoints, such as swimming ability and physiological or biological changes, can be used to assess the toxicity of organisms. Some endpoints related to fish embryos can be measured within a week, which is considered by experts to be acute or chronic toxicity, and the relevant basis for the decision should be documented.

3.5. Japan

The Ministry of the Environment Government in Japan presented the guidance on the derivation of WQS for protecting aquatic life from 2003 [5]; aquatic organisms are mainly focused on fish or shellfish, which are considered as seafood resources, and feed organisms consumed by seafood organisms are also objects for consideration [5]. The process of WQS derivation for aquatic life is separately conducted in seafood and feed organisms, and this study reviews the fish-related contents. Targeted fish data are collected in freshwater or oceanic groups; the top 20 catch-and-release fish species or indigenous species used in the international test methods, such as the Japanese medaka (O. latipes) in freshwater, and the top 20 hauling fish in the marine environment, are subjected to toxicity testing.

The acute effects on fish include the signs of survival, swimming, and growth inhibition after 48–96 h of exposure to chemicals or targeted contaminants. Related test guidelines are the acute fish tests (OECD TG 203 and EPA OPPTS 850.1075) and fish life cycle toxicity test (EPA 850.1500) [9,24,40]. The results of acute fish toxicity assessments are calculated as LC₅₀ or EC₅₀ and used to derive WQS [5].

Chronic effects are related to maturation, reproduction for adult fish, and survival or growth inhibition at embryonic- or larval-staged organisms. Exposure durations are specified to cover the embryo stage to the larval stage or pre-mature to reproductive stages of fish. The observative results of reproduction, mortality, immobilization, and growth inhibition are used to calculate the toxicity values (lowest observed effect concentration, LOEC, NOEC; maximum allowable toxic concentration, MATC). Related test methods include fish early-life-stage toxicity assays (OECD TG 210, EPA OPPTS 850.1400) [5,10,13]. Based on the acute and chronic toxicity to fish, the ACR is calculated to confirm the final chronic toxicity value; each final value of chronic toxicity in seafood or feed organism group is compared to the others; the lowest value is confirmed as the WQS value to protect the overall aquatic eco-receptor [5].

3.6. Korea

Korea has been developing a standard procedure to derive the environmental criteria for the protection of aquatic organisms following the guidelines of other countries [47,48,49,50,51]. The Ministry of Environment in Korea and the National Institute of Environmental Research (NIER) have been conducting a project for deriving water quality standards for protecting aquatic ecosystems since 2017 [47,48,49,50,51]; the number of substances for which WQS values have been derived has increased steadily [48,49,50,51]. With no established toxicity guidelines for researching ecotoxicity data or tests in fish species, this study analyzed the fish toxicity test methods proposed by the NIER and the Rural Development Administration (RDA) [16,52]. The NIER suggests an acute toxicity test and three chronic toxicity tests using various fish species. Acute toxicity tests could be conducted with fish embryos for 96 h with survival observation. Embryos are exposed to chemicals until their larvae could have free feeding activity, being assessed for the fertilization rate, hatching, survival, abnormality, and body length or weight. Juveniles weighing 0.05–0.1 g are exposed to chemicals for 4 weeks, and their survival rate, abnormal symptoms, and growth (weight) are recorded. The RDA has two acute toxicity tests using adults of three test species (Cyprinus carpio, O. latipes, Misgurnus anguillicaudatus), with observations of abnormalities, mortality, length, and weight. For exposure at the embryonic stage for 30 days, the chronic test involves recording hatching, survival, abnormalities, and behavioral effects [16]. In the fish life-stage toxicity test, fish at the embryonic or adult stage are exposed for the duration of the next generation, and the fertilization rate, hatching, survival, abnormalities, sex ratio, length, and weight are measured (Table 4).

3.7. Comparison of Acute and Chronic Toxicity for Fish and Priority Substances in WQS

Based on the guidelines for deriving the WQS to protect aquatic eco-receptors, each organization presented different standards for classifying the acute and chronic toxicity in fish (Table 5). While most fish test guidelines follow a 4-day acute exposure without developmental distinction [1,2,3,4,5], Canada, Australia, and New Zealand clearly distinguish the exposure period by developmental stage, with common criteria of 7 days for fish embryos and larvae and 21 days for juvenile and adult fish [2,5]. The fish species used in toxicity assays have rapid growth rates, so different developmental stages have different sensitivities to toxicants and pollutants [1,2,4]. These aspects eventually confirm the application of the appropriate exposure period at each developmental stage of the fish.

Fish are vertebrates, and fish tests using juveniles or adults can be unethical assessments for animal testing [4,6,7,8]; these should be replaced by alternative test methods using fish embryos or larvae. Furthermore, fish embryo- and larval-stage organisms are not considered to be regulated by the ethics of animal testing [6,7,8], so fish embryo assays are comparatively utilized in toxicity assessment. According to the well-organized distinction between acute and chronic toxicity to fish based on the developmental stages [2,4], the 7-day exposure in the fish embryo or larval test can be regarded as a discriminative baseline for chemical toxicity in early developmental stages of fish. Fish assays using embryos or larvae for less than 7 d are considered for acute toxicity testing. For the estimation of chronic toxicity, fish embryos or larvae should be exposed for at least 7 d, depending on the developmental stage. As suggested in the guidelines of each country and organization, chronic toxicity tests are recommended, so the early-stage or developmental-stage fish assay could be used as alternative tools to derive toxicity data for chemicals or toxicants for WQS.

The EQS for waterbodies (WQS) considers various indicators, including the acute and chronic toxicity of aquatic eco-receptors, and physico-chemical and microbiological indicators. There are different regulations applied to derive the WQS in each country or organization, with multiple indicators considering the impacts on the aquatic organisms caused by the characteristics of specific substances. Biochemical oxygen demand, chemical oxygen demand, total organic carbon, suspended solids, dissolved oxygen, pH level, and total coliforms directly affect living organisms in aquatic environments [1,2,3,4,5]; these can have significant impacts on the WQS derivation. By comparing the maximum acceptance level of common priority substances in each country and organization [53,54,55], different values for the chemical species were described (Table 6). These indicate that the WQS derivation systems have different calculation protocols considering various indicators, which result in different values for certain priority substances.

4. Comparison of Toxicity in Adult Fish and Embryo Assay

Previous research has tried to validate the fish embryo test as an alternative test method to adult fish tests [56,57,58,59,60,61,62,63,64,65]. Based on zebrafish toxicity assays, various chemical species were exposed to fish embryos, and their resultant toxicity values were compared with those for adult fish tests and compared to those for other fish species; this research was applied to reduce the unnecessary sacrifice of adult fish by animal testing.

To evaluate the applicability of the FET (fish embryo test) in chemical toxicity testing, Lammer et al. [56] compared and analyzed the fish and embryo toxicity data for 143 chemicals and identified the toxicity correlation for overall chemicals between adult fish and fish embryos. The lethal and sub-lethal endpoints were also observed during the chemical exposure. Compared to zebrafish embryo toxicity, all the fish embryo data, including P. promelas, O. latipes, and C. gariepinus, showed similar toxicity trends; this proximity confirmed that the zebrafish embryo toxicity data can be considered as representative of toxicity for chemical exposure among various fish species, except for high-molecular-weight surfactants. Furthermore, the adult fish assay has similar toxicity trends derived via FET, so FET can be a suitable alternative for adult fish test.

Knöbel et al. [57] proposed the FET as an alternative tool for adult fish assays by comparing the toxicity data of chemical species. The toxicity value of the FET showed similar sensitivity to those of fish adult tests, except for permethrin and allyl alcohol. Belanger et al. [58] also compared the fish toxicity data of chemical groups in fish embryos and adults and found that large functional groups of chemicals such as industrial organic compounds, pesticides, and surfactants showed similar toxicity trends, and these trends were also confirmed upon interspecies comparison. Unfortunately, some neurotoxic compounds (aldicarb, azinphos methyl, cyanizine, dieldrin, diquat dibromide, endosulfan, esfenvalerate, and picloram) have low embryonic toxicity in fish compared to adult fish toxicity [59,60]. The toxic impacts of movement in larval fish corresponded to lethality in adult fish, and the neurotoxic impacts can be predicted by determining the motility of larval fish [60]. These observations suggest that the fish toxicity data for organic compounds or non-ionic chemicals could contain uncertainties for extrapolation to ionic chemicals or inorganic compounds of different chemical functional groups. Furthermore, the uncertainty in the neurotoxic mode of action encourages the use of FET as a weight of evidence approach to gather the toxic impacts of narcotic substances [59,60,61].

To confirm the utility of FET by comparing the toxicity between species, Jeffries et al. [62] conducted a larval growth and survival (LGS) assay of P. promelas and FET for 3,4-dicholoraniline and ammonia. Toxicity sensitivity was found in 3,4-dicholoraniline; the FET can be used as a replacement method for the LGS assay for P. promelas. As well as chemical toxicity testing, the toxicity of the simulated wastewater treatment plant effluent in P. promelas and zebrafish embryos was also observed and the P. promelas assay included the chronic effects. The FET assay can be used with sensitive endpoints, such as the gene expression of developmental abnormalities, to improve the predictability of chronic toxicity [61,63].

As the FET has a high correlation with the adult fish assay, this relationship makes it a viable alternative tool for compliance with the 3R principles [64,65]. When collecting data for WQS derivation, the FET tool has been shown to reduce the unnecessary sacrifice of adult fish by 37.7–88% [64]. Although the FET data do not completely match the adult fish data, the more sensitive impacts of the fish embryo assay can provide more conservative predictions of chemical toxicity than the adult fish toxicity [65].

Consequently, toxicity information obtained from fish embryos can reflect the toxicity of adult fish, and this means that the fish embryo assay can be a good alternative to predict the toxic effects of contaminants or chemicals in adult fish. Researchers can promote reflection on toxic impacts on normal adult fish by controlling uncertainty with the extension of endpoints.

5. Conclusions

Fish toxicity information is used to derive water quality standards, and various fish assays have been applied. The study of fish embryo toxicity as a screening test could be used for alternatives to chronic assays, with the common test duration being longer than 5 days after hatching from embryos; these resultant values can be considered as indicative of chronic toxicity. Given their high reliability and concerns over experimental ethical issues, fish embryo toxicity tests with a suitable exposure duration could effectively substitute adult fish toxicity tests.

Author Contributions

Conceptualization, L.K. and Y.-J.A.; methodology, L.K.; investigation, L.K.; writing—original draft preparation, L.K.; writing—review and editing, Y.-J.A.; supervision, Y.-J.A.; project administration, Y.-J.A.; funding acquisition, Y.-J.A. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figure and Tables

Figure 1. Research trends of fish toxicity tests using 19 fish species. (A) Numbers of studies found in Google Scholar research engine, and three most frequently used fish species were ‘Cyprinus carpio’, ‘Danio rerio (zebrafish)’, and ‘Oncorhynchus mykiss’. (B) Relative ratios of fish embryo test in fish toxicity tests in 19 test species implied that Zebrafish and Oryzias latipes showed highest rates of fish embryo assay compared to other test species. Blue bars indicate the research ratio for D. rerio and Oryzias latipes which exceed 5% of embryo assay from total fish assay.

Figure 2. Development of zebrafish (A–D) and Oryzias latipes (E–H). (A) Embryo, (B) 2cdays post-fertilization (dpf) sac-fry, (C) 7-dpf larvae, (D) 2-month-old juveniles of zebrafish and (E) embryos, (F) 12-dpf sac-fry, (G) 16-dpf larvae, and (H) 2-month-old juveniles of O. latipes.

Table 1

Ecological effects test guidelines for fish, according to United States Environmental Protection Agency.

Acute/Chronic	TestGuideline	Test Species	Exposure Stage	TestDuration	Endpoints
Acute	OPPTS 850.1085 [21]	Oncorhynchus mykiss, Lepomis macrochirus, Pimephales promelas	Juvenile	4 days	Survival
	OSCPP 850.1075 [22]	Salmo salar, Menidia menidia, Lepomis macrochirus, Salvelinus fontinalis, Ictalurus punctatus, Oncorhynchus kisutch, Cyprinus carpio, Pimephales promelas, Poecilia reticulata, Poecilia reticulata, Oryzias latipes, Oncorhynchus mykiss, Cyprinodon variegatus, Menidia peninsulae, Danio rerio	Juvenile	4 days	Survival
Not classified	OPPTS 890.1350 [25]	Pimephales promelas	Juvenile	21 days	Survival, growth (length/weight), organ damage, behavior, fecundity, sex steroids
Chronic	OPPTS 850.1500 [9]	Pimephales promelas, Cyprinodon variegatus	At least one life cycle(from P0 embryos to F1 embryo)		Survival, abnormality, behavior, reproduction, fecundity
	OPPTS 885.4200 [26]	Pimephales promelas, Lepomis marochirus	Young fish 1	≥30 days	Survival, abnormality, behavior, reproduction, fecundity
	OPPTS 885.4700 [24]	Salvelinus fontinalis, Pimephales promelas, Lepomis marochirus, Ictalurus punctatus, Cyprinodon variegatus, Menidia menidia	Larvae	≥30 days(lifespan < 1 year)	Survival, abnormality, growth, behavior, reproduction, fecundity
			Larvae	≥90 days(lifespan ≥ 1 year)
	OCSPP 890.2200 [23]	Oryzias latipes	12–16 wpf 2	19 weeks	Survival, growth (length/weight), organ damage, fecundity, reproduction
	OSCPP 850.1400 [10]	Oncorhynchus mykiss	Embryo	60 dph 3	Survival, hatching, growth (length/weight), abnormality, behavior
		Lepomis macrochirus		32 d
		Pimephales promelas		28 dph 3
		Oryzias latipes, Danio rerio		30 dph 3

¹ Not applicable for non-feeding, spawning fish. ² Weeks post-fertilization (wpf). ³ Days post-hatching (dph).

Table 2

Biological test method for fish in Canada (published by Environment Canada and Environment and Climate Change Canada).

Acute/Chronic	Test Guideline	Test Species	Exposure Stage	Test Duration	Endpoints
Acute	Biological test method; reference method for determining acute lethality using three-spine stickleback [28]	Gasterosteus aculeatus	Juvenile/adult	4 days	Survival
	Biological test method; acute lethality test using rainbow trout [29]	Oncorhynchus mykiss	Swim-up fry *Fingerling life	4 days	Survival, appearance, behavior
Chronic	Biological test method; Toxicity tests using early life stages of salmonid fish (rainbow trout) [11]	Oncorhynchus mykiss	Embryo	7 days	Nonviability
			Embryo/larvae	7 days **	Nonviability, abnormality (hatching delay, deformation)
			Embryo/larvae/juvenile	30 days ***	Nonviability, abnormality (hatching delay, deformation), behavior
	Biological test method; test of larval growth and survival using fathead minnow [30]	Pimephales promelas	Larvae	7 days	Survival, growth (biomass)

* A young, yolk-sac larval with feeding activity. ** 7 days after half of embryos in control group hatched. *** 30 days after half of survival fish in control group showed swimming behavior.

Table 3

Test guidelines for fish in Organisation for Economic Co-operation and Development used in the derivation for Environmental Standard Quality recommended by European Commission.

Acute/Chronic	TestGuideline	Test Species	Exposure Stage	Test Duration	Endpoints
Acute	TG 203 [40]	Danio rerio	Adult	4 days	Survival, abnormality
	TG 236 [12]	Danio rerio	Embryo	4 days	Survival, abnormality, heartbeat
Chronic	TG 210 [13]	Oncorhynchus mykiss	Embryo	60 dph 1	Hatching, survival, abnormality, behavior, length, weight
		Pimephales promelas		28 dph1
		Danio rerio, Oryzias latipes		30 dph 1
	TG 212 [14]	Cyprinus carpio	Embryo	8–9 days	Hatching, survival, abnormality, behavior, growth (length, weight)
		Oncorhynchus mykiss		50–55 days
		Pimephales promelas		8–9 days
		Brachydanio rerio		8–10 days
		Oryzias latipes		13–16 days
	TG 215 [42]	Oncorhynchus mykiss, Danio rerio, Oryzias latipes	Juvenile	28 days	Abnormality, growth (length, weight)
	TG 234 [15]	Oryzias latipes, Danio rerio, Gasterostreus aculeatus	Embryo	60 dph 1	Hatching, survival, growth (length, weight), organ damage, sex ratio
	TG 240 [43]	Oryzias latipes	12–16 wpf 2	19 weeks	Survival, growth (length/weight), organ damage, fecundity, reproduction
Screening tool for chronic toxicity	TG 229 [41]	Pimephales promelas	18–22 wpf 2	21 days	Survival, appearance, behavior, fecundity, sex ratio
		Danio rerio, Oryzias latipes	14–18 wpf 2
	TG 230 [44]	Pimephales promelas	18–22 wpf 2	21 days	Survival, appearance, behavior, sex ratio
		Danio rerio, Oryzias latipes	14–18 wpf 2

¹ Days post-hatching (dph). ² Weeks post-fertilization (wpf).

Table 4

Test guideline for fish in Korea (published by National Institute of Environmental Research and Rural Development Administration).

Acute/Chronic	Test Guideline	Test Species	Exposure Stage	Test Duration	Endpoints
Acute	Freshwater fish acute toxicity test [16]	Cyprinus carpio, Oryzias latipes, Misgurnus anguillicaudatus	Adult	4 days	Mortality, abnormality, growth (length/weight)
	Freshwater fish effect test [16]	Cyprinus carpio, Oryzias latipes, Misgurnus anguillicaudatus	Adult	4 days	Mortality, abnormality, growth (length/weight)
	Fish acute toxicity test [51]	Cyprinus carpio, Oryzins latipes, Poecilia reticulata, Danio rerio, Lepomis macrochirus, Oncorhynchus mykiss	Embryo	4 days	Mortality, abnormality
Chronic	Fish early life stage toxicity test [16]	Oryzias latipes	Embryo	30 days	Mortality, hatching, abnormality, behavior
	Fish life cycle toxicity test [16]	Oryzias latipes, Oryzias sinensis	One life cycle(from P0 embryos to F1 embryo or P0 adult to F1 adult)		Mortality, abnormality, growth (length/weight), sex ratio, fecundity, reproduction
	Freshwater fish effect test [16]	Cyprinus carpio, Oryzias latipes, Misgurnus anguillicaudatus	Adult	30 days	Mortality, abnormality, growth (length/weight), sex ratio, fecundity, reproduction
	Fish early life cycle toxicity test [51]	Oryzias latipes, Oncorhynchus mykiss, Pinephales promelas, Danio rerio, Cyprinodon varieqatus, Cyprinus carpio, Oncochychus kisutch, Oncochychus tschawytscha, Salmo salar, Salmo trutta, Salvelinus fontinalis, Salvelinus namaycush, Lepomis macrochirus, Ichtalurus punctatus	Embryo	Difference in test species 1
	Fish embryo and sac fry toxicity test [51]	Oryzias latipes, Cyprinus carpio, Oncorhynchus mykiss, Pinephales promelas, Danio rerio, Carrassius auratus, Lepomis macrochirus	Embryo	Larvae 2	Mortality, abnormality, growth (length/weight), sex ratio, fecundity, reproduction
	Fish juvenile growth test [51]	Oryzias latipes, Oncorhynchus mykiss, Danio rerio	Juvenile 3	28 days	Mortality, abnormality, growth (length/weight)

¹ Test duration minimum reaching to larval stage without feeding activity. ² Larval stages right before the absorption of chorion. ³ Juveniles weighted at 0.05–0.1 g for O. latipes and D. rerio and 1–5 g for O. mykiss.

Table 5

Classification of acute and chronic toxicity of fish, depending on the developmental stages used for deriving water quality standards for protecting aquatic ecosystems in each country or continental union.

Country	Acute Toxicity		Chronic Toxicity		GuidelineReference
	Stage	Duration	Stage	Duration
United States	Juvenile/adult	4 days	Embryo, 48 hpf larvae *	≥28 days	[1]
			Juvenile	≥28 days
			Embryo → larvae → early juvenile	28–32 days
Canada	Not stated	4 days	Embryo/larvae	≥7 days	[2]
			Juvenile/adult	≥21 days
European Union	Embryo	4 days	Embryo	28–32 days	[3]
	Adult	4 days	Embryo → sac-fry	16 days
Australia & New Zealand	Embryo/larvae	<7 days	Embryo/larvae	≥7 days	[4]
	Juvenile/adult	<21 days	Juvenile/adult	≥21 days
Japan	Adult	2–4 days	Embryo/larvae	28–32 days	[5]

* 48 h post fertilization (hpf).

Table 6

The freshwater quality standards of maximum allowable concentrations (µg/L) for priority substances in US EPA, Canada, and European Chemical Agency (ECHA).

Substance	US EPA [53]		Canada [54]		ECHA [55]
	Freshwater		Freshwater		Emission Medium
	Short-Term	Long-Term	Short-Term	Long-Term	Inland Surface Water	Surface Water *
4,4-DDT	1.1	0.001	—	—	0.01	0.01
Acrolein	3	3	—	—	—	—
Aldrin	3	—	—	0.004	0.01	0.005
alpha-Endosulfan	0.22	0.056	0.06	0.003	0.01	0.004
Arsenic	340	150	—	5	—	—
Atrazine	—	—	—	1.8	—	—
beta-Endosulfan	0.22	0.056	0.06	0.003	0.01	0.004
Boron	—	—	29,000	1500	—	—
Cadmium	1.8	—	—	—	0.9	1.5
Carbaryl	2.1	2.1	3.3	0.2	—	—
Chlordane	2.4	0.0043	—	0.006	—	—
Chloride	860,000	230,000	640,000	120,000	—	—
Chlorine	19	11	—	0.5	—	—
Chlorpyrifos	0.083	0.041	—	0.002	0.1	0.1
Chromium (III)	570	74	—	8.9	—	—
Chromium (VI)	16	11	—	1	—	—
Copper	—	—	—	—	—	—
Cyanide	22	5.2	5	—	—	—
Demeton	—	0.1	—	—	—	—
Diazinon	0.17	0.17	—	—	—	—
Dieldrin	0.24	0.056	—	0.004	0.01	0.005
Endrin	0.086	0.036	—	0.0023	0.01	0.005
gamma-BHC (Lindane)	0.95	—	—	—	—	—
Guthion	—	0.01	—	—	—	—
Heptachlor Epoxide	0.52	0.0038	—	—	0.0003	0
Heptachlor	0.52	0.0038	—	0.01	0.0003	0
Iron	—	1000	—	300	0	0
Lead	65	2.5	—	—	14	14
Malathion	—	0.1	—	—	—	—
Mercury	1.4	0.77	—	0.026	0.07	0.07
Methoxychlor	—	0.03	—	—	—	—
Mirex	—	0.001	—	—	—	—
Nickel	470	52	—	—	34	34
Nonylphenol	28	6.6	—	1	2	2
Parathion	0.065	0.013	—	—	—	—
Pentachlorophenol	19	15	—	0.5	1	1
PFOS	71	0.25	—	—	36	7.2
PFOA	3100	100	—	—	—	—
PCBs	—	0.014	—	0.001	—	—
Selenium	—	—	—	1	—	—
Silver	3.2	—	—	0.25	—	—
Sulfide-HydrogenSulfide	—	2	—	—	—	—
Toxaphene	0.73	0.0002	—	0.008	—	—
Tributyltin	0.46	0.072	—	0.008	0.0015	0.0015
Zinc	120	120	—	—	—	—

* Surface water other than inland surface waters.

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