Proposing Effective Ecotoxicity Test Species for Chemical Safety Assessment in East Asia: A Review

East Asia leads the global chemical industry, but environmental chemical risk in these countries is an emerging concern. Despite this, only a few native species that are representative of East Asian environments are listed as test species in international guidelines compared with those native to Europe and America. This review suggests that Zacco platypus, Misgurnus anguillicaudatus, Hydrilla verticillata, Neocaridina denticulata spp., and Scenedesmus obliquus, all resident to East Asia, are promising test species for ecotoxicity tests. The utility of these five species in environmental risk assessment (ERA) varies depending on their individual traits and the state of ecotoxicity research, indicating a need for different applications of each species according to ERA objectives. Furthermore, the traits of these five species can complement each other when assessing chemical effects under diverse exposure scenarios, suggesting they can form a versatile battery for ERA. This review also analyzes recent trends in ecotoxicity studies and proposes emerging research issues, such as the application of alternative test methods, comparative studies using model species, the identification of specific markers for test species, and performance of toxicity tests under environmentally relevant conditions. The information provided on the utility of the five species and alternative issues in toxicity tests could assist in selecting test species suited to study objectives for more effective ERA.


Introduction
Choosing suitable ecotoxicity test species is a critical initial step in effect assessment for environmental risk assessment (ERA), as responses at different levels of biological organization to chemical exposure can serve as predictive evidence of chemical impacts [1].Sub-individual level responses, known as biomarkers, are useful in effect assessment as they can function as early warning signals for adverse outcomes [2].Additionally, responses at the organism level or higher, such as mortality, behavior changes, and a decrease in population, can provide direct evidence for assessing chemical effects on the environment [1,2] (Figure 1).Specifically, the responses of test species native to the environment under study can be crucial evidence in ERA.This is because the physiological traits of these species adapted to the region can be helpful for the prediction of the chemical effects on that region, especially if site-specific information is required, and if a few key species determine the function and structure of the aquatic ecosystem [3].
Nevertheless, challenges exist in applying these species to assessment of chemical effects.One issue is interspecies variability in chemical toxicity.Research has demonstrated that each species in an ecosystem may respond differently to chemical exposure [4].While uncertainty factors and species sensitivity distributions have been applied to ERA to address this, the lack of toxicity mechanisms that explain interspecies differences introduces uncertainty [1,4].Thus, identifying test species with diverse traits in response to chemical exposure is crucial.Moreover, as an ecosystem comprises various species interconnected by food chains, it would be more effective to select multiple species from different trophic levels rather than relying on a single species.In summary, to perform effective ERA for chemicals, there is a need to develop diverse ecotoxicity test species, taking into account interspecific differences and roles within food chains.Nevertheless, challenges exist in applying these species to assessment of chemical effects.One issue is interspecies variability in chemical toxicity.Research has demonstrated that each species in an ecosystem may respond differently to chemical exposure [4].While uncertainty factors and species sensitivity distributions have been applied to ERA to address this, the lack of toxicity mechanisms that explain interspecies differences introduces uncertainty [1,4].Thus, identifying test species with diverse traits in response to chemical exposure is crucial.Moreover, as an ecosystem comprises various species interconnected by food chains, it would be more effective to select multiple species from different trophic levels rather than relying on a single species.In summary, to perform effective ERA for chemicals, there is a need to develop diverse ecotoxicity test species, taking into account interspecific differences and roles within food chains.
East Asia is a significant player in global industrial chemical production.In particular, South Korea, Japan, and China, situated in East Asia, lead the world's industrial chemical sales markets.From 2011 to 2021, China, Japan, and South Korea ranked first, fourth, and fifth in chemical sales, collectively accounting for 51% of global market sales in 2021 [5].As the chemical market expands in these countries, the emphasis on chemical safety management is increasing.Consequently, the governments of South Korea, Japan, and China have enacted chemical regulatory acts/laws, including South Korea's Act on Registration and Evaluation of Chemicals and Consumer Chemical Products and Biocides Safety Control Act, Japan's Chemical Substance Control Law, and China's Ministry of Ecology and Environment Order No. 12 [6].Based on the principle of "no data, no market," these acts require all manufacturers or importers of chemicals to register or obtain approval for their products from regulatory authorities, providing proof of safety to humans and the environment through human risk assessment and ERA.For ERA, regulatory authorities require manufacturers/importers to submit ecotoxicity test data conducted following international standard test guidelines (e.g., OECD test guidelines), which recommend several model/test species for ecotoxicity testing.
In these guidelines, most ecotoxicity study protocols have been conducted using only a single or a few model species.Typically, toxicity tests for ERA recommend model species such as Danio rerio, Daphnia magna, Pseudokirchneriella subcapitata, among others [7,8].The rationale for using these model species is that they are cost-effective as they have been cultured under laboratory conditions for several years.Furthermore, the test methods for these species are typically better established compared with those for non-model species, and they are more easily integrated into toxicity evaluations [3].However, most of these East Asia is a significant player in global industrial chemical production.In particular, South Korea, Japan, and China, situated in East Asia, lead the world's industrial chemical sales markets.From 2011 to 2021, China, Japan, and South Korea ranked first, fourth, and fifth in chemical sales, collectively accounting for 51% of global market sales in 2021 [5].As the chemical market expands in these countries, the emphasis on chemical safety management is increasing.Consequently, the governments of South Korea, Japan, and China have enacted chemical regulatory acts/laws, including South Korea's Act on Registration and Evaluation of Chemicals and Consumer Chemical Products and Biocides Safety Control Act, Japan's Chemical Substance Control Law, and China's Ministry of Ecology and Environment Order No. 12 [6].Based on the principle of "no data, no market," these acts require all manufacturers or importers of chemicals to register or obtain approval for their products from regulatory authorities, providing proof of safety to humans and the environment through human risk assessment and ERA.For ERA, regulatory authorities require manufacturers/importers to submit ecotoxicity test data conducted following international standard test guidelines (e.g., OECD test guidelines), which recommend several model/test species for ecotoxicity testing.
In these guidelines, most ecotoxicity study protocols have been conducted using only a single or a few model species.Typically, toxicity tests for ERA recommend model species such as Danio rerio, Daphnia magna, Pseudokirchneriella subcapitata, among others [7,8].The rationale for using these model species is that they are cost-effective as they have been cultured under laboratory conditions for several years.Furthermore, the test methods for these species are typically better established compared with those for non-model species, and they are more easily integrated into toxicity evaluations [3].However, most of these model species inhabit temperate climates and originate from North America, South Asia, and Europe.For East Asia, there are only a few recommended model species (e.g., Oryzias latipes).Therefore, there is a need for increased efforts to identify resident test species in East Asia that would be useful for ERA.
In this context, this study aims to propose promising test species, each possessing a variety of traits useful for ERA in East Asia, and to analyze the utility of these species for ERA.Additionally, by examining trends in ecotoxicity tests focusing on alternative approaches in ERA, this study identifies and suggests challenges and future research directions for enhancing the utility of test species in ERA.This research will be beneficial for identifying and applying reliable test species for effect assessment in ERA.

Selection Criteria for Freshwater Test Species of East Asia
By summarizing existing literature, 9 selection criteria are chosen as follows: (1) clear taxonomy, (2) geological distribution in East Asia, (3) a role in a trophic level, (4) habitat type, (5) well-analyzed morphology and physiological traits such as life cycle, reproduction pattern, ( 6) applicability to a laboratory study such as ease of manipulation/culture, small size, short life cycle and others, (7) the specific traits of a species, (8) references for chemical ecotoxicity in diverse biological organization levels, (9) sensitivity to chemical exposure [1][2][3].Using these selection criteria, promising test species were selected (Figure 2).In total, 40 species were randomly chosen and analyzed.Among them, 5 species were selected as a test species (in preparation).

Mining Promising Test Species
In this section, the traits of each species are analyzed based on selection criteria, and a state of toxicity researches.Then, applicability/utility of each species in effect assessment of ERA is discussed.

Life Cycle and Physiological Characteristics
In order to observe the chemical effects in the level of individual or higher, the information for taxonomy, geological distribution, habitat type, a role in a trophic level, phys-

Mining Promising Test Species
In this section, the traits of each species are analyzed based on selection criteria, and a state of toxicity researches.Then, applicability/utility of each species in effect assessment of ERA is discussed.

Life Cycle and Physiological Characteristics
In order to observe the chemical effects in the level of individual or higher, the information for taxonomy, geological distribution, habitat type, a role in a trophic level, physiological traits and so forth of five species were required.Also, to establish proper test conditions of a toxicity test for a specific chemical, with chemical properties, the information of species physiological and ecological traits are necessary.The information of the five species are noted as follows.
Scenedesmus obliquus (NCBI taxonomy ID: 3088) are one of the unicellular green s, involved in genus Scenedesmus that are distributed in the freshwater and brackish water environment, being found in China, South Korea, Japan and so on [9][10][11][12].And they have the traits of rapid growth, CO 2 fixation efficiency, growth in the wastewater (e.g., Harsh environment), ease to manage, lipid accumulation, removing metal, having diverse antioxidant [9,10,13].S. obliquus keep growing at the temperature in a range of 15 • C-40 • C [10].In 25 • C, 150 µmol/(m 2 •s) light intensity, pH 10, the growth rate of S. obliquus showed the highest increase level [10,14].But in another study, at pH 8, the optimization of a growth rate was reported [10].S. obliquus cells are non-mortal and observed in the form of coenobia (microcolonies) of four cells produced by one parent cells, even though under multiple fission mode of reproduction, 8-16 cells can be produced by a cell.Besides asexual reproduction mode, S. obliquus have a sexual mode of cell cycle [14].
Macrophytes, including Hydrilla verticillata (NCBI taxonomy ID: 51024), have contributed to the improvement of water quality, sediment stabilization and others [15].H. verticillata play an important role in the freshwater ecosystem through the regulation of the nutrient as well as a carbon cycle.Also, as a primary producer, they acts as a habitat provider and energy supplier for other organisms [15,16].Their habitats involve temperate and tropical regions, and major habitats are ditches, spring, leks, marshes, rivers, tidal zones, channels, quarries, shallow reservoirs [15,17].Monoecious type is supposed to be originated from Korea [17,18].Nowadays, they are distributed in Europe, Asia (e.g., Korea, Japan, China), Central Africa, North America, South America, and others [15,17,19].In reproduction, they are either monoecious or dioecious and the flowers are unisexual.Their flowers and fruits are produced in May to October [17].Their reproduction is mainly by fragments of stems, but it also can be reproduced through growth of subterranean tubers and turions (axillary buds), suggesting high level of plasticity [15].
Aquatic plants and algae are one of the important components of the ecosystem.Thus, the study for them in response to environmental pollutants have attracted the attention of regulatory authorities such as US-EPA, Korea-MOE.In general, among freshwater plants and algae, microalgae are selected as a toxicity test species, since their high sensitivity to municipal and industrial wastewater, easy manipulation in a laboratory.Thus, OECD test guideline 201 and ISO 8692 test guideline for microalgae were established [20].Moreover, in the OECD 221, ISO 20079, floating rootless flowering plants such as Lemna minor were applied to an ecotoxicological test.However, several toxicants did not cause effects upon microalgae, L. minor, because of precipitating properties and others, resulting in raising necessary for a submerged macrophyte (rooted plants) such as H. verticillata [20].
Neocardina denticulata ssp.including Neocaridina denticulata sinensis (Kemp, 1918, NCBI ID: 274643) and Neocaridina denticulata denticulata (De Hann, 1844, ID: 436129) are classified as a subgroup of Neocaridina denticulata (De Hann, 1844.ID: 274642).These are a member of atyidae and native species to Asia including Korea, China, Taiwan, Vietnam, Japan [21].Their habitats are described as lentic and lotic waters [22] or ponds, rivers, agricultural canals, mountain streams, reservoirs [23].They have diverse colors and they eat detritus and micro-organisms upon macrophyte roots, and immersed sub-stances as an omnivore.Their total length is 2-3 cm and carapace length of 6.4-7.8 mm in female [22,24].In other study, carapace length of female and male were 5.1 ± 1.6 mm and 4.4 ± 1.5 mm, respectively [25].They can live in diverse environmental conditions involving pH (6.5-8.04),temperature (24-29 • C), oxygen contents (5-7 mg/L), ammonia (0.1-1.9 mg/L), nitrate (0.1-10 mg/L), so they can be tolerant to environmental changes, being world-widely distributed even though they are native to East Asia including Korea, Japan, China, Taiwan [23,24,26].The diameter of eggs is 0.57-1.19mm on average.Embryonic development lasts for 15 days at the temperature of 27 • C [21,24].The hatched larvae has a total length of about 2-3.3 mm on average.The number of larvae produced by a female are 21-51 individuals.After about 60 days, the larvae grows to a juvenile stage (total length: 1.2-1.5 cm) and 15 days later, they reach an adult stage [21,23].Based on the previous study, from a larvae stage, ovigerous females were found in five months, then they can carry the embryos for approximately 30 days [25].Through 2-3 hatching events, their larva were released.The maximum life span was about 1.3-1.4years, and main spawning time is Jun-July [22].Relative to a field, in the laboratory, a slower rate of the growth of this species was observed [25].The initial growth rate of male was similar with females but with time passed, males grew more slowly, becoming smaller than females according to the previous study [25].N. denticulata ssp.have advantages such as size, availability, ease of culture, and so on.They showed resistance to bacterial infection [27].They have a transparent exoskeleton, which make possible to carry out noninvasive monitoring of internal organs and tissues, facilitating the investigation of reproduction, molting.What is more, this trait can provide an advantage of large-scale mutant selection [24].
Zacco platypus (NCBI taxonomy ID: 80810) that is called freshwater minnow, pale bleak, or pale chub, are freshwater fish and distributed to Asia broadly involving South Korea, North Korea, Japan, China, northern Vietnam.They inhabit in the subtropical climate of 10 • C-22 • C [28].At an adult stage, commonly they are observed in rivers and streams with rapid water flow, whereas they are not found in deep or stagnant waters.They feed on zooplankton, small crustaceans, macroscopic algae, small fish, and detritus [28].As benthopelagic fish, they fit to toxicity study for both waterborne as well as precipitating/absorbing chemicals.Maximum total length of them is about 22.5 cm and common length of them is about 13 cm.Based on an estimation model (life-history tool), generation time is 1.8 years, Age at the first maturity is 1.8 years (1.4-2.3 years), intrinsic rate of increase 2.56 years [28].They have about 5 years of life span [29].
Misgurnus anguillicaudatus (NCBI taxonomy ID: 75329) are found in Korea, China, India, Thailand, Laos, Siberia (Tugur and Amur drainages), Japan, Vietnam, Taiwan, Cambodia [30].Moreover, they are introduced into Australia, USA, Germany (Rhine), Italy (Ticino drainages, north of Milano), Aral sea basin and others.They are benthic species and their prominent habitats include ditches, rice paddy fields, streams, and mud places, ponds and they can live without water for a short period, hiding into the mud until water can be available [28,30].As a benthic species, they are subject to chemicals absorbed upon sediment/debris/soil/submerged plant (e.g., triclosan), having high Koc value and pesticides introduced to rice paddy fields or ditches than other conditions.They live in a subtropical environment.They feed on insects, worms, snails, ostracods, cladocerans, fish eggs, algae, detritus and other small aquatic organisms.Especially, mosquito larvae can be consumed by them [30].In terms of reproduction, the male M. anguillicaudatus can be mature within a year, while female fish can be mature within one or two years [30,31].Their spawning season is from mid-April to early October and within the period, they spawn multiple times and their fecundity increased in proportion to a body size [30,31].Female fish yields 1800-15,500 eggs per batch, surviving average 2000 eggs per batch [32].They inhabit in the subtropical climate of 5 • C-25 • C [28].Based on an estimation model (life-history tool), their generation time is estimated to be 2.8 years and age at first maturity is 2.6 years.Intrinsic rate of increase is 1.8 years.Their common length is about 15 cm in total length, while maximum length reach 28 cm [28].Their size in an adult stage is no more than 20 cm, so a laboratory test is plausible.Genetically, they have diverse polyploidy population from 2n to 6n [30].Triploid cell lines were considered to have superior flesh than diploid, attracting industrial attention [33].

Toxicity Study Status
S. obliquus as microalgae have been studied in diverse biological organization levels and endpoints.For the oxidative stress assessment, S. obliquus were exposed to nanomaterial, metals, copper sulfate, herbicide, fungicide, nitrates and wastewater, in which ROS contents, antioxidative enzyme (e.g., CAT, SOD) activity, antioxidant (e.g., GSH) contents, lipid peroxidation contents and other oxidative stress markers were significantly changed, suggesting S. obliquus is suitable test species for detecting oxidative stress [11,[34][35][36][37][38][39].But in these cases, the oxidative stress profiles were significantly affected by humic acid and other chemicals co-treatment.
Oxidative stress (e.g., ROS contents), metaolomic analysis using GC-MS, light quantum yield (Y(II)), electron transfer rate (ETR) Growth inhibition [39] In a population level, S. obliquus showed a significant growth rate decline in responses to metals, biocide, flue gas, dioxin, nanomaterials, in which the rate decline was accompanied with oxidative stress and photosynthesis disruption.Also, natural organic compound such as humic acid affected the effect occurrence [11,34,35,46].
H. verticillata have been studied in diverse biological organization levels and endpoints under a laboratory condition.In an individual level, when H. verticillata were exposed to 2-methyl-4-chlorophenoxy acetic acid (MCPA), perchlorate, metals, fluoride, nanoparticle, they showed the effects such as growth inhibition, leaf dead zone increase, brownish green leaf, hard and thin texture of a leaf, brown stem color, decrease of stem thickness, internode distance and others in both mature and young leaves as well as perforation in surface cuticle of young leaves and others [16,[48][49][50][51] (Table 2).
Zacco platypus have been studied in diverse biological organization levels and endpoints.In a laboratory test condition, Z. platypus development stages were either a juvenile stage or an adult stage.In a juvenile stage, average length was 5.5 ± 0.3-7.7 ± 1.04 cm and in an adult stage, ave.length was distributed from 10-20 cm.In juvenile fish ave.weight of 6.49 ± 2.92-9 g, while in an adult stage, average weight was 15.72 ± 4.46 g.Commonly, during 2 w-6 m, they are acclimatized prior to a test in a laboratory.Tests were performed in an acute condition within 96 h-14 d, whereas until now a chronic toxicity test was not conducted.In the laboratory test, water temperature was set at about 19-22 • C and pH of water was 6.8-7.4.Research target tissues included liver, muscle, gill, skin, kidney, muscle, brain (Table 4).They have shown the potential as a promising test species in a sub-organism/organism level.Precisely by exposure of benzo(a)pyrene, cadmium, copper, wastewater contaminated with municipal chemicals (Cd, Co, Cr, Mn, Ni), Z. platypus showed oxidative stress occurrence (increase of MDA content, CAT and SOD activity), enhanced biotransformation (CYP1A and CPR activation), genetic toxicity (DNA adduct formation, nuclear abnormality), stress responses (metallothionine expression, heat-shock protein 79/90), endocrine disruption (E2 hormone level, GSI), neurotoxicity (AChE activty) [71][72][73][74][76][77][78][79][80][81].In addition, Ammonia treatment caused decrease of a survival rate and a hatching rate as well as deformed alevins increase [75].

Characteristics Useful for Chemical Toxicity Tests
S. obliquus have diverse traits that can be used as markers in ecotoxicity studies.Scenedesmus sp. have plenty of diverse metabolites.Astaxanthin is one of the carotenoid compounds acting as an antioxidant, enhancing preventing UV light influence [9].Mycosporine-like amino acids that were bound to a chromophore play an important role in protecting UV radiation were exploited for cosmetic skin-care products for UV protection [9].The expression level of these metabolites would act as a marker for chemical-induced oxidative stress or UV effect analysis.S. obliquus have the ability to inhabit in wastewater, so that it can be used in toxicity test under municipal wastewater.Moreover, they can be used in removing pollutants such as metals, ammonium, phosphate and by producing oxygen, reducing chemical oxygen demand [92].These characters mean that the metabolism enzymes of S. obliquus for the pollutant biotransformation can be a useful biomarker to metals, ammonium, phosphate contamination [93].In addition, S. obliquus can form a defense colony in response to zooplankton grazing cues and this colony formation is modulated by cadmium exposure and salinity, suggesting that the colony formation can be markers for cadmium exposure and salinity changes [94].
In diverse previous studies, Z. platypus showed useful traits for ERA.Precisely, male fish reveals a body color change around the time of breeding in June every year [98].Also, at that time, a nuptial organ is found in the mouth, gills, rear fin of male fish.These types of traits can be used as an indicator for sexual differentiation.Also, Z. platypus show behavioral traits such as shorter latency in escape, swimming speed increase under predation [99].If the change of neurotoxicity biomarker such as AChE activity were associated with the locomotive traits, the mechanism of chemicals bringing about neurotoxicity would be understood precisely.For CYP1A, a previous study reported the tissue distribution and amount of it in Z. platypus [73].In 10 tissues, CYP1A mRNA expression was significantly induced in response to β-naphthoflavone and the expression level were in order of Liver > Gill ≈ Kideny ≈ Intestine > Brain ≈ Gonad > Muscle > Skin ≈ Eye ≈ Heart.CYP1A and CPR mRNA expression showed time-dependent fluctuation tendency, where mRNA expression of them reached a maximum level at early exposure (1-4 days) time while as exposure time passed the expression level decreased [77].
Several studies investigating traits of M. anguillicaudatus showed they are a promising test species.On the analysis of M. anguillicaudatus eye, the genes involving sox2, msi1, bmi1 that control the neural stem cell differentiation were found.The eye of M. anguillicaudatus have proliferative and pluripotent neural stem cells which supply new cells during development.Here, in the immunohistochemistry analysis, peroxisome proliferator activated receptors (PPARα, γ) were observed, suggesting that PPARs have a role in neurogenesis of M. anguillicaudatus eye.These traits would be useful information for a chemical-induced neurotoxicity study [100].During a drought, M. anguillicaudatus put itself into mud and they can live for weeks or months without water.Under an air-breathing condition, an ammonia concentration in intracellular space would increase, so for survival, M. anguillicaudatus have several survival strategies including reducing ammonia production by reducing protein/amino acid catabolism, detoxifying ammonia to glutamine, reducing ammonia production by leading to alanine formation and others [101].These tolerance traits of them to reducing ammonia contents by diverse molecular mechanisms could be applied to assessing ammonia effect upon target-aquatic environment.

Applicability of 5 Species to ERA
In the environment media, by physicochemical properties of chemicals, there are different chemical fates and behavior and this difference can cause different chemical effects on organisms.For example, chemicals easily absorbed to sediment would be more threatened to benthic organisms than pelagic organisms in the aquatic environment.Moreover, By biomagnification through food-chain, the easily bioaccumulated chemicals would be more risk to high level consumer species than producer or primary consumer species.Taken together, in the environment, by diverse factors affecting chemical effects, there can be various environmental chemical exposure scenarios causing different chemical effects on organisms.Thus, to predict precise chemical effects in the environment under a laboratory condition, the application of multi-species having diverse traits covering the chemical environmental exposure scenarios would be reasonable.
This study showed that the traits of five species complement each other in chemical effect assessment on diverse exposure scenarios.M. anguillicaudatus live in bottom habitat of water body so they are more suitable in assessing chemical effects having high Koc.H. verticillata are sub-merged plants, so they are useful to analyze effects of chemicals precipitating in water than floating plant or algae.As a producer in the food-chain, S. obliquus and H. verticillata would be sensitive test species in assessing chemicals causing photosynthesis disruption.As a high-level consumer in the food-chain, Z. platypus and M. anguillicaudatus would be useful in study chemical effects having high Kow than producer or primary consumer.N. denticulata ssp.would exhibit chemical effects upon crustacea development and primary consumer.
Moreover, five species are well-studied in life cycle, physiological/ecological characteristics, habitats and they have studied in assessing effects for diverse chemicals such as metal, PFAS, nanoparticles, pharmaceuticals, biocides, PAH and others under a laboratory condition.The effects were studied in diverse biological level endpoints in sub-individual level (e.g., oxidative stress, photosynthesis disruption, xenobiotic transformation, endocrine disruption, immune disruption), individual level (e.g., mortality, reproduction, development, behavior change), population level (e.g., population growth rate) (Table 6).In summary, the collective application of the species would be useful in predicting diverse chemical effects upon the East Asia ecosystem under studies in diverse chemical exposure scenarios (Figure 2).

Remaining Issues in the Study of Test Species
In this section, current ecotoxicity test trends focusing on alternative approaches for regulatory purposes are analyzed.In each paragraph, by diverse alternative approaches, emerging issues/topics are introduced.Then, to increase their utility/applicability in ERA, further study directions for the test species are discussed.

Alternative Testing Methods
Due to rising attention for animal welfare, increase of chemical toxicity information and development of new analysis technology, the requirement for alternative methods reducing/eliminating animal (vertebrate) use was augmented [2,102,103].In an aquatic toxicity study, the predominant application of a fish toxicity test for a regulatory purpose was reported, relative to other species [104].Indeed, the UK annual National Statistics report exhibits that fish tests are the most frequently performed tests in non-mammal (birds, reptiles, amphibians, rats, fishes) tests and the fish test number (15%) take second place next to mice (54%), being followed by birds (14%), rats (11%) [105].Fish and mammal aquatic organisms are classified as vertebrates, so it is necessary to replace/reduce the traditional test as an alternative test.Even though the aquatic invertebrates (D. magna), plant (H.verticillata) and others are not classified to a vertebrate animal, alternative approaches such as in vitro, in silico, in chemico assay would be helpful to unveil chemical toxicity mechanism in the invertebrates and plants.
A representative in silico test is the test conducted with a program of quantitative structure activity relationship (QSAR) that is a kind of a mathematical model describing the relationship between chemical structure and biological activity.Using a QSAR program, we can obtain information for biological activity, ADME (absorption, distribution, metabolism and excretion), toxicity of a test chemical without animal sacrifice.It is cost-effective, and rapid relative to in vivo tests and it can be used in filling the data gap in existing toxicity information [106].In existing literature, there have been diverse trials to develop QSAR.Based on the pEC50 values from Photobacterium phosporeum and Selenastrum capricornutum, structural features of individual chemicals and their mixture were modeled according to the OECD guidelines.The model was approved to have applicability to predict non-tested chemicals [107].A numerous endpoint data set for toxicity to algae, Daphnia, fish in response to 3680 chemicals were used to develop a model following OECD principles [108].But, in this study, only one case of a QSAR study was found for S. obliquus.By combining the EC50, n-octanol/water coefficient (logP), frontier orbital energy gap (∆E), the equation for the single toxicity of substituted phenols, anilines, and mixtures were established [47].Considering interspecies difference, there would be diverse results for the prediction of chemical effects upon the ecosystem, so the application of test species having diverse traits for developing of QSAR is required.
An in vitro test can be used not only as an alternative to in vivo tests but also, as a screening tool for potential hazard of chemicals [109].Relative to an in vivo test, an in vitro test has several advantages such as being cost-effective, simple performance, less time consuming, small scale, suitability for evaluating mode of action/for high-throughput test, satisfying ethics requirements and efficiency [109,110].Thereby, for regulatory purposes, the submission of in vitro test reports is permitted under the regulation of the EU-REACH program.
A fish or invertebrate embryo has been regarded to be one of practical in vitro systems.Fish in an embryo stage is not classified as a vertebrate animal by NIH [111].For this, fish embryo toxicity tests have been recommended as an alternative test method.In a comparative ecotoxicity study, fish embryo, D. magna or Alga were assessed for 233 chemical compounds, in which lowest-observed effect concentrations (LOECs) was obtained.
Therein, the early life stage test based on OECD test guideline 210 indicated 10-fold higher sensitivity of a fish embryo than other systems, suggesting that in some modes of action (MOA) by chemicals, a fish embryo system could be a sensitive tool for detecting chemical toxicity [112].However, a fish embryo test has some problems.First, a fish embryo test has the lack of endpoints for unveiling the sublethal chemical effects.Second, in some chemical treatments, a fish embryo test showed lower sensitivity than an adult fish test [113,114].Thus, a previous study suggests the markers such as snout-vent length (≥14% length reduction) and pericardial area (≥3.54 fold pericardial area increases) as an alternative marker.The markers in a fish embryo were predictive of mortality, indicating it would be available as the alternative to a mortality test [114].In this study, the toxicity test using embryos of Z. platypus and M. anguillicaudatus were not found to our best knowledge, Thus, considering the advantages mentioned above, the effort to characterize embryos of the species, develop/validate test methods, apply embryo systems to toxicity assessments, and evaluate the sensitivity of embryo systems relative to other species are necessary to increase utility of test species.
Since an invertebrate such as crustacea is not classified as a vertebrate animal, an invertebrate embryo test does not attract the attention relative to a vertebrate case.But there are several advantages of an embryo toxicity test such as utility in the understanding of the toxicity mechanism to development process, reproduction and analysis of chemical effects upon life cycle of test species, so profound studies for it need.In fact, previous studies showed that an invertebrate embryo system could be used to assess chemical toxicity.Embryos of Marisa cornuarietis were found to be a suitable system for evaluate endocrine disruption, development toxicity by chemicals.And sensitivity of the embryos is equal or higher than zebrafish embryos [115].But in this work, no studies using an embryo system of N. denticulata ssp.are found.
Cellular systems including primary cell, cell lines, stem cell have been used as an important alternative tool in ecotoxicity test for environmental chemicals.In fish and shellfish, numerous studies using primary cells showed it is a useful system to assess chemical ecotoxicity such as endocrine disruption, oxidative stress, apoptosis and immune disruption [116][117][118][119]. Cell lines also have been applied to an in vitro toxicity test [104].So far, several cell lines including RTL-W1, RTgill-W1, RGT2 of Oncorhynchus mykiss, PLHC-1 of Poeciliopsis lucida, and CHSE-14 of Oncorhynchus tshawytscha have been applied to an ecotoxicity test, which includes [104].Cell lines have advantages including homogeneity, ease to culture, and being suitable for a high-throughput test.A cytotoxicity test using a gill cell line of O. mykiss indicated an approximately 1:1 correlation with a fish acute toxicity test result in responses to organic chemicals [120].Interlaboratory reproducibility of a test method using a fish gill cell line (RTgill-W1 cell line) was demonstrated [121].This suggests that fish gill cell lines can be a useful alternative tool for in vivo toxicity.Additionally, a stem cell system has received attention because of useful traits.It has characteristics such as self-renewal, differentiation to other cell types, forming tissue originated from the same cell, which are not found in cell line and primary cell systems.Therefore, the problems including availability limitation, functional variability, genetic alterations could be eased [65].
In terms of this work, in vitro tests using a cell line, primary cell, stem cell, embryos were not detected in these species except for a cellular level study for M. anguillicaudatus.As mentioned above, those systems have diverse advantages in chemical ecotoxicity assessment and the application of the systems is encouraged in future ecotoxicity tests.Thus, not only development, but also applicability the evaluation of the systems originated from a test species would be helpful for elevating the species utility.

Other Alternative Approaches
Omics is a discipline of biology that deals with genomics, proteomics, transcriptomics, metabolomics [122].Since omics is based on total information of the gene, transcript, protein, or metabolite produced in an organism, the information on it can enhance the understanding of total response profiles to chemical exposure [123].For example, under PFOS exposure, analysis using omics technology presented neurological function change, oxidative stress, energy metabolism disruption, axonal deformation, neuroinflammatory stimulation, calcium ion signaling dysregulation, suggesting that omics analysis can be a tool to broaden understanding of a toxicity mechanism [124].However, in this review, a few case studies evaluating chemical toxicity using omics technology were found in terms of S. obliquus, H. verticillata, N. denticulata spp., whereas no study exist for Z. platypus, M. anguillicaudatus.National Center for Biotechnology Information (NCBI) database showed that whole genome sequence of M. anguillicaudatus (assembly ID: HAU_Mang_1.0/GCA_027580225.1 (reference genome)) and S. obliquus (7 assemblies including UMN_S.PABB004_v1) are published, suggesting that they can be used in studies such as RNA sequencing analysis and other omics technologies for unveiling chemical toxicity mechanism [125].
When using omics technology, mining of diverse genes and proteins useful for chemical effect assessment can be possible.Among those, the genes relevant with xenobiotic biotransformation, development, endocrine control, reproduction, immune system as well as house-keeping genes are important in the understanding of chemical effect.Xenobiotic biotransformation is the process that transform lipophilic chemicals to hydrophilic chemicals, eliminating them out of body.In model species such as Danio rerio, genes and enzymes related with xenobiotics biotransformation are well evaluated [126].But, in the five species of this work, lack of information for the genes was found.For example, in case of Z. platypus, cytochrome P450 systems are characterized, whereas the characterization of cytochrome P450 systems of M. anguillicaudatus and N. denticulata spp.was not reported [73,77].To detect a background level within an organism, in case of transcript/translation analysis, housekeeping (reference) gene/protein expression levels are used, since their expression are maintained at a constant level in most of the organs and the expression level cannot be changed significantly [127].In case of plant, Arabidopsis pumila showed about 10 genes that were recommended as reference genes by specific stress conditions.The application of UBQ9 (Polyubiquitin 9) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) in heat stress, ACT1 (Actin 1) and GAPDH in salt stress, UBC35 (Ubiquitin conjugating enzyme 35) and GAPDH in cold stress, ACT1 and GAPDH in comparison with different tissues were suggested [128].In D. magna, exposed to ibuprofen, GAPDH, UBC (Ubiquitin conjugating enzyme) showed stability relative to other candidates such as 18s, 28s genes [129].Among the species reviewed in this work, well-known housekeeping genes such as βactine (ACTB), elongation factor 1 alpha (EF-1a), GAPDH, 18S ribosomal RNA (18S rRNA) were analyzed and expression profiles in terms of gender difference, tissue type, different developmental stages were studied in Misgurnus anguillicaudatus [85].Z. platypus beta-actin gene expression was used as a reference gene and protein [77,79].In N. denticulata, betaactin was used as an internal control [62].This suggests that the identification/evaluation of the reference genes under a different condition is required.By extension, the genes related to endocrine system, reproduction, development, immune system and others can be significantly related with adverse outcome [2].Thus, profound study for searching the genes using omics technology are required.
Most of dossiers submitted to regulatory authorities (e.g., EU-ECHA) for proving chemical safety are mainly composed of in vivo test reports performed at the individual or higher level [130].The reason for this is that relative to an in vitro test, an in vivo test is considered to have higher ecological relevance [104].Adverse outcome pathway (AOP) is a conceptual framework composed of molecular initiation events, adverse outcome [2].It can be used for the prediction of adverse outcome in an individual or higher level by using the information for molecular level events.For this, AOP could augment trustworthy/utility of in vitro test, in silico test by increasing ecological relevance, finally reducing animal sacrifice.In this sense, to establish AOP to chemical exposure, the information for chemical effects in diverse biological organization levels is required (Figure 1).Especially, nuclear receptor activation (e.g., estrogen receptor) can act as molecular initiation event (MIE) that play a role as a starting point in AOP, since nuclear receptor activation can trigger cell signaling and physiological changes followed by chemical-induced reproduction disruption, growth rate inhibition and others [2,131,132].This suggests that chemical toxicity assessment with nuclear receptor activation would be reasonable in establishing AOP.However, this study showed that the toxicity endpoints for five species were concentrated upon specific biological organization levels.For example, the molecular level study for H. verticillata were concentrated on oxidative stress and photosynthesis disruption.Also, in five species, little information for molecular/cellular/organ level responses involving nuclear receptors activation were available.
The perfect replacement of animal tests with an alternative test is not practicable due to the complexity of a biological system [103].Rather, an alternative test can provide a piece of information, and thus, the collective application of the results from alternative test (e.g., in vitro test) and in vivo test might be practical to assess risk of chemicals.For these reasons, integrated approaches to testing and assessment (IATA) by OECD, accelerating pace of chemical risk assessment (APCRA), EU ToxRisk are developed [103,133].Among them, IATA is a type of method for characterizing chemical hazard and assessing chemical safety by integrating multiple sources of information and producing new data for regulatory decision-making about chemical hazard/risk [134].This suggests that the application of diverse research results using the test species based on alternative test methods as well as conventional methods would increase utility of test species for regulatory purposes.

Traits Specific Issues to Given Species
The specific markers found in a specific test species can influence test species utility.For example, male fish of Z. platypus showed a nuptial organ on their mouth, gills, rear fin.Changes of these traits can be a marker of endocrine disruption, sexual development.Also, the molecular level events for development of the organ would be used as a biomarker showing chemical induced developmental effects.M. anguillicaudatus show tolerance to ammonia exposure by suppression of amino acid metabolism.H. verticillata can mitigate the metal contamination by absorbing it through their root.These properties mean that the molecular level changes to the ammonia, metals exposure could be a sensitive earlywarning signal.S. obliquus have a range of metabolites such as astaxanthin that can be used as a biomarker for oxidative stress and they inhabit in wastewater, so they can be cultivated/tested in municipal wastewater growth medium.S. obliquus form a defense colony when they are exposed to zooplankton grazing cues, while this system is impaired by cadmium exposure and a salinity change.These traits indicate that an integrity of defense colony system of S. obliquus can be a marker suggesting cadmium exposure and salinity increase [94].Additionally, the male G. holbrooki size works as an advantage in mating success with female, paternity, sperm quantity, sperm quality, suggesting that change profiles of male fish size could be used as a reproduction toxicity marker [135].
Through comparison of test species with model species, utility/potential of them can be proved in toxicity research area in which the utility of model species is wellevaluated.P. ramosa showed higher sensitivity to six chemicals, when they are compared to D. magna [136].D. similis involved in the same genus with D. magna showed significantly different response profiles to environmental chemical exposure with D. magna, suggesting their utility relative to D. magna [137].CYP450 1A gene expression of Z. platypus gills was compared to the expression of gills of O. latipes, C. carpio, D. rerio in response to benzo(a)pyrene.Wherein, highest sensitivity was observed in the Z. platypus gills.N. denticulata showed more sensitive responses to pain relief drugs such as acetaminophen and biocide dichlorooctylisothiazolinone exposure than D. japonica.Following glyphosate exposure, H. verticillata showed a significant increase of oxidative stress and pigment contents, while V. natans don't show reverse responses/no responses [138].By triclosan, M. anguillicaudatus showed the most sensitive response in terms of acute mortality compared to insects (C.plumosus), shrimp (N.denticulata sinensis), Daphnia magna, P. parva, Annelids (L.hoffmeisteri), R. Limnocharis [84].M. anguillicaudatus showed higher tolerance to ammonia exposure than P. parva, A. liaoningensis, C. giurinus.S. obliquus showed a higher ammonia removal rate than Chlorella vulgaris.Considering utility of model species in an ecotoxicity study, this type of comparison can be an effective approach to improve test species utility in ERA.
Prior to a comparative study, the definition of the model species is important prerequisite step.By the US-EPA aquatic toxicity methods, in invertebrates, Daphnia magna, Daphnia pulex, Ceriodaphnia dubia, and as a shrimp-like crustaceans, Mysids (Mysidopsis bahia, Holmesimysis costata), Brine shrimp (Artemia salina) were listed in the test guideline.For a fish, Pimephales promelas, O. mykiss, Salvelinus fontinalis, Cyprinodon variegatus, Silversides: inland silverside (Mendia beryllina), Atlantic silverside (Mendia menidia), Tidewater silverside (M.Peninsulae) were listed (US-EPA, 2002).In addition to the species listed above, as a supplemental organism, freshwater vertebrates living in warmwater including Cyprinella leedsi, Lepomis macrochirus, Ictalurus punctatus, freshwater invertebrates living in cold water including Pteronarcys spp., Pacifastacus leniusculus, Baetis spp., Ephemerella spp., freshwater invertebrate living in warmwater including Hyalella spp., Gammarus lacustris, G. fasciatus, G. pseudolimnaeus, Hexagenia limbate, H. bilineata, Chironomus spp.were in test species list of EPA-guideline.The precise information for life span, culture and toxicity test methods of each species is well-noted in each test guideline [7,139] and other brief information such as species identity and whole genome information are summarized in Table 7.

Ecological Relevance
To predict environmental chemical effects upon the ecosystem in a laboratory, conducting an ecotoxicity study under ecological conditions is required.Indeed, numerous studies showed that there was a significant change of chemical effects under ecological conditions such as chemical mixture treatment, multi/trans-generation, coexistence with predator/prey, flow-through exposure and others.For example, PFOS exposure with pentachlorophenol inhibited growth of S. obliquus, while atrazine and diuron increased the growth, suggesting that coexistence of other chemicals can cause chemical toxicity changes [44].By the chemical exposure through multi-generations, different effects such as intersex occurrence relative to one generation chemical exposure condition were caused in an offspring [140,141].Under the co-existent condition with a predator, Z. platypus showed a behavioral change such as swimming speed increase and shorter latency in case of an escape response [99].Similarly, with predators, D. similis showed inducible morphological defenses [142].Under the competition with R. raciborskii, S. obliquus showed increase of oxidative stress [38].With the food (Rseudokirchneriella subcapitata), 48 h-acute toxicity of D. similis was suppressed [143].These findings suggest that co-existence of predator, competitor, prey could induce changes of chemical ecotoxicity.However, a few case toxicity studies were found on ecological conditions using S obliquus, N. denticulata (Tables 1 and 3).
In other cases, the applicability of test species in chemical effect prediction can be evaluated in a field, microcosm, mesocosm condition.In the study using H. verticillata, instead of a field study, the conditions such as microcosm and mesocosm that mimic the ecosystem, were applied to assess chemical toxicity [54].In addition, in the field contaminated with perchlorate, H. verticillata showed diverse decay in leaf, and stem (Table 2).In the study using M. anguillicaudatus, the effects of wastewater under the condition that they were caged in a field were analyzed (Table 5).By contrast, in case of Z. platypus, field and laboratory test are carried out in diverse chemical effect analyses.Indeed, biomarkers related to oxidative stress, genetic toxicity, biotransformation that were assessed for metal exposure in a laboratory condition are proved to be applicable in a field (Table 4) [74,[77][78][79].
Also, to predict chemical effects upon the ecosystem, species habitat type should be considered.Test species ecological traits such as a habitat type (e.g., benthic/pelagic habitat of aquatic animals and submerged habitat/water surface habitat of aquatic plants/algea) can cause different responses to chemicals exposure.Submerged plant and benthic species can be easily exposed to chemicals that are precipitated or absorbed to sediment, whereas floating/waterborne chemicals are more hazardous to pelagic species or floating plants/algea.As such, this review suggests benthic species M. anguillicaudatus and submerged plant H. verticillata with benthopelagic species Z. platypus and floating algea, S. obliquus.However, no comparison studies considering chemical fate and behavior in an environmental medium by physico-chemical properties (e.g., Koc, Kow, solubility) were observed in the collective application of them.

Standadization of Recommended Species
Finally, to develop or enroll these test species in international test guidelines, establishing a standard culture methods in laboratory and standardizing the recommended species should follow.Also, to update the diverse international test guidelines, proposing new test draft guideline, discussing and proofreading of the draft, meeting with international experts relevant to the field of ecotoxicity testing, and interlaboratory cross-checking the draft guidelines should be carried out in the future.

Conclusions
Based on the test species selection criteria in existing literature, S. obliquus, N. denticulata, Z. platypus, M. anguillicaudatus, H. verticillata distributed in East Asia are suggested as promising test species.As an analysis result for them, this study showed that their utility in ERA varied by their physiological/ecological traits and a research state, suggesting that there would be no species fit for all ERA objectives, so multiple species application to ERA is necessary.Especially, even though five species exhibit the overlapped geological distribution, they have differences in habitat, ecological traits (e.g., trophic level, habitat type), physiological traits (e.g., life cycle, sensitivity to chemicals), a state of an ecotoxicity study.Thus, five species can be a battery in chemical effect assessment under diverse exposure scenarios.
By analyzing recent ecotoxicity test trends, this review shows that alternative approaches are important emerging issues in a study for increase of test species utility in ERA.In detail, test species application to QSAR development, cellular system development, conducting toxicity test using embryos are suggested as further study issues.This work shows that using omics technology, mining housekeeping genes and biomarker genes relevant with xenobiotic biotransformation, endocrine disruption, and neurotoxicity would be future study areas.Also, for the development of AOP, chemical effect analysis in various biological organization levels is suggested as another further study issue for utility increase of test species.
As a non-alternative approach, a comparison study to find interspecies difference (e.g., sensitivity), development of markers specific to a test organism, ecotoxicity test under ecological conditions, and others are suggested as the remaining issues.Accordingly, an integrative study of data from diverse alternative approaches as well as conventional ecotoxicity test would assist in test species utility increase.
The utility information of five species and further study issues discussed in this review would be helpful not only in the application of the five species to ERA, but also in mining a promising test species and increasing their utility in ERA, resulting in effective performance of ERA for regulatory purposes.

Toxics 2024, 12 , 2 of 31 Figure 1 .
Figure 1.Conceptual diagram of chemical effect assessment and ecotoxicity test species role in the assessment.

Figure 1 .
Figure 1.Conceptual diagram of chemical effect assessment and ecotoxicity test species role in the assessment.

Figure 2 .
Figure 2. The schematic framework for the ecotoxicity test species selection and utility analysis.

Figure 2 .
Figure 2. The schematic framework for the ecotoxicity test species selection and utility analysis.

Table 1 .
Test condition and endpoints for ecotoxicity test using Scenedesmus obliquus.

Table 2 .
Test condition and endpoints for ecotoxicity test using Hydrilla verticillata.

Table 3 .
Test condition and endpoints for ecotoxicity test using Neocaridina denticulata ssp.

Table 4 .
Test condition and endpoints for ecotoxicity test using Zacco platypus.

Table 5 .
Test condition and endpoints for ecotoxicity test using Misgurnus anguilicaudatus.

Table 6 .
Summary of selected promising ecotoxicity test species.

Table 7 .
Information for several model ecotoxicity test species listed in international test guideline.