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Harnessing Ascidians as Model Organisms for Environmental Risk Assessment

Amalia Rosner

^* and

Baruch Rinkevich

National Institute of Oceanography, Israel Oceanography and Limnological Research, Tel-Shikmona, P.O. Box 9753, Haifa 3109701, Israel

Author to whom correspondence should be addressed.

Environments 2024, 11(11), 232; https://doi.org/10.3390/environments11110232

Submission received: 26 August 2024 / Revised: 17 October 2024 / Accepted: 19 October 2024 / Published: 23 October 2024

(This article belongs to the Special Issue Environmental Risk Assessment of Aquatic Ecosystem)

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Figure 1
Phylogenetic tree based on 18S ribosomal RNA (18S rRNA) gene fragments of ascidian species reviewed in this study. A few species are not represented due to the absence of their 18S gene sequences in the database. The taxonomic order of the species is depicted, illustrating the clustering of species within the same order. The analysis was conducted using the EMBL-EBI T-Coffee program [<a href="#B33-environments-11-00232" class="html-bibr">33</a>]. The NCBI GenBank accession numbers for the 18S rRNA gene sequences used are as follows: Ascidiella scabra (AB811928.1), Botrylloides leachi (JN573237.1), Botrylloides_violaceus (AY903927.1), Botryllus_schlosseri (FM244858.1), Ciona intestinalis (AB013017.1), Ciona savignyi (LC547329.1), Didemnum molle (AB211071.1), Didemnum vexillum (JF738071.1), Halocynthia roretzi (AB013016.1), Herdmania momus (AF165827.1), Microcosmus exasperates (XR005567858.1), Molgula manhattensis (L12426.2), Phallusia nigra (FM244845.1), Polycarpa mytiligera (FM244860.1), Styela clava (XR_005567858.1), Styela_plicata (L12444.2). "> Figure 2
Modes of life cycles in two ascidian model species: (a) a solitary ascidian (Ciona spp.) revealing classical sexual reproduction progressions of a broadcasting species; (b) asexual (outer cycle in the diagram) and sexual phases in a colonial ascidian (Botryllus schlosseri) highlighted by weekly astogenic rounds of zooidal life and death (each cycle is called blastogenesis). Each blastogenic cycle is divided into four stages (A–D) where three generations of colonial modules (the functional zooids and two cohorts of developing buds, primary and secondary buds) coexist side by side, depicting highly synchronized developmental statuses as the colony progresses through blastogenesis. At stage D, the functional zooids start their degeneration, first by closing the siphons, where simultaneously all zooids undergo cell apoptosis and phagocytosis processes within the next 24–36 h and are morphologically absorbed, while the primary buds mature to the zooidal level of development. Subsequently, stage A of the following blastogenic cycle begins as primary buds complete their development into zooids by opening the inhalant siphons and resuming water filtration, while secondary buds (budlets) develop to the primary bud’s state, starting the generation of new sets of secondary buds. Gametogenesis is highly synchronized with the blastogenic cycle among modules of the same generation. The sexual cycle is typified as brooding, where gametes start differentiation within the budlets. Gametes maturation and egg fertilization occur within the zooids (at the onset of stage A). Embryos differentiation is synchronized with the blastogenic stage and continues within the zooids (inner cycle; showing for each blastogenic stage a single large zooid with its bud/s and budlet/s), culminating in the release of the larvae into the surrounding waters at late blastogenic stage C. The larvae swim for a very short period until they settle, undergo metamorphosis starting with the absorption of the tail, and new juveniles (oozooids) are formed, and a colony is formed by repeated blastogenic cycles. "> Figure 3
Pie charts depicting taxa percentages for search hits in the ‘Web of Science’ and ‘Google Scholar’ databases, filtered by the terms ‘environmental risk assessments’ and ‘marine’ environment, as compared to the total hits for ‘environmental risk assessment’ (marine and terrestrial). Each taxon (a group of different bioindicators) is represented by a specific-colored descriptor detailed in the caption. The black sections encompass the residual hits for taxa with the smaller number of hits. The magnified pie sections on the right provide a detailed breakdown for some of these taxa. <a href="#app1-environments-11-00232" class="html-app">Supplementary Table S1</a> details the specific queries used to obtain the data for each taxon. "> Figure 4
Commonly used model ascidians: the solitary ascidians (a) Ciona robusta and (b) Ciona intestinalis (by John Bishop from the Marine Biological Association of the United Kingdom), once considered as a single species; (c,d) different color morphs of the colonial ascidian Botryllus schlosseri. (c) A colony reared in the laboratory at the Israel Oceanography and Limnological Research, Haifa, and maintained at a constant temperature of 20 °C with a regimen of 12:12 light:dark hours. This colony is a descendant of the Monterey, California, population; (d) a colony from New Zealand reared on a glass slide. "> Figure 5
Pictures of additional solitary (a–c) and colonial ascidian (d–f) used in toxicity, environmental pollution monitoring tests, and biological invasions. (a) Phallusia spp.; (b) Polycarpa spp.; (c) Halocynthia spp.; (d) Botrylloides spp.; (e) Didemnum spp.; (f) Didemnum vexillum. "> Figure 6
Pie charts depicting taxa percentages for search hits in ‘Google Scholar’ databases, filtered by the terms: (a) ‘environmental risk assessment’ and ‘marine’ environment, and ‘invasion’ (61.5% of hits) as compared to the total hits for ‘environmental risk assessments’ and ‘invasion’ (100%). The total number of hits involving individual taxa exceeds the number of hits for ‘environmental risk assessment’, ‘marine’ environment, and ‘invasion’, as many publications examine multiple taxa; (b) ‘environmental risk assessment’ and ‘marine’ environment, and ‘biodiversity’ (62.6%) as compared to the total hits for ‘environmental risk assessments’ and ‘biodiversity’ (100%). The total number of hits involving individual taxa exceeds the number of hits for ‘environmental risk assessment’ and ‘marine’ environment and ‘biodiversity’, as many publications examine multiple taxa. Each taxon is represented by a specific-colored descriptor detailed in the caption. The magnified pie sections on the right are for categories with smaller numbers of hits. <a href="#app1-environments-11-00232" class="html-app">Supplementary Table S2</a> details the specific queries used to obtain the data for each taxon. "> Figure 7
A graphical representation illustrating the potential applications of ascidian-based bioassays within the framework of ERA. The bioassays utilize both solitary and colonial ascidians, which may offer unique advantages for studying different environmental impacts. MCR—multiple clonal ramets; Blue arrows indicate bioassays that have already been successfully employed in toxicity testing; Red arrows represent potential applications that have yet to be widely explored. ">

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Abstract

Environmental Risk Assessment (ERA) often relies on a restricted set of species as bio-indicators, introducing uncertainty when modeling complex environmental variables. This may lead to oversimplified or erroneous risk assessments. Ascidians, marine filter-feeding sessile chordates, are valuable models for scientific research in various biological fields such as stem cell biology, embryogenesis, regeneration, innate immunity, and developmental biology. Their global distribution, sensitivity to pollutants, high abundance, mass sexual reproduction, and habitation in coastal areas impacted by anthropogenic pollution make them excellent indicators for monitoring marine pollution and global environmental changes, including biological invasions and species diversity diminution cases. Despite their potential as environmental bioindicators, ascidians remain underutilized in ERAs (≤0.13% of ERA studies), particularly in the field of chemical pollution impact assessment, primarily due to a lack of standardization. This underrepresentation poses a challenge for accurate modeling, especially in models relying on a broad range of species (e.g., Species Sensitivity Distributions). Given these constraints, expanding the use of ascidians in ERAs could improve the comprehension and precision of environmental changes and their assessments. This underscores the necessity for future research to establish standardized testing protocols and choose the most suitable ascidian species for inclusion in ERAs.

Keywords:

ascidians; biodiversity; Botryllus schlosseri; Ciona spp.; Didemnum vexillum; ERA; invasive species; non-model organisms; pollution; toxicity

1. Introduction

The traditional understanding of environmental risk assessment (ERA) is generally associated with methodologies recommended by regulatory bodies to gauge the likelihood of specific adverse effects on populations and communities of organisms within an ecosystem. Grounded in science-based methodologies, ERA employs a well-defined, hypothesis-based approach to assess environmental risks. Unlike basic research, ERA is distinguished by its structured process, designed to address precise inquiries regarding the risk posed by a particular threat [1]. Further, the ERA’s structural process is clearly divided into three major phases: problem formulation, analysis, and risk characterization [2], and the connection of ecological components of significance, such as species or habitats, with the likely impacts of various pressures. Subsequently, this framework plays a crucial role in pinpointing indicators, measuring reference conditions, and assessing management options [3]. There is also increasing recognition that integrating science, policy, and management through ERA schemes is an effective method for addressing a diverse range of ecological and environmental challenges [4].

Unlike terrestrial ecosystems, ERA protocols for addressing various detrimental impacts on the marine environment (including the formulation of regulations) have yet to be developed. This encompasses a wide range of environmental concerns (including the transport, distribution levels, and biological fates) from compounds released into the environment, such as pharmaceuticals and drugs [5,6], genetically modified crops [1], herbicides and pesticides and other agrochemicals [7,8,9] and more. Further, the portrayal of multiple drivers and the intricate interplay among various detrimental activities and their pressures are still inadequately addressed, and some pressures might go unnoticed, despite their significant impacts on the ecosystem [3,10,11]. When devising new sets of ERAs and/or refining existing ERA practices, several key issues must be considered, with the first challenge revolving around “the choice of representative test species” [12].

Ascidians (phylum: Chordata, Class: Ascidiacea) are cosmopolitan soft-body marine filter-feeding invertebrate species that thrive in marine environments with salinities exceeding 2.5%. Most ascidian species are ecologically important benthic organisms found in shallow water environments, and they are particularly prevalent in regions affected by pollution, including ports, harbors, and industrial zones [13]. A few species, like Dicopia antirrhinum [14], live in the deep sea, down to about 4500 m depth, where they become macrophagous. Numerous ascidian species are classified as invasive species [15], a characteristic often attributed to their fouling ability [16].

There are about 3000 accepted extant species of ascidians [17,18] with over 1600 species being solitary, and the rest are compound or colonial organisms [19]. Both solitary and colonial ascidians are distributed worldwide across various marine environments, ranging from temperate to tropical regions and from shallow coastal zones to deep-sea habitats. Solitary ascidians generally tend to inhabit deeper, more stable habitats like rocky areas, coral reefs, and submerged man-made structures and are commonly found along the coasts of Asia, Europe, North America, and the Indo-Pacific region. In contrast, colonial ascidians thrive in more dynamic and shallower environments, such as coastal areas, estuaries, shallow reefs, and rocky shorelines, with significant population sizes in the Atlantic, Caribbean, Mediterranean, and throughout the Indo-Pacific. This broad distribution allows both solitary and colonial ascidians to play crucial roles in marine ecosystems, though they can also become invasive species when introduced to new areas, primarily through human activities like shipping and aquaculture [19].

All ascidians are distinguished by an outer protective layer, the tunic, composed primarily of polysaccharides (tunicin) and containing various types of proteins, specialized cells, and symbionts [20]. Based on its composition, the tunic can be classified as gelatinous (soft, e.g., Molgula manhattensis) or leathery (tough, e.g., Styela clava), a characteristic that may influence ascidian habitat preference, predation risk, and the efficiency of food, gas, and pollutant absorption. The classification of ascidians as chordates stems from their short-lived (12–24 h [21]) swimming larval stage, which possesses several key characteristics of chordates, including a notochord, a dorsal tubular nerve cord, and pharyngeal gill slits [22]. These chordate features disappear when the larvae settle onto a substrate, absorb their tails, and metamorphose into juveniles. Any sessile mature ascidian represents a sac-shaped body plan with two siphons: an oral siphon responsible for drawing water into the body and an atrial siphon through which the filtered water is spelled out. On the gross morphology level, ascidians can be either colonial/compound, consisting of a few to many tightly or loosely genetically identical units (each called a zooid) or solitary, ranging from 0.5 to 20 cm in length [23], although the largest species, such as Pyura pachydermatina, can grow up to 100 cm long [24]. Ascidians are hermaphrodites, of which the solitary species are broadcast spawners that reproduce through external fertilization, while the colonial species are typically ovoviviparous [23].

The body of an adult ascidian, or each zooid in colonial ascidians, contains several distinctive organs, including (besides the tunic and the reproductive system) a branchial sac that occupies the majority of the body’s trunk, a digestive system, including the pharynx, stomach, and gut, a heart, and an open circulatory system with hemolymph containing various types of blood cells. The branchial sac governs the filtration and bioaccumulation abilities of ascidians. Its walls are perforated by numerous stigmata and covered with ciliated epithelium. The ciliary action generates water flow from the oral siphon along the branchial sac, resulting in rapid filtering of the seawater. This filtration is essential for respiration, trapping particulate matter, and transporting it to the esophagus. On the ventral side of the branchial sac is located the endostyle, a ciliated groove that secretes mucus important for particulate trapping and which is believed to be homologous with the thyroid gland of vertebrates, though differing in function [25]. The branchial sac also contains a variety of hemocytes, including granular amoebocytes, that express immune-related genes like TNF-α and the complement component C3 [26] and stem cells [27]. The branchial sac structure is also a taxonomic characteristic that distinguishes the three orders within the class Ascidiacea: aplousobranchia with a simple branchial sac; phlebobranchia with longitudinal blood vessels; and stolidobranchia with a folded branchial sac (Figure 1 [19,28]).

Ascidians also possess a nervous system composed of peripheral nerve cells, a central nerve tube (consisting of the sensory vesicle, the neck, the visceral or tail ganglion, and the caudal nerve cord), a large ganglion located at the connective tissue between the two siphons, and an exocrine gland. Other features include muscle bands and an enclosed epidermal layer [29]. Zooids in colonial ascidians are further interconnected by blood vessels, ensuring synchronization between the modules within a colony. Some groups exhibit intricate astogeny (creation of colonial forms) and remarkable regenerative abilities, capable of regenerating from even minute bodily fragments [30,31,32], enabling the animal’s survival after major damage. A schematic representation of the general morphology and internal anatomy of representative ascidians is presented in Vanni et al. [32].

Figure 1. Phylogenetic tree based on 18S ribosomal RNA (18S rRNA) gene fragments of ascidian species reviewed in this study. A few species are not represented due to the absence of their 18S gene sequences in the database. The taxonomic order of the species is depicted, illustrating the clustering of species within the same order. The analysis was conducted using the EMBL-EBI T-Coffee program [33]. The NCBI GenBank accession numbers for the 18S rRNA gene sequences used are as follows: Ascidiella scabra (AB811928.1), Botrylloides leachi (JN573237.1), Botrylloides_violaceus (AY903927.1), Botryllus_schlosseri (FM244858.1), Ciona intestinalis (AB013017.1), Ciona savignyi (LC547329.1), Didemnum molle (AB211071.1), Didemnum vexillum (JF738071.1), Halocynthia roretzi (AB013016.1), Herdmania momus (AF165827.1), Microcosmus exasperates (XR005567858.1), Molgula manhattensis (L12426.2), Phallusia nigra (FM244845.1), Polycarpa mytiligera (FM244860.1), Styela clava (XR_005567858.1), Styela_plicata (L12444.2).

Each solitary ascidian, such as the model species Ciona intestinalis, follows a typical developmental sequence of a multicellular organism, including egg fertilization and embryonic and larval development, leading to a sessile juvenile that progresses into the adult stage (Figure 2, [34]). In colonial ascidians, once the founder zooids are established, they undergo astogeny through various asexual reproduction processes and phenomena [35,36]. One such phenomenon, typified to the subfamily Botryllinae (family Styelidae; Figure 2), or botryllid ascidians, is blastogenesis (Figure 2). This phenomenon involves weekly recurrent cycles of death and life (under a 20 °C regimen) that continue throughout the animal’s lifespan. During these blastogenesis cycles, the number of modules in the colony increases, with species like Botryllus schlosseri and Botrylloides leachi potentially growing to several hundreds of zooids. Using other asexual growth processes, ascidian colonies can reach major sizes. For example, colonies of the genus Didemnum can expand to cover several square meters of substrate ([37,38], and references therein). While discussing ascidians as a group, this overview focuses on two model species, the solitary ascidian C. intestinalis (Figure 2) and the colonial form B. schlosseri (Figure 2).

Ascidians are key organisms in numerous marine ecosystems, commonly found in coastal waters, coral reefs, and intertidal zones, where they attach to rocks, coral reef substrates, and other hard surfaces. While they predominantly inhabit shallow environments, some ascidian species have adapted to deep-sea conditions. As filter feeders [39,40], ascidians play a crucial role in maintaining water quality by removing plankton, bacteria, and organic particles, contributing to nutrient cycling [41]. Their filtration trait has even been proposed for cleaning particle pollutants from ambient water, such as microbes [41,42], heavy metals [43], and microplastics [44]. As part of the epifauna, ascidians form biogenic habitats due to the dense colonies they establish on various surfaces, providing shelter and protection for marine life, including small fish, crustaceans, and other invertebrates [45]. Certain taxa, like the ascidicolous copepods, are exclusive to these ecosystems [46]. Unfortunately, these biogenic habitats often become contaminated by metals, hydrocarbons (PAHs), and chlorinated compounds that accumulate there [45]. Ascidians also serve as a vital food source for a wide range of predators, including fish, sea stars, and certain crabs. Furthermore, some ascidian species like Halocynthia roretzi (Sea Pineapple) and Styela clava are consumed by humans [47]. Recent research has highlighted ascidians as potential sources of valuable sources of nutrients and pharmaceutical compounds, showing antioxidant, anti-inflammatory, antidiabetic, antimicrobial, and anticancer properties [48].

Regardless of the ERA protocol applied, all ERA are linked with risk identification or assessment and require specific tests (such as acute, chronic, and biodiversity assays). These tests often rely on animal models, commonly referred to as bioindicators. Bioindicators are organisms, or groups of organisms, used to assess the quality of the environment [49].

As a group, ascidian species exhibit a wide range of traits that make them ideal bioindicators, including: (1) a widely documented global distribution [50,51,52]; (2) standardized protocols for ex-situ mariculture of mature individuals and their reproductive products [53,54,55]; (3) sequenced genomic databases for selected species [56,57]; (4) established advanced tools such as gene editing [58]; (5) solitary ascidian species release large quantities of eggs and sperm simultaneously into the water column, facilitating the rapid and synchronous development of a substantial number of embryos in vitro. This reproductive trait, combined with their relatively simple larvae, aligns well with the embryonal-larval tests recommended for ERA-standardized protocols for other species; (6) the ascidians unique life cycle featuring both sessile and planktonic (free-floating) stages, providing them with exposure to a broad array of environmental contaminants; (7) being filter feeders, ascidians exhibit heightened sensitivity to pollutants present in the water column, resulting in the accumulation of these substances within their bodies through bioaccumulation processes; (8) the documented relative tolerance of ascidians to abiotic factors, such as variations in salinity and temperature, makes them suitable for studying pollutant impacts under different environmental conditions, as well as adaptive responses to fluctuating conditions; (9) the sampling ease of abundant sessile species, advantageous for studies requiring a large number of specimens; (10) their high regenerative capacity contributes to their suitability for various toxicological ERAs [59]; (11) as their phylogeny relationship as the closest relatives to the vertebrate subphylum [60], with key vertebrate-like features, complex organs, and many cell types, ascidians provide a valuable model for studying the effects of pollutants on humans and vertebrates in general, while also addressing ethical concerns and research restrictions typically associated with vertebrate models; (12) the scientific literature recognizes ascidians as sensitive to various pollutants, recommending them as bioindicators [59,61,62]; (13) invasive ascidians may provide valuable insights into the mechanisms of biological invasion. Collectively, the above-listed traits underscore the considerable potential of ascidians as bioindicators and their importance as research subjects in both environmental and biological studies.

2. Risk Assessments of Chemical Pollution

Despite the numerous advantages of using ascidians as models in ERA for marine environments, including their potential for regulatory compliance scenarios, and their successful implementation when properly implemented (as will be discussed below), their current utilization in ERA remains constrained.

2.1. Marine Chemical Hazard Assessment: Regulations, Monitoring, and Challenges

The Water Framework Directive (WFD), the Marine Strategy Framework Directive (MSFD), and the Registration, Evaluation, Authorization, and Restriction of Chemicals policy (REACH) collectively mandate an expedited approach to hazard assessment for both new and existing chemicals [63]. This necessitates the development of rapid, sensitive, and economically efficient tests, imperative within the framework of existing international regulations. Concurrently, the “3Rs” principles, which advocate for the replacement, refinement, and reduction of animals used in tests, strongly encourage the adoption of in vitro and in silico model-based tests, and whenever possible, prioritize the use of invertebrates over vertebrates [64,65]. In response to these guidelines, international agencies such as the OECD, USEPA, NOAA, and ISO have standardized a few marine invertebrates as bioindicators in numerous tests [66]. These organizations, which have provided the framework for various ERA protocols [67,68,69,70,71,72,73,74,75] often recommend tests that focus on limited sets of invertebrates. This focus may potentially neglect the critical ecosystem roles and the suitability of a bioindicator for specific ecosystems [66,76], potentially leading to the overrepresentation of a few taxa in marine ERA tests (Figure 3).

2.2. Ascidians in Toxicological ERAs

ERA typically follows tiered protocols, where testing progresses from simple to complex and to site-specific assessments at each tier [77]. A common method employed in these assessments is the hazard quotient (HQ) approach that compares each environmental concentration of a contaminant to its effect, like the median lethal concentration (LC50) or predicted no-effect concentrations (PNEC). These HQ values are based on single exposure and effect levels and offer preliminary indications of potential risks or relative safety. Yet, marine ecosystems pose challenges for the ERA validity (short-term and long-term effects) as many pollutant types are unidentified or interact synergistically, in addition to the need for consideration of abiotic factors that may influence the outcomes [78,79,80]. To address these challenges, there is a growing need to develop a broader range of tests employing diverse, sensitive animal models and to incorporate statistical and modeling systems into predictive processes [76,78,80,81].

To examine the extent to which ascidians are used in ERA that focus on marine environments, we conducted a search for the term ‘environmental risk assessment’ in the databases ‘Google Scholar’ and ‘Web of Science’ which yielded 236,000 and 5993 hits, respectively (Figure 3; Supplementary Material: Table S1). The results revealed that hits related to the marine environment made up only a small fraction of the total results (27.8% and 17% of the databases, respectively), despite the significant size of the marine ecosystems on earth. This discrepancy could be linked to the historical background of ERA combined with challenges such as the high costs associated with monitoring of the marine environment. Ascidians, commonly found in heavily polluted coastal areas such as ports, are notably underrepresented in ERA studies compared to other marine organisms. In the ‘Google Scholar search’, fish account for 15.08% of all ERA hits, mollusks and corals represent 2.43% and 1.94%, respectively, while ascidians make up only 0.13% of the hits (Figure 3). Similar trends were observed in the ‘Web of Science’ search. About one-third of the ascidians’ hits in Google Scholar deal with ERAs that are associated with biological invasion, while the rest relate to pollution, toxicology, and biodiversity. Yet, among the latter, most papers do not directly deal with either ERA analysis (see part 2.4).

2.3. Exploring Pollution Beyond Standard Models: Non-Standardized Tests with Ascidians

Although ascidians are not yet standardized in officially established tests, numerous scholars [82,83,84,85,86] have proposed them as highly effective bioindicators for toxicity assessments and even incorporated them into various toxicity tests and bioassays applicable to different ERAs. However, the number of tests conducted on ascidians is significantly lower compared to other taxa (Figure 3), leading to a lack of comprehensive toxicological metrics to compare their sensitivity to a broad range of pollutants. Despite this, the available data demonstrate that the sensitivity of ascidians is comparable to that of standardized species. For instance, C. intestinalis exhibits an EC50 of 0.721 mg/L for Cd²⁺, indicating higher sensitivity compared to Mytilus galloprovincialis (EC50 = 1.925 mg/L) and Paracentrotus lividus (EC50 = 0.230–9.240 mg/L). For Cu²⁺, C. intestinalis shows an EC50 of 0.036–0.054 mg/L, compared to P. lividus at 0.045–0.068 mg/L. Similarly, for Hg²⁺, C. intestinalis has an EC50 of 0.044 mg/L, compared to P. lividus at 0.08–0.04 mg/L [87]. Further comparisons of the sensitivity of Ciona spp. embryos to various inorganic and organic compounds with the sensitivity of Paracentrotus spp. and Mytilus spp. revealed similar sensitivity levels [64]. In other cases, Ciona spp. embryos were found to be even more sensitive than Dicentrarchus labrax (fish) juveniles when exposed to certain dispersants [84]. In this context, it is important to emphasize that sensitivity to pollutants can be species-specific. Embryos of closely related organisms, such as Ciona robusta and C. intestinalis (Figure 4a,b), may exhibit similar sensitivity to certain contaminants, such as Bisphenol A, or display markedly different sensitivities to others, such as tris(1-chloro-2-propyl) phosphate, depending on the specific pollutant being tested [88]. This phenomenon is not unique to ascidians but is common across species [89].

Two ascidian species (the solitary Ciona spp. and the colonial B. schlosseri) account for 82.7% of the ascidian-related ERA hits according to ‘Google Scholar’ (66% with Ciona spp. and 16.7% with B. schlosseri, respectively). C. intestinalis (Table 1; [82]), previously considered as a single taxon, is now recognized as a clade of four distinct species, collectively referred to as Ciona spp. ([90]).

Embryo and larval tests [86] are vital components of ERA, specifically designed to evaluate the potential impacts of various substances or environmental stressors on the early life stages of aquatic organisms. In particular, Ciona spp. are ideal candidates for such studies due to the high number of eggs produced, which can be collected from mature adults in a laboratory setting, combined with fast gametogenesis and embryogenesis. The above features make the various early life stages of Ciona (Figure 2) excellent tools for developing fast, high-throughput tests. The tests, including gonadotoxicity, fertilization toxicity, larval hatching rates, larval swimming behaviors, metamorphosis, larval settlement, morphological abnormalities, and juvenile survival, can be performed by just adding the tested chemical to their growing medium and may serve as initial screening tools for chemicals, aligned with current legislation and first-tier ERA tests [63]. Although not yet part of established ERA protocols, gametes-embryo-larval tests using Ciona spp. have been successfully used to measure the impacts of pollutants like heavy metals [91,92,93,94], pharmaceutical drugs and their metabolites [95], dispersants [84,85], and endocrine disruptors [96] like bisphenol A (BPA)-contaminated microplastics [97,98,99], polycyclic aromatic hydrocarbons (PAHs) [100], BPA along with tributyltin [101], and nanoplastics [102,103]. On the other hand, the ovoviviparous colonial ascidian B. schlosseri is less suitable for embryo tests due to its internal fertilization and the lower numbers of released embryos (Figure 2). Yet, toxicological assessments using B. schlosseri larvae have proven effective in evaluating the effects of antifouling paints and biocides (Table 1, [104]), in addition to the use of assays on adult organisms.

Further, while most ascidian larvae do not feed [105], they may absorb pollutants through their tunic. Adult ascidians, both solitary and colonial forms, not only absorb soluble material through their tunic but also actively filter masses of seawater daily through their branchial sac, resulting in augmented bio-accumulating of pollutants [39,106,107], that include, among others, heavy metals [108], microplastics, plasticizers [109], endocrine-disrupting materials, and pharmaceutical products. Bioindicator assays developed on such adults include differential gene expression tests [66,110], biotransformation assessments [111], oxidative stress [112,113], hemocyte functions [114], comet assays [115], and microbiome analyses [116,117] and others.

Adult colonial ascidians offer distinct experimental advantages due to their ability to produce multiple clonal ramets (MCRs) from a single genotype, a feature shared only with some basal taxa. Ramets are individual units produced asexually by detaching from a single parent organism (the genet) in clonal species. In appropriate species, multiple ramets can be experimentally induced by cutting the parent organism into sections and placing each section on a separate substrate. Each ramet then continues to live and propagate independently. The MCR approach enables the creation of genetically and epigenetically identical testable replicates, allowing for the simultaneous evaluation of multiple chemicals/conditions [113] or the examination of a single substance under diverse environmental conditions [118]. This approach can provide valuable insights into less understood phenomena such as hormesis [119], tolerance [120,121], and chemical sensitization [122]. Additionally, colonial organisms, which reproduce asexually through budding and display communal behaviors, offer unique insights to study collective behaviors, coordination, and communications among individual units; those features can also serve as endpoints in chemical testing. The ability of adult colonial ascidians to regenerate entire bodies from minute blood vessel fragments [31,123,124] or following torpor [118], presents another valuable aspect for pollution assessment endpoints.

Table 1. Common toxicity tests based on the model ascidians Ciona spp. and Botryllus schlosseri used as bioindicators.

Test	Ciona spp.	Botryllus schlosseri
Sperm toxicity	[82,94,125]	N/A
Oocyte toxicity	[82,94,125]	N/A
Fertilization	[82,125]	N/A
Embryotoxicity	[59,126,127,128]	N/A
Embryo development impairment	[91]	N/A
Larval hatching	[91,92,102,126]	N/A
Alterations in larvae behavior (e.g., spontaneous swimming, shadow response)	[99,102]	N/A
Larval settlement	[91,92,126]	[104]
Larva development	[95,97,98,102]	[104]
Larvae mortality	N/A	[104]
Juvenile	[84,85,100]	N/A
Transcriptome profiling	[103]	N/A
Mature colony
Reproductive physiology	[94]	N/A
Bioaccumulation	[96,129]	N/A
Oxidative stress tests	[112]	[113]
Heat shock protein expression	N/A	[130]
Detoxification enzyme expressions	[131]	[132]
Transcriptome profiling	[133]	N/A
Functional responses of hemocytes: Viability, morphology, lysosomal membrane stability, phagocytic activity, apoptosis, enzyme activities	N/A	[114,132,134,135]
Comet assay (genotoxicity test)	N/A	[115]
Microbiome composition	[116,117]	[116,117]
Colony growth	N/A	[136]
Colony recovery	N/A	[136]
Pollution-induced differential gene expression	[110]	[66,130]
Biotransformation	[111]	N/A
Immunotoxicity	[137]	[132,138,139,140,141,142,143]
Phenotypic changes	N/A	[144]
Mortality (survival; LC50)	N/A	[144]

N/A—not available.

In addition to Ciona spp. and B. schlosseri (Figure 4a–d), a wide range of other solitary and colonial species were also used in toxicity and environmental pollution testing (Figure 5a–e). However, these species were rarely incorporated in ERA. These include species of the solitary genus Phallusia (Figure 5a), which were utilized to evaluate the teratogenic effects of Bisphenol A (BPA) on larvae, evidenced by the disruption of pigmented organ differentiation [145,146,147]. LC50, sperm viability, and effects on fertilization, as well as analyses of teratogenic impacts conducted using light microscopy and immunohistochemistry with anti-tubulin antibodies, were used for assessing pesticide impacts on embryos and larvae [148]. Additionally, specialized software called Toxicosis was developed for high-content image analysis, illustrating morphological endpoints following exposure to various classes of toxicants [147].

Heavy metal bioaccumulation in Phallusia nigra and Microcosmus exasperatus showed species-dependent preferences for the tissues (tunic or body) where heavy metals tend to accumulate, as well as a pollution-dependent Hepato-Somatic Index, which was lower in polluted regions [149,150]. Similarly, bioaccumulation of heavy metals by the ascidian Styela plicata has been documented [151], leading to reduced cell proliferation, decreased viability, and diminished cytotoxic activity of hemocytes [152,153]. Hemocyte morphological alterations and cell death were also observed in ‘in vitro’ systems [154]. Moreover, acute exposure of animals to diesel oil resulted in behavior changes, inflammatory responses, oxidative stress, and activation of the apoptotic pathway [155]. Additionally, the quantification of ten polycyclic aromatic sulfur heterocycles accumulated in Phallusia nigra (Figure 5a) was performed using ‘green’ extraction methods followed by gas chromatography and mass spectrometry. This analysis showed a higher concentration of these compounds in the branchial basket compared to the tunic [156].

Differential protein expression in Herdmania momus was detected following the accumulation of the pharmaceutical compound Carbamazepine administered to the animal under laboratory conditions [157]. Similarly, differential protein expressions were detected in M. exasperates collected from five sites along the Israeli Mediterranean coast and Polycarpa mytiligera (Figure 5b) collected from four sites along the Red Sea coast in response to various stressors present at those sites [158]. Although proteins from similar families were detected in both tested species (e.g., stress-related and synthesis proteins), the results revealed a significant difference in the number of deregulated proteins in the two species analyzed (193 and 13, respectively), which might reflect species-specific differences or varying levels of pollution within the tested sites. These results underscore the challenge of identifying reliable biomarkers for detecting environmental stressors and indicate that further calibration is needed before such biomarkers can be recommended.

Further research supports the usefulness of ascidians as models in toxicology tests. Various concentrations of heavy metals, including chromium (Cr), copper (Cu), and zinc (Zn), have been found to affect oxygen consumption and filtration rates of the solitary ascidian Halocynthia spp. (Figure 5c [159]). Additionally, Halocynthia roretzi has been shown to accumulate microplastics [160]. Similarly, the colonial ascidian Didemnum molle (Figure 5e) has been proposed as a potential biomarker for microplastics accumulation [161]. Other studies have investigated the effects of pollutant mixtures in defined regions, like ports or marinas, on species quantity and variety. These, along with additional research, have shown reduced species diversity, the emergence of resistant groups, and the widespread proliferation of invasive species, many of which are ascidians [162]. Similarly, Johnston et al. [122] investigated the impacts of repeated copper exposure on sessile marine invertebrate assemblages at two sites in Port Phillip Bay, Victoria, Australia. The results revealed significantly altered species assemblages at both sites, primarily due to direct negative effects on the densities of large space-dominating ascidian populations.

2.4. ERAs Performed with Ascidians

While providing valuable insights, some of the aforementioned protocols diverge from standardized toxicity tests routinely used in ERAs. This lack of standardization poses a major challenge, as it limits the comparability of results across regulatory bodies. Nonetheless, some ERAs integrate data from both standardized and non-standardized tests, including the use of ascidians as bioindicators. For example, Martins et al. [163] conducted an ERA on 14 organic and organo-metallic antifouling agents, using data collected from acute and chronic literature tests on 57 and 33 marine (including the solitary ascidians C. intestinalis and S. clava) and freshwater species, respectively, and employing the probabilistic Species Sensitivity Distribution (SSD) method to estimate PNECs. Data for S. clava was available only for an acute test with a single material, while for C. intestinalis, acute tests existed for six materials and chronic tests for two materials. For example, Martins et al. [163] revealed that in an acute toxicity test for cybutryne (Irgarol-1051) on C. intestinalis embryos (48 h), the EC50 value of 2115 µg/L falls within the range observed in other marine invertebrates. Certain well-studied and standardized model organisms were even less sensitive to Irgarol-1051 than C. intestinalis. For example, Crassostrea virginica, which is used in standardized chemical testing (e.g., EPA OPPTS 850.1025 [164]; EPA OCSPP 850.1710 [165]; EPA OCSPP 850.1055 [166]; ASTM E724-98 [167]; ASTM E1022-94 [168]), reveals a 48 h EC50 value of 3200 μg/L for Irgarol-1051 exposure. Similarly, the polychaete Hydroides elegans (standardized in ASTM E1562-00 [169]) shows a 48 h LC50 value of 2600 μg/L, and the copepod Nitocra spinipes (standardized in ISO/DIS 14669 [170]) has a 96 h LC50 value of 4500 μg/L. Above ERA examples on the toxicity of Irgarol-1051 further highlight the necessity of conducting SSD tests across diverse ecological groups, encompassing both model and non-model organisms, such as ascidians. An ERA performed on the impacts of the antifouling agent Chlorothalonil on fertilization and offspring of the invasive species Ciona spp. [171] has revealed a potential risk of this antifouling material to aquatic species. Another ERA study [172] assessed the risk for linear alkylbenzene sulfonates (LAS) on 15 freshwater and 14 marine species from North Sea estuaries and coastal areas, including the ascidians B. schlosseri and Botrylloides spp. (Figure 5d). This ERA has concluded that LAS poses no threat to local populations, further revealing that short-term exposures resulted in no significant differences between freshwater and marine organisms, in contrast to higher sensitivities posed by chronic LAS exposures. The above and other studies [173,174] indicated uncertainties regarding the development of an effective model organism to predict various types of pollution. This point is further related to some accepted standards (e.g., [175]) that recommend tests to be performed on a limited number of species, thus presenting a constraint on comprehensive assessment.

3. Ecological Risk Assessments

We then conducted a secondary analysis focused specifically on ERAs assigned to either species invasions or biodiversity (Figure 6). This analysis revealed that 61.5% and 62.6% of ERAs on species invasions and biodiversity, respectively, address the marine environments. This contrasts sharply with the overall trend observed in the total ERAs (Figure 3), suggesting that the focus on chemical pollution may contribute to the lower overall number of ERAs conducted in marine settings. In these marine ERAs, fish are the most prominently represented taxon, accounting for 48.3% in invasion studies and 45.84% in biodiversity studies, whereas ascidians rank as the least utilized among the tested taxa, with 1.47% in invasion studies and 0.77% in biodiversity studies. It is worth noting, however, that the use of ascidians in biological invasion studies is significantly higher compared to that in pollution assessments, even though their overall representation remains relatively low compared to other taxa. One notable finding is the relatively elevated percentage of ERAs on corals, accounting for 7.84% in invasion studies and 8.88% in biodiversity studies. Despite not being standardized species, corals hold significant importance as ecosystem builders and are appropriately considered when addressing risks, such as species invasions and biodiversity loss.

3.1. Biological Invasion

Bio-invasion stands out as a prominent catalyst of worldwide environmental shifts, posing significant threats to marine biodiversity [176]. While estimates diverge, the general consensus discloses that marine non-indigenous species (NIS) and primarily marine invasive species exact significant costs on the world’s economy and at the same time present threats to nonmarket environmental resources (including biodiversity and some ecosystem services) and public health [177]. It is also evident that ecological invasion varies between 10% and 70% for ecosystems in tropical America, Africa, and Neo-tropical regions [178]. Biological invasions in the marine arena reflect a wide range of global shipping vectors, mainly through ballast water or via biological fouling on surfaces. Yet, the vast majority of NIS-related risk assessment is focused on the management of ballast water through a targeted species approach, which evaluates three major elements in the ballast water’s invasion profile that include uptake, transit survival, and discharge [179]. The evolved ERAs involve the identification of NIS impacts and then the quantification of their consequences while accounting for uncertainties associated with the assessment process [176]. Given the constraints of limited economic resources, it is suggested to prioritize the list of invasive species for proper management [180].

In contrast to ERAs associated with chemical spills, ERAs linked to biological invasions are engaged with prolonged timetables while entailed with extended intervals between introduction, establishment, and successful colonization and are further influenced by both physical and biological parameters [181]. It is also emerged that ERAs developed for non-agricultural ecosystems receive dwindled scientific and little legislation attention [182] and that they are established on few well-studied invasive cases while many other potential NIS are considered harmless [183]. These are some of the reasons for the claim that the regulation of biological invasions rests on “risk assessment procedures that are narrowly focused, subjective, often arbitrary and unquantified, and subject to political interference” [182], supporting the claims for the use of robust and well-defined methodologies [177].

At present, although there are some invasive risk scoring systems for NIS in place, they lack comprehensive integration of fully quantitative impact metrics [180], and there is no sufficiently validated method for assessing the risk of many taxa, contrasting the established approaches for few, more studied taxa. Furthermore, it remains uncertain whether traits associated with invasiveness in one taxon can be extrapolated to other taxonomic groups due to discrepancies in the traits associated with invasiveness [184], such as the trait of propagule pressure [185], and formal analyses and validations are required to identify traits predictive of invasiveness in taxa beyond established model cases (such as nuisance freshwater fish; [186,187]). Thus, extending an established ERA framework initially designed for one taxon might not effectively encompass the potential for invasiveness in other taxa.

Ascidians are regarded as a model taxon for evaluating biological invasions given that they represent one of the most active groups in bio-invasion reports and are known to invade coasts of all continents except Antarctica, with northern to southern hemisphere invasions [15,188,189]. An important biological trait characterizes ascidian larvae as having short planktonic phases that varied from a few minutes to a few hours [190,191], limiting their natural dispersals to short distances in close proximity to parental colonies. Thus, the trans-oceanic dispersal of notorious invasive ascidian taxa, such as B. schlosseri [50,192], Botrylloides violaceus [193,194], C. intestinalis [195], S. clava [196], and Didemnum vexillum (Figure 5f) [197,198], can only occur through human-mediated transport via attachment to ship hulls and floating objects, with >80 ascidian species listed as NIS [15]. Clearly, environmental alternations caused by global climate changes, such as increasing seawater temperatures [199], and the ongoing development of coastal areas, ports, marinas, and sea farms, play pivotal roles in facilitating the worldwide ascidian colonization [200,201]. Concerning ERA approaches, ascidians thus offer a distinctive system for examining the extent to which human-mediated dispersal (aided by climate change drivers) contributes to the geographical distributions of invasive species in the marine environment.

Invasive ascidians typically exert ecological impacts on benthic communities due to their robust competitive abilities, as evidenced by numerous studies indicating a reduction in species richness and alterations in biodiversity within ascidian-invaded habitats [16,188,189,197,202]. Once established in new environments, invasive ascidians have the potential to overgrow, outcompete native species, and eventually emerge as dominant members of communities [16] or affect various ecological processes by reducing benthic-pelagic coupling [203]. Economically, the aquaculture industry reports on negative impacts attributed to invasive ascidians, typically resulting in reduced harvests and increased production costs due to competition and biofouling on cultured species, necessitating additional maintenance efforts (e.g., [204]). Yet, despite the resultant environmental and economic impacts imposed by invasive ascidian species, there is a notable scarcity of ERAs that integrate predictive models of environmental suitability and/or evaluations of propagule pressure for upcoming invasions. Only very few ERA attempts have established estimates on the local scales (e.g., [52,205,206,207]) and even fewer on the global scale [208].

Several studies have used various biological traits to predict the risk of ascidian invasions, as research results revealed that invasive ascidians may tolerate wide spectrums of salinities and temperatures, may hybridize with closely related species in targeted sites, may reveal specific adaptive genomic variations, and/or carry potential pathogens, parasites, and associated biota. Using this approach for biological traits, studies (e.g., [209]) indicated the high-risk probabilities of tropical ascidian species to invade subtropical zones. These capabilities are varied, as ecological risks presented by C. intestinalis were deemed high to moderate for various Canadian Pacific and Atlantic coast sites, resembling the genetic risk for hybridization with the congener C. savignyi, while exhibiting moderate risk for the delivery of pathogens/parasites or associated biota [210]. Under scenarios of future climate change on two clusters of the Chinese invasive ascidian Molgula manhattensi, two distinct models (gradient forest and species distribution models) revealed conflicting results for the northern Chinese cluster (lower/higher invasion risks, respectively), yet integration of both models highlighted the higher invasion risks for the southern cluster [211]. This is an important outcome as corroborating with other invasive species, such as the invasive clade A in B. schlosseri [212] and the invasive clade A of D. vexillum [213], which are the only clades documented outside the species natal distributions. Ecological Niche model approach for Ascidiella scabra, a new ascidian invader into an Argentinian port, highlighted the risk for invasions into additional SW Atlantic coast sites and other sites worldwide, far away from its native distribution within the NE Atlantic, North Sea, and Mediterranean Sea [214].

In an earlier ERA regional study (Atlantic Canada), Locke [206] has used environmental conditions, biogeography data (species distributions), and vessel ballast water tracks to predict future invasions of listed 57 candidate ascidians species. Nine absent species in Atlantic Canada were excluded from the analysis due to insufficient distribution data; 7 species were already present, whereas the remaining 41 species were found to co-occur in one or more bioregions with species already established in Atlantic Canada. By examining distributions relative to shipping patterns, additional species were removed from the list since they were not found in areas with significant shipping traffic to Atlantic Canada or that they were restricted to subtropical or tropical waters, resulting in a “watch list” of 17 candidate ascidian species deemed most likely to successfully invade Atlantic Canada.

Using the data available for the worldwide invasions of 19 ascidian species across 15 coastal regions, Lins et al. [208] applied ensemble niche modeling, powered by three algorithms (Random Forest, Support Vector Machine, MaxEnt), to forecast ecologically suitable areas for ascidian’s invasions that were further evaluated using independent (area under the curve) and dependent (true skill statistics) thresholds. For environmental variables, the authors considered the maximum and range of sea surface temperature, mean salinity, and maximum chlorophyll. The results revealed that each of the 15 coastal studied regions exhibits areas conducive and interconnected to receive new introductions of ascidians or to facilitate the spread of already established species. They also found that the Central Indo-Pacific, Northwest Pacific, Mediterranean, and West Indo-Pacific coastal regions are the most vulnerable regions to the expansion of established invasive species, results that may put prioritization for species of concern in future control measures. When comparing the almost 15-year period between Locke [206] and Lins et al. [208] ERAs, it is interesting to note that 6 out of the 17 candidate species in the Locke [206] study were listed in Lins et al. [208], with four of them already reaching the northeastern United States and carrying a high likelihood of near-future spreading towards Canada, while one additional species posed a high risk of primary introduction and one presented a low risk. There were at least two species not considered in the earlier study that were flagged with high invasive probability into Atlantic Canada. The continuous increase in seawater temperatures [199] will clearly add potential species that were excluded before, following climate zone filtering.

3.2. Biodiversity and Ecosystem Services

Historically, the ERA discipline has primarily focused on human health and the immediate environment, but it is now expanding to include broader environmental considerations, such as biodiversity and ecosystem integrity, reflecting growing societal interest [215]. One major challenge is that the protection objectives outlined in legislation are frequently overly broad and too general for direct application in ERAs [216]. Additionally, different ecosystem types, such as ‘rare‘ ecosystems, highly diverse ecoregions, ecosystems in significant decline, those hosting endangered species, those facing imminent threats, and ecosystems projected to disappear in the near future, may require distinct ERA attention [215]. There is also a gradual shift from evaluating adverse health impacts on the small scales to assessing impacts on entire ecosystems, spanning ecologically meaningful landscapes [4]. Moreover, increasing research is being devoted to integrating ecosystem services and biodiversity [217] within ERA practices to better inform and support regulatory environmental protection goals. This, in turn, strengthens the policy framework and the relevance of environmental risk assessment processes [218]. Although ERAs are commonly employed to assess biodiversity and ecosystem service deteriorations, this sub-chapter will not address the biodiversity impacts of contaminants (discussed in Section 2), nor will it cover ERAs related to genetically modified organisms, which are outside the scope of ascidian studies.

Species distribution models, integrated with climate projections, are essential for assessing extinction-risk assessments and biodiversity-associated ERAs. Yet, the reliability of future climate-change projections hinges heavily on thorough evaluations of the performance and uncertainties associated with global climate models [219]. Given the variability regarding projections of species’ range shifts and extinction risks among different climate models, it is important to recognize that no single model reigns supreme in forecasting climatic features. Nonetheless, ecologists are increasingly acknowledging the benefits of utilizing multi-model ensemble-averaged climate forecasts to address climate-model uncertainties inherent in climate models. Additionally, integrating species distribution models with metapopulation dynamics models is gaining recognition among ecologists for its potential benefits [220]. Moreover, the existing ERA methodologies that assess the local, regional, and global impacts of human activities on biodiversity and ecosystem services often lack comprehensive assessments, highlighting the need for the integration of additional methods [221]. This includes incorporating life cycle assessments into ERAs [222] and adopting trait-based approaches that consider the physical features, ecological niche, and functional role of species within ecosystems [223] to provide more comprehensive evaluations.

As previously outlined, biological invasion poses a significant threat to marine biodiversity [16,188,189,197,202] and to ecological services, a consensus supported by numerous studies [217]. Despite this recognition, there has been a significant lack of comprehensive ERA efforts quantifying the impacts of biological invasion on biodiversity/ecological services and the pathways of introduction. Utilizing data from over 350 databases and sources to compile information on 329 marine invasive species, including their distribution, biodiversity impacts, and introduction pathways, Molnar et al. [224] revealed that only 16% of marine ecoregions did not report instances of marine invasions. They also identified international shipping (see also [201]) and aquaculture as the primary introduction pathways that pose the greatest potential threat to biodiversity.

Indeed, the standout ascidian ERA in the fields of biodiversity and ecological services is biological invasion [16,176,177,188,189,197,202,208,210,217]. While most biological marine invasions are linked to direct anthropogenic involvement, there are also ERAs that consider natural invasive phenomena, such as those that followed the Great Tsunami of 2011, where marine debris landings facilitated the migration of Japanese marine sedentary species (including ascidians) to Pacific North America and Hawaii [225]. In a similar way, climate change factors, primarily seawater warming [226], have initiated ascidians’ poleward range expansions [226,227] and equatorward range expansions [228]. Continuous range expansions of about 16 km per year were accounted for tunicates [229], while another study revealed a more dynamic figure that also involves cycled range expansions and contractions in response to altered abiotic and biotic conditions [230].

4. Discussion

Ascidians serve as common model organisms for studying diverse phenomena, such as biological invasion, evolutionary biology, allorecognition, innate immunity, stem cell biology, mechanisms of aging, regeneration, embryonic development, germ cell sequestration, cell fate determination, and the production of secondary metabolites, some of which may have pharmaceutical potential [15,38,83,231]. Their abundance, accessibility, sensitivity to different toxins [83] and filter-feeding behavior [232] render ascidians highly sensitive to environmental fluctuations. This responsiveness makes them valuable indicators of environmental health and suitable candidates for studies investigating the impacts of environmental pollutants and stressors on marine ecosystems, e.g., [59,82]. Moreover, the adaptability of ascidians to unstable and varying environmental conditions and the high invasive nature of some species make them excellent candidates for studying issues related to species invasions and biodiversity.

Despite their remarkable versatility as models, the use of ascidians in standardized ecotoxicological tests and ERAs is surprisingly limited, primarily restricted to ERAs dealing with invasive species [15,208,233]. While they are not presently incorporated into ERAs that deal with environmental pollution and other hazards, their potential as environmental bioindicators is demonstrated in this review paper (Figure 7). The underutilization of ascidians in environmental testing represents a missed opportunity to leverage their unique cellular, immunological, and developmental biology capabilities for a more comprehensive understanding of marine health and for developing effective ERA protocols.

A particular type of ERA with a specific focus involves providing early warnings of ecosystem deterioration due to anthropogenic pressures before significant impacts, such as observed mortality, reduced species diversity, or population decline, manifest. Such ERAs are based on in vitro bioassays that utilize normal animal physiological adaptation and defense mechanisms, such as changes in gene expression (e.g., oxidative stress-related genes [234]) in response to low-level, chronic, sub-lethal environmental pressures. Such bioassays are already integrated into national environmental monitoring programs as biomarkers [64]. The potential of similar ascidian-based bioassays can be inferred from a few existing studies. One such study, conducted at various coastal sites along the Mediterranean and Red Seas, revealed alterations in the expression of stress-related proteins [158]. In another study, Tasselli et al. [130] examined the expression and function of oxidative stress-related genes in B. schlosseri colonies immersed in two sites within the Venice Lagoon for 22 days. Their findings showed increased expression of most of the tested stress-related genes compared to control colonies maintained under laboratory conditions. Notably, the expression of one gene, glutathione peroxidase (GPX), was also documented as a significant indicator of environmental oil pollution in a separate study testing pollution at Haifa Port and in environmental control setups [66]. Collectively, these results underscore the potential of ascidian models in ERAs, including those of early warning bioassays based on their unique physiology. However, further research is needed to strengthen their application.

Throughout scientific literature, there is no debate regarding the reproducibility and repeatability of toxicity tests using ascidians as model organisms. Recent publications continue to endorse ascidians as valuable tools in environmental assessments [84]. However, the number of publications in this field is limited, and comprehensive data from repeated tests across different laboratories using identical protocols is lacking, preventing statistical validation. Consistency between endpoints does exist, as demonstrated by Eliso et al., who used two endpoints, transcriptome profiling and phenotypic assessments, to develop an ‘Adverse Outcome Pathway’ for polystyrene nanoparticle toxicity [103]. Yet, as is true for all species, different tests may vary in the degree of sensitivity, emphasizing again the need for a few specific, multi-purpose standardized protocols. The question remains: why have ascidians not been included in standardized protocols? Ascidians are not unique in this regard. Only a few species have been standardized for environmental assessments. Historically, models related to human health and its immediate environments (terrestrial and freshwater) have been preferred, while marine environments have been underrepresented, as shown in Figure 3. Additionally, ascidian research is supported by a small scientific community, and laboratory cultivation requires specialized facilities with access to seawater, which may lead standardization agencies to prioritize other models. However, such considerations should not replace ecological relevance. On the contrary, organizations like the EPA recommend using non-model organisms when appropriate [235].

The limited presence of ascidian species in ERAs that use marine organisms poses a challenge. In addition to fish, researchers often focus on a few invertebrate model species recommended in standardized tests, typically from the phyla Arthropoda, Mollusca, Annelida, and Echinodermata [66]. This further emerged when searching for comparability across different ERAs [236], revealing fragmented efforts that ignore other taxa, sometimes better fitting the questions asked, especially when testing novel risks or unique regional ecosystems. This traditional way of using few model species further leads to (a) redundant data from an ERA standpoint [237,238,239], and (b) the use of species that are less sensitive for specific pollutants (further discussed in Section 2.4) [240]. Therefore, despite recognizing the benefits of non-model organisms, many ERAs prioritize standardized species. While standardization bodies may address this issue by recommending the use of additional model organisms, such as ascidian species, this has not been conducted. Yet, the employment ascidians for standardized ERAs would streamline research efforts and facilitate the development of widely applicable protocols for ERAs [241] and quantitative ERAs [242] that can be based on more fitted new model organisms. It will also satisfy the emerging needs for formulating effective ERAs for specific scientific fields, like the impacts of nano-materials [243], microplastics [244], and endocrine active substances [245].

The outcomes of the present review suggest that future research endeavors should change directions for improved ERAs by focusing on: (1) formulating standardized test protocols that incorporate novel model organisms, such as ascidians, as environmental bioindicators and integrating them into appropriate ERA protocols; (2) exploring the molecular mechanisms underlying unique phenomena in ascidians, like regeneration [31,123,124], immunology [246,247,248], colonial astogeny and torpor [35,249,250,251,252], for potential implications in environmental testing. Molecular tests utilizing ascidians are rarely used for environmental assessment but hold significant potential as fast, high-throughput methods for estimating sublethal effects. As ascidians dwell in highly polluted or heavily invasive regions, developing a variety of molecular tools that can be associated with environmental stress could serve as an early warning system for the deterioration of aquatic regions as well as to study less understood phenomena like hormesis, tolerance, and chemical sensitization; (3) understanding the ecological ramifications of invasive ascidian species and devising control strategies within ERA frameworks; (4) investigating ascidian population diversity to discern patterns of adaptation and resilience amidst the dynamics in a changing marine environment.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/environments11110232/s1, Table S1: Queries performed on ‘Google scholar’ and’ Web of science’ for preparation of Figure 3; Table S2: Queries performed on ‘Google Scholar’ for preparation of Figure 6.

Author Contributions

Conceptualization, A.R. and B.R.; writing—original draft preparation, A.R. and B.R.; writing—review and editing, A.R. and B.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

No new data were created or analyzed in this study.

Acknowledgments

We would like to thank Guy Paz for the artistic work, John Bishop from the Marine Biological Association of the United Kingdom for the Ciona figures, and Shai Shafir from Oranim Academic College, Israel, for the solitary ascidians’ figures.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 2. Modes of life cycles in two ascidian model species: (a) a solitary ascidian (Ciona spp.) revealing classical sexual reproduction progressions of a broadcasting species; (b) asexual (outer cycle in the diagram) and sexual phases in a colonial ascidian (Botryllus schlosseri) highlighted by weekly astogenic rounds of zooidal life and death (each cycle is called blastogenesis). Each blastogenic cycle is divided into four stages (A–D) where three generations of colonial modules (the functional zooids and two cohorts of developing buds, primary and secondary buds) coexist side by side, depicting highly synchronized developmental statuses as the colony progresses through blastogenesis. At stage D, the functional zooids start their degeneration, first by closing the siphons, where simultaneously all zooids undergo cell apoptosis and phagocytosis processes within the next 24–36 h and are morphologically absorbed, while the primary buds mature to the zooidal level of development. Subsequently, stage A of the following blastogenic cycle begins as primary buds complete their development into zooids by opening the inhalant siphons and resuming water filtration, while secondary buds (budlets) develop to the primary bud’s state, starting the generation of new sets of secondary buds. Gametogenesis is highly synchronized with the blastogenic cycle among modules of the same generation. The sexual cycle is typified as brooding, where gametes start differentiation within the budlets. Gametes maturation and egg fertilization occur within the zooids (at the onset of stage A). Embryos differentiation is synchronized with the blastogenic stage and continues within the zooids (inner cycle; showing for each blastogenic stage a single large zooid with its bud/s and budlet/s), culminating in the release of the larvae into the surrounding waters at late blastogenic stage C. The larvae swim for a very short period until they settle, undergo metamorphosis starting with the absorption of the tail, and new juveniles (oozooids) are formed, and a colony is formed by repeated blastogenic cycles.

Figure 3. Pie charts depicting taxa percentages for search hits in the ‘Web of Science’ and ‘Google Scholar’ databases, filtered by the terms ‘environmental risk assessments’ and ‘marine’ environment, as compared to the total hits for ‘environmental risk assessment’ (marine and terrestrial). Each taxon (a group of different bioindicators) is represented by a specific-colored descriptor detailed in the caption. The black sections encompass the residual hits for taxa with the smaller number of hits. The magnified pie sections on the right provide a detailed breakdown for some of these taxa. Supplementary Table S1 details the specific queries used to obtain the data for each taxon.

Figure 4. Commonly used model ascidians: the solitary ascidians (a) Ciona robusta and (b) Ciona intestinalis (by John Bishop from the Marine Biological Association of the United Kingdom), once considered as a single species; (c,d) different color morphs of the colonial ascidian Botryllus schlosseri. (c) A colony reared in the laboratory at the Israel Oceanography and Limnological Research, Haifa, and maintained at a constant temperature of 20 °C with a regimen of 12:12 light:dark hours. This colony is a descendant of the Monterey, California, population; (d) a colony from New Zealand reared on a glass slide.

Figure 5. Pictures of additional solitary (a–c) and colonial ascidian (d–f) used in toxicity, environmental pollution monitoring tests, and biological invasions. (a) Phallusia spp.; (b) Polycarpa spp.; (c) Halocynthia spp.; (d) Botrylloides spp.; (e) Didemnum spp.; (f) Didemnum vexillum.

Figure 6. Pie charts depicting taxa percentages for search hits in ‘Google Scholar’ databases, filtered by the terms: (a) ‘environmental risk assessment’ and ‘marine’ environment, and ‘invasion’ (61.5% of hits) as compared to the total hits for ‘environmental risk assessments’ and ‘invasion’ (100%). The total number of hits involving individual taxa exceeds the number of hits for ‘environmental risk assessment’, ‘marine’ environment, and ‘invasion’, as many publications examine multiple taxa; (b) ‘environmental risk assessment’ and ‘marine’ environment, and ‘biodiversity’ (62.6%) as compared to the total hits for ‘environmental risk assessments’ and ‘biodiversity’ (100%). The total number of hits involving individual taxa exceeds the number of hits for ‘environmental risk assessment’ and ‘marine’ environment and ‘biodiversity’, as many publications examine multiple taxa. Each taxon is represented by a specific-colored descriptor detailed in the caption. The magnified pie sections on the right are for categories with smaller numbers of hits. Supplementary Table S2 details the specific queries used to obtain the data for each taxon.

Figure 7. A graphical representation illustrating the potential applications of ascidian-based bioassays within the framework of ERA. The bioassays utilize both solitary and colonial ascidians, which may offer unique advantages for studying different environmental impacts. MCR—multiple clonal ramets; Blue arrows indicate bioassays that have already been successfully employed in toxicity testing; Red arrows represent potential applications that have yet to be widely explored.

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Rosner, A.; Rinkevich, B. Harnessing Ascidians as Model Organisms for Environmental Risk Assessment. Environments 2024, 11, 232. https://doi.org/10.3390/environments11110232

AMA Style

Rosner A, Rinkevich B. Harnessing Ascidians as Model Organisms for Environmental Risk Assessment. Environments. 2024; 11(11):232. https://doi.org/10.3390/environments11110232

Chicago/Turabian Style

Rosner, Amalia, and Baruch Rinkevich. 2024. "Harnessing Ascidians as Model Organisms for Environmental Risk Assessment" Environments 11, no. 11: 232. https://doi.org/10.3390/environments11110232

APA Style

Rosner, A., & Rinkevich, B. (2024). Harnessing Ascidians as Model Organisms for Environmental Risk Assessment. Environments, 11(11), 232. https://doi.org/10.3390/environments11110232

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