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Biological Reviews - 2023 - Rosner

September 2023
Biological reviews of the Cambridge Philosophical Society

September 2023

Authors:

Amalia Rosner

Israel Oceanographic and Limnological Research Institute (IOLR)

Pedro Martínez

University of Barcelona

(A) Daphnia sp. (B) Gammarus fossarum. (C-H) Whole zooplankton community crude-oil acute toxicity (LC50) test using experimental microcosms. (C) Experimental setup. (D-H) Zooplankton species exposed to crude oil in concentrations of 50-1000 ppm (v/v) for 48 h. (D) Sapphirina sp. (E) Oithona nana. (F) Oithona plumifera. (G) Pseudoevadne tergestina. (H) Cirripedia nauplius. Black scale bar = 100 μm; red scale bar = 5 mm.

…

(A-C) Mollusca. (A) Mytilus edulis. (B) Pecten maximus. (C) Crassostrea gigas. (D-F) Annelida. (D) Polychaeta, Eunicidae. (E) Polychaeta, Opheliidae. (F) Leech, Hirudo verbana. (G-K) Nematoda. (G) Vasostoma sp., head. (H) Vasostoma sp., tail in female. (I) Vasostoma sp. (J) Thershelingia sp. (K) Dorylaimopsis sp. am, amphid; bc, buccal cavity; cs, cephalic setae; mo, mouth opening; rp, reproductive pore; ut, uterus. Black scale bar = 1 cm; red scale bar = 100 μm.

…

Representative echinoderm experimental models used in ecotoxicological research. (A) The starfish Asterias rubens; scale bar = 2 cm. (B) The sea cucumber Holothuria tubulosa on a muddy substrate; scale bar = 2.5 cm. (C) The sea-urchin Paracentrotus lividus feeding on Posidonia oceanica; scale bar = 1 cm. (D) A pluteus larvae of P. lividus; scale bar = 25 μm. (E) Culture of P. lividus immune cells; scale bar = 30 μm. (F) The crinoid Antedon mediterranea; scale bar = 2 cm. (G) Arm regeneration (arrow) of A. mediterranea 2 weeks post-amputation; scale bar = 1 mm. (H) The brittle star Ophiactis virens undergoing regeneration (arrow) after asexual reproduction (fission); scale bar = 1.5 mm.

…

Non-standard models. (A, B) Sponges. (A) Halichondria panicea. (B) Aplysina cavernicola. (C-F) Cnidarians. (C) Stylophora pistillata. (D) Pocillopora damicornis. (E) Nubbins (small fragments) of Stylophora pistillata portraying horizontal spread on the substrate. (F) Coral nursery. (G) Nematostella vectensis. (H-K) Flatworms. (H) Schmidtea mediterranea. (I) Dugesia japonica. (J) Dugesia tigrine. (K) Macrostomum lignano.

…

Ascidians. (A) Diagram of a typical larva. Bv, brain vesicle; gd, gastrodermis; ep, epidermis; mc, mesenchyme; ms, muscle; nc, nerve chord; nt, notochord; oc, ocellus; ot, otolith; vg, visceral ganglion. (B) A typical solitary ascidian Ciona robusta. (C-F) Solitary ascidian life stages used in (eco)toxicological tests.

…

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A broad-taxa approach as an important

concept in ecotoxicological studies and

pollution monitoring

Amalia Rosner

*,Loriano Ballarin

,Stéphanie Barnay-Verdier

,Ilya Borisenko

Laura Drago

,Damjana Drobne

,Maria Concetta Eliso

6,7

,Zoya Harbuzov

1,8

Annalisa Grimaldi

,Tamar Guy-Haim

,Arzu Karahan

,Iseult Lynch

Maria Giulia Lionetto

12,13

,Pedro Martinez

14,15

,Kahina Mehennaoui

Elif Oruc Ozcan

,Annalisa Pinsino

,Guy Paz

,Baruch Rinkevich

Antonietta Spagnuolo

,Michela Sugni

and Sébastien Cambier

16,

Israel Oceanographic and Limnological Research, National Institute of Oceanography, PO 2336 Sha’ar Palmer 1, Haifa 3102201, Israel

Department of Biology, University of Padova, via Ugo Bassi 58/B, Padova I-35121, Italy

Sorbonne Université; CNRS, INSERM, Université Côte d’Azur, Institute for Research on Cancer and Aging Nice, 28 avenue Valombrose, Nice

F-06107, France

Faculty of Biology, Department of Embryology, Saint Petersburg State University, Universitetskaya embankment 7/9, Saint Petersburg 199034,

Russia

Department of Biology, Biotechnical Faculty, University of Ljubljana, Veˇ

cna pot 111, Ljubljana 1111, Slovenia

Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples 80121, Italy

Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy

Leon H. Charney School of Marine Sciences, Department of Marine Biology, University of Haifa, 199 Aba Koushy Ave., Haifa 3498838,

Israel

Department of Biotechnology and Life Sciences, University of Insubria, Via J. H. Dunant, Varese 3–21100, Italy

Middle East Technical University, Institute of Marine Sciences, Erdemli-Mersin PO 28, 33731, Turkey

School of Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham, B15 2TT, UK

Department of Biological and Environmental Sciences and Technologies, University of Salento, via prov. le Lecce –Monteroni, Lecce I–73100, Italy

NBFC, National Biodiversity Future Center, Piazza Marina, 61, Palermo I–90133, Italy

Department de Genètica, Microbiologia i Estadística, Universitat de Barcelona, Av. Diagonal 643, Barcelona 08028, Spain

Institut Català de Recerca i Estudis Avançats (ICREA), Passeig de Lluís Companys, Barcelona 08010, Spain

Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Science and Technology (LIST), 41, rue du Brill, Belvaux

L-4422, Luxembourg

Faculty of Arts and Science, Department of Biology, Cukurova University, Balcali, Saricam, Adana 01330, Turkey

National Research Council, Institute of Translational Pharmacology (IFT), National Research Council (CNR), Via Ugo La Malfa 153, Palermo

90146, Italy

Department of Environmental Science and Policy, University of Milan, Via Celoria 26, Milan 20133, Italy

ABSTRACT

Aquatic invertebrates play a pivotal role in (eco)toxicological assessments because they offer ethical, cost-effective and

repeatable testing options. Additionally, their signiﬁcance in the food chain and their ability to represent diverse aquatic

ecosystems make them valuable subjects for (eco)toxicological studies. To ensure consistency and comparability across

*Authors for correspondence: A. Rosner (Tel.: +972 4 8565233; E-mail: amalia@ocean.org.il) and S. Cambier (Tel.: +352 275 888 5018;

E-mail: sebastien.cambier@list.lu).

Biol. Rev. (2023), pp. 000–000. 1

doi: 10.1111/brv.13015

studies, international (eco)toxicology guidelines have been used to establish standardised methods and protocols for data

collection, analysis and interpretation. However, the current standardised protocols primarily focus on a limited number

of aquatic invertebrate species, mainly from Arthropoda, Mollusca and Annelida. These protocols are suitable for basic

toxicity screening, effectively assessing the immediate and severe effects of toxic substances on organisms. For more com-

prehensive and ecologically relevant assessments, particularly those addressing long-term effects and ecosystem-wide

impacts, we recommended the use of a broader diversity of species, since the present choice of taxa exacerbates the lim-

ited scope of basic ecotoxicological studies.

This review provides a comprehensive overview of (eco)toxicological studies, focusing on major aquatic invertebrate

taxa and how they are used to assess the impact of chemicals in diverse aquatic environments. The present work sup-

ports the use of a broad-taxa approach in basic environmental assessments, as it better represents the natural popu-

lations inhabiting various ecosystems. Advances in omics and other biochemical and computational techniques

make the broad-taxa approach more feasible, enabling mechanistic studies on non-model organisms. By combining

these approaches with in vitro techniques together with the broad-taxa approach, researchers can gain insights

into less-explored impacts of pollution, such as changes in population diversity, the development of tolerance

and transgenerational inheritance of pollution responses, the impact on organism phenotypic plasticity, biological

invasion outcomes, social behaviour changes, metabolome changes, regeneration phenomena, disease susceptibility

and tissue pathologies. This review also emphasises the need for harmonised data-reporting standards and

minimum annotation checklists to ensure that research results are ﬁndable, accessible, interoperable

and reusable (FAIR), maximising the use and reusability of data. The ultimate goal is to encourage integrated and

holistic problem-focused collaboration between diverse scientiﬁc disciplines, international standardisation organisa-

tions and decision-making bodies, with a focus on transdisciplinary knowledge co-production for the One-Health

approach.

Key words: animal model, ecotoxicology, environmental risk assessment, freshwater and marine invertebrates, innovative

methods.

CONTENTS

I. Introduction .........................................................................3

II. Use of standardised testing in chemical risk assessment and water quality control ....................4

III. Key aquatic invertebrates in standardised aquatic ecotoxicology/monitoring ........................6

(1) Arthropoda ..................................................................... 6

(2) Mollusca ....................................................................... 8

(3) Annelida ...................................................................... 10

(4) Nematoda ..................................................................... 11

(5) Echinodermata ................................................................. 12

(6) Standardised models –overview .................................................... 14

IV. Non-standard models .................................................................14

(1) Porifera ....................................................................... 14

(2) Cnidaria ...................................................................... 16

(3) Platyhelminthes ................................................................. 18

(4) Tunicata ...................................................................... 20

(a) Solitary ascidians ............................................................. 20

(b) Colonial ascidians ............................................................ 21

(5) Non-standard models –overview ................................................... 24

V. New options for bio-monitoring using non-standard models following the development of innovative

methods .................................................................................25

VI. Integration of data for reuse in (eco)toxicology and environmental risk assessment ...................26

VII. Certainties and uncertainties in assessing aquatic ecotoxicology .................................27

VIII. Discussion ..........................................................................28

IX. Conclusions .........................................................................30

X. Acknowledgements ...................................................................30

XI. References ..........................................................................30

XII. Supporting information ................................................................46

2Amalia Rosner and others

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I. INTRODUCTION

Invertebrates account for the largest number of species in

marine and freshwater ecosystems. According to current

estimates, of the 6.7 million invertebrate species, 16%

(1.1 million) live in marine and 2% (150,000) in freshwater

environments, where they have colonised a large spectrum

of ecological niches (Collier, Probert & Jeffries, 2016). This

colonising ability is closely linked to their vast diversity,

accompanied by unique biological characteristics, including

possession of large numbers of pluripotent adult stem cells

(ASCs; Rinkevich et al., 2022) and the ability to regenerate

lost body parts, even up to whole-body regeneration in

some cases (Rinkevich et al., 2007b). Molluscs and crustaceans

are the richest taxa in terms of species numbers in the marine

environment (Appeltans et al., 2012), while insects are the

richest invertebrate taxon in freshwater habitats. Aquatic

invertebrates perform critical ecological functions in all

aquatic ecosystems (Palumbi et al., 2009; Macadam &

Stockan, 2015). They provide a range of ecosystem services,

including ﬁltering water (Ostroumov, 2005), processing

organic matter (Bart et al., 2020), ecosystem engineering

(Howell et al., 2016; Angiolillo & Canese, 2018), recycling

nutrients (Lohrer, Thrush & Gibbs, 2004; Atkinson et

al., 2017), participating in the carbon cycle (Tue et al., 2017),

and playing key roles in mitigating natural risks [e.g. dissipa-

tion of wave energy that might impact shorelines (Ferrario et

al., 2014;Wiberget al., 2019)]. In addition, they are sources

of ‘blue food’(edible aquatic organisms), biomolecules and

biomaterials of economic relevance, especially in the pharma-

ceutical industry (Datta, Talapatra & Swarnakar, 2015).

Anthropogenic pollutants ultimately reach the aquatic

environment and cause a deleterious impact on freshwater

and marine ecosystems (Häder et al., 2020), leading to

large-scale biological impacts that may culminate in the

extinction of some species (Baines et al., 2021). Eventually,

these pollutants may reach human populations through the

food chain (Adeel et al., 2017; Lecomte et al., 2017). The

effects of legacy pollutants [e.g. polycyclic aromatic hydro-

carbons (PAHs), dioxins, polychlorinated biphenyls (PCBs),

pesticides, and heavy metals], and newly emerging contami-

nants [e.g. pharmaceuticals, and microplastics and nanoplastics

(MNPs)] are usually investigated with in vitro and in vivo toxic-

ity tests, which may be performed on single-species model

organisms or at community levels. These tests are necessary

for setting accurate toxicity thresholds and identifying toxic-

ity mechanisms or pathways. They form part of the prospec-

tive and retrospective assessment tools required for the

market authorisation of new compounds or characterisation

of the causes of adverse effects. Commonly used in vitro tests

have recently been reviewed by Rosner et al.(

2021).

The immense diversity and abundance of aquatic

invertebrates, their relatively simple body organisation,

small size, reduced genetic complexity, and high sensitivity

to a broad spectrum of chemical compounds have resulted

in their widespread use in ecotoxicological studies (Lagadic

&Caquet,1998) and their inclusion in international guidelines

for ecotoxicity testing. In addition, apart from cephalopods,

they are not included in EU legislation on animal research,

such as Directive 2010/63/EU on the protection of animals

used for scientiﬁc purposes. Furthermore, the ease of rearing

many aquatic invertebrates under controlled laboratory

conditions, their high fecundity, and their short lifespans

enable rapid in vivo testing and the potential for multi-genera-

tional tests (e.g. to investigate epigenetic effects on unexposed

offspring). Furthermore, the rearing of these animals in

laboratories reduces the use of natural populations (Collier

et al., 2016). Therefore, numerous biomarkers based on

aquatic invertebrates have been developed for (eco)toxicity

testing (Tosti & Gallo, 2012;Lopez-Pedrouso et al., 2020;

Trestrail, Nugegoda & Shimeta, 2020). Aquatic invertebrates

also can successfully replace vertebrates in various tests, as

demonstrated by their integration into national and interna-

tional guidelines. Short-term toxicity tests on invertebrates

are mandatory under regulation 1907/2006/EC, better

known as the REACH (Registration, Evaluation, Authorisation

and Restriction of Chemicals) regulation, and in long-term tox-

icity studies for substances for which production exceeds

100 tons/year (Tarazona et al., 2014).

There are, however, important shortcomings in terms of

environmental relevance associated with the use of laboratory-

bred aquatic invertebrates. Speciﬁcally, extrapolation from

controlled laboratory conditions to natural environments is

often difﬁcult as many parameters are not taken into account.

These include: (i) inter-species variation in toxicity sensitivity

associated with substance movement through food webs and

potential biomagniﬁcation and biotransformation; (ii) syner-

gistic impacts, which might also be affected by the alteration

of physical attributes of the experimental animals; (iii) life

traits associated with previous exposure or the transgenera-

tional inheritance of epigenetic signatures due to exposure

of previous generations. Additional doubts can be raised

regarding which species should be considered ‘gate-keepers’

(and thus used for different trophic-level ecotoxicity tests)

due to species-speciﬁc sensitivity to pollutants (Chaumot

et al., 2014). These considerations, together with legislation

that minimises animal use [e.g. REACH regulations, 2006;

bans on animal testing, 2013; the ‘3Rs’principle of

Replacement, Reduction and Reﬁnement of animal use in

research (Burden et al., 2015)], encourage the development

of alternatives to in vivo toxicity testing based on multiple

model organisms and endpoints (Rosner et al., 2021). In

addition, the use of omics and high-throughput techniques

that allow extrapolation from responses at the cellular level

to effects on whole organisms, populations, ecosystems or

among species, must be accompanied by computational

models (Brockmeier et al., 2017;Vinkenet al., 2017;

Sakuratani, Horie & Leinala, 2018). Approaches that pre-

dict endpoint information for one substance using

data from another structurally similar substance (source

substance[s]), termed ‘read-across’, are also increasingly

available and accepted for regulatory purposes. Most model

organisms for both in vivo and in vitro tests are chosen to rep-

resent the many phyla and ecosystems in the aquatic

A broad-taxa approach as an important concept 3

environment. The aquatic invertebrate models used most

widely in the various regulatory test guidelines and reported

in ecotoxicological studies include molluscs, arthropods, cni-

darians, and annelids. Other taxa such as sponges,

ﬂatworms, nematodes and tunicates are poorly represented

(Fig. 1). In this review, we describe the common aquatic inver-

tebrate taxa used in ecotoxicological studies and pollution

monitoring, emphasising the value that underrepresented taxa

could add to provide a more comprehensive understanding of

the impact of pollution on the aquatic environment.

II. USE OF STANDARDISED TESTING IN

CHEMICAL RISK ASSESSMENT AND WATER

QUALITY CONTROL

Work on the safety of pesticides and chemicals began in

1971, following establishment of the Organisation for

Economic Cooperation and Development (OECD) Environ-

ment Committee. The OECD Mutual Acceptance of Data

(MAD) agreement on the assessment of chemicals is an essen-

tial component of the worldwide standardisation of

methodologies for chemical safety (OECD, 1981). The

OECD test guidelines (TG) programme develops guidelines,

which are published online (Rasmussen et al., 2019). An

advantage of MAD has been a reduction of unnecessary ani-

mal testing because substances only need to be tested once

rather than in every country of notiﬁcation. The 3Rs princi-

ples for animal experimental usage (Russell & Burch, 1959)

was inﬂuential on EU legislation for regulations to protect

animals used in research and other scientiﬁc purposes in

1986. The 3Rs subsequently was made a legal requirement

in Directive 2010/63/EU on the protection of animals used

for research purposes. A key aspect of the resulting replace-

ment of vertebrates with invertebrates has been a dramatic

increase in the use of the latter in research and chemical

safety assessments (see Table 1for the OECD TGs related

to using invertebrates in the testing and assessment of chemicals).

There are few ethical guidelines governing the use of inverte-

brates in science (Drinkwater, Robinson & Hart, 2019), other

than their ability to feel pain or demonstration of advanced

cognition. Scientiﬁc organisations like the Association for

the Society of Animal Behaviour publish some rules for using

invertebrates in research (ASAB, 2018). However, there

remain differences among countries in the legal protection

Fig. 1. Phylogenetic tree showing the number of marine (yellow numbers) and freshwater (white numbers) species in the various taxa

retrieved, from WoRMS (2022)andBalianet al.(2008), respectively; the total number of publications in the domains of

ecotoxicology, toxicology and pollution (green numbers) in the last 10 years (see Q1 in Table S1 for search string); and the number of

publications based on omics technologies in the domains of ecotoxicology, toxicology and pollution (black numbers) in the last

10 years (see Q2 in Table S1 for search string). Data were obtained from a search of Web of Science for the search terms listed in Q1

and Q2 on 31 December 2021.

4Amalia Rosner and others

Table 1. Standardised regulatory tests utilising aquatic invertebrates.

Phylum Species Tests Guidelines References

Arthropoda Chironomus dilutus Acute toxicity test OECD Test No. 218–219 OECD (2018)

Sediment-water life-

cycle toxicity test

OECD Test No. 233 OECD (2005)

Bioaccumulation test ASTM E1688-19 ASTM (2020)

Multigeneration test for

assessment of

endocrine-active

chemicals

OECD Test No. 218-219 OECD (2018)

Daphnia magna Acute toxicity test EPA OCSPP 850.1010 EPA (2016a)

Immobilisation OECD Test No. 202 OECD (2004)

Chronic toxicity test EPA OPPTS 850.1300 EPA (2016f)

EC50 ASTM E1193-20 ASTM (2016)

Daphnia magna,Daphnia pulex Chronic toxicity test:

reproduction test

(male induction)

OECD Test No. 211 OECD (2018)

Gammarus fasciatus Acute toxicity test EPA OCSPP 850.1020 EPA (2016b)

Gammarus pseudolimnaeus Acute toxicity test EPA OCSPP 850.1020 EPA (2016b)

Gammarus lacustris Acute toxicity test EPA OCSPP 850.1020 EPA (2016b)

Hyalella azteca Toxicity test ISO 16303 ISO (2013)

Leptocheirus plumulosus Chronic toxicity test ASTM E1367-03 ASTM (2014)

Rhepoxynius abronius Chronic toxicity test ASTM E1367-03 ASTM (2014)

Ampelisca abdita Chronic toxicity test ASTM E1367-03 ASTM (2014)

Eohaustorius estuarius Chronic toxicity test ASTM E1367-03 ASTM (2014)

Mysidae Acute toxicity test EPA OCSPP 850.1035 EPA (2016c)

Penaeidae Acute toxicity test EPA OCSPP 850.1045 EPA (2016d)

Amphiascus tenuiremis (Copepoda) Acute lethal toxicity ISO/DIS 14669 ISO (2007)

Nitocra spinipes (Copepoda) Acute lethal toxicity ISO/DIS 14669 ISO (2007)

Tisbe battagliai (Copepoda) Acute lethal toxicity ISO/DIS 14669 ISO (2007)

Acartia tonsa (Copepoda) Acute lethal toxicity ISO/DIS 14669 ISO (2007)

Mollusca Crassostrea virginica Acute toxicity test (shell

deposition)

EPA-OPPTS 850.1025 EPA (1996)

Acute toxicity test

(embryo-larval)

EPA OCSPP 850.1055 EPA (2016e)

Bioconcentration

factors

EPA OCSPP 850.1710 EPA (2016g)

Acute toxicity test

(embryo)

ASTM E724-98 ASTM (1998)

Bioconcentration test ASTM E1022-94 ASTM (1994)

Crassostrea gigas Acute toxicity test

(embryo-larval)

EPA OCSPP 850.1055 EPA (2016e)

Acute toxicity test

(embryo)

ASTM E724-98 ASTM (1998)

Bioconcentration test ASTM E1022-94 ASTM (1994)

Mercenaria mercenaria Acute toxicity test

(embryo-larval)

EPA OCSPP 850.1055 EPA (2016e)

Acute toxicity test

(embryo)

ASTM E724-98 ASTM (1998)

Mytilus edulis Acute toxicity test

(embryo-larval)

EPA OCSPP 850.1055 EPA (2016e)

Mytilus edulis Acute toxicity test

(embryo)

ASTM E724-98 ASTM (1998)

Mytilus edulis Bioconcentration test ASTM E1022-94 ASTM (1994)

Pecten sp. Bioconcentration test ASTM E1022-94 ASTM (1994)

Unionidae Toxicity test (glochidia

and juvenile)

ASTM E2455-05 ASTM (2005)

Bivalvia In-situ ﬁeld bioassays ASTM E2122-01 ASTM (2001)

Potamopirgus antipodarum Reproduction test OECD, Test No. 242 OECD (2016a)

Lymnaea stagnalis Reproduction test OECD, Test No. 243 OECD (2016b)

Annelida Lumbriculus variegatus Toxicity test using

spiked sediment

OECD, Test No. 225 OECD (2007a)

(Continues on next page)

A broad-taxa approach as an important concept 5

afforded to invertebrates used in research. For example, the

UK does not have the same laws regarding the euthanasia

of crustaceans as New Zealand (Ministry for Primary

Industry, 2017).

In parallel with chemical legislation, pollution regula-

tion and environmental protection legislation were also

developed. In October 2000, the Directive 2000/60/

EC, also known as the Water Framework Directive

(WFD; https://ec.europa.eu/environment/water/water-

framework/info/intro_en.htm), was adopted, forming a

framework for community action in the ﬁeld of water policy.

The WFD also places various international obligations on

member states regarding the protection of sea waters from

pollution, including compliance with: (i) the Conventions

on the Protection of the Baltic Sea Environment (signed in

Helsinki on 9 April 1992 and conﬁrmed by Council resolu-

tion 94/157/EEC); (ii) the Convention on the Protection of

the Northeast Atlantic Marine Environment, signed in Paris

on 22 September 1992 and conﬁrmed by Council Decision

98/249/EEC; (iii) the Convention on the Protection of the

Mediterranean Sea against Pollution, approved by Council

Decision 77/585/EEC signed in Barcelona on 16 February

1976, and the Convention against pollution from land-based

sources signed in Athens on 17 May 1980 [83/101/EEC

(WFD 2000)].

In addition, the maritime policy of the EU is included in

the EC Marine Strategy Framework Directive (Directive

2008/56/EC, June 2008), which established a framework

for the domain of community action in maritime environ-

mental policy (Marine Strategy Framework Directive, 2008).

The United Nations 2030 Agenda for Sustainable Development

also addresses water quality and pollution via Goal 14, which

aims to establish the conservation and sustainable use of the

oceans, seas, and marine resources (https://sdgs.un.org/

goals/goal14). Goal 14.1 aims ‘by 2025, [to] prevent and

signiﬁcantly reduce marine pollution of all kinds, in particular

from land-based activities, including marine debris and

nutrient pollution’.

To fulﬁl the goals of both chemical legislation and the

WFD, there is an urgent need for tools for the assessment of

environmental hazards and risks to support responsible deci-

sions that enable sustainable development. Invertebrate tests,

both those standardised through OECD TGs (see Table 1),

and those using non-standard species, are required to cap-

ture the breadth of potential effects and impact of pollutants,

and to span the diversity of species and ecosystems impacted

by the >85,000 chemicals currently available in commercial

markets, only a small fraction of which has been evaluated by

US regulators (Gross & Birnbaum, 2017). Similarly, in the

EU, many of the 27,000 registered chemicals (i.e. those pro-

duced or imported at volumes >1 tonne/annum) have yet to

be evaluated. By 2027, the European Chemicals Agency’s

(ECHA) Integrated Regulatory Strategy aims to clarify

which registered compounds are low priority for additional

regulatory action and which are high priority for regulatory

risk management or data collection (ECHA, 2021).

III. KEY AQUATIC INVERTEBRATES IN

STANDARDISED AQUATIC ECOTOXICOLOGY/

MONITORING

(1) Arthropoda

Over 80% of known animal species on Earth are arthropods,

occupying crucial positions in aquatic ecosystems as

detritivores, herbivores, omnivores, and carnivores

(Ødegaard, 2000). They are vital components of healthy

ecosystems and are key indicators of environmental

change and pollution (Chakravarthy & Sridhara, 2016).

Arthropods are highly recommended for aquatic pollution

studies due to their short life cycle, high reproductive

Table 1. (Cont.)

Phylum Species Tests Guidelines References

Acute toxicity tests ASTM E729-80 279–280 ASTM (1996)

Bioaccumulation tests EPA-823-R-00-001 (v. 1);

EPA-823-R-00-002 (v2)

EPA (2000)

Tubifex tubifex Bioaccumulation tests EPA-823-R-00-001 (v.1);

EPA-823-R-00-002 (v.2)

EPA (2000)

Polychaeta spp. Acute, chronic, and

lifecycle aquatic

toxicity tests

ASTM E1562-00 ASTM (2000)

Polychaeta spp. Sediment toxicity tests ASTM E1611-07 ASTM (2007)

Nereis spp. Bioaccumulation tests EPA-823-R-00-001 (v. 1);

EPA-823-R-00-002 (v.2)

EPA (2000)

Echinodermata Sea urchin Fertilisation test EPA-821-R-02-014 EPA (2002)

Sand dollar Fertilization test EPS 1/RM/27 Environment

Canada (2011)

Sea urchin Short-term toxicity test ASTM E1563-21 ASTM (2021)

Nematoda Caenorhabditis elegans Growth, fertility and

reproduction

ISO 10872:2020 ISO (2020)

6Amalia Rosner and others

potential, wide distribution, and representativeness of

plankton and benthic fauna in the littoral and intertidal

zones where most chemical spills occur (Lee, 1977). Their

exoskeleton-moulting process, which is hormone dependent, is

an important endpoint for ecotoxicological studies, serving as

a good indicator of endocrine-disrupting/reproductive disor-

ders induced by chemicals such as industrial chemicals and

pesticides (Peterson, Kashian & Dodson, 2001;Zou,2005;

Mensah, Muller & Palmer, 2012; OECD, 2018).

Among the arthropods, insects (around 76,000 species;

Balian et al., 2008) and crustaceans (67,000 species; Ahyong

& Huang, 2020) are the most prominent groups in aquatic

environments. Freshwater aquatic insects live all or part of

their lives in lentic (still) or lotic (running) water systems (Starr

& Wallace, 2021). The EPT (Ephemeroptera, Trichoptera,

Plecoptera) index, based on pollution-intolerant taxa, is

widely used in freshwater environmental studies (Lenat &

Penrose, 1996) as a common metric for environmental

health, water quality, ecosystem integrity, and response to

pollution (Blöcher et al., 2020) Additionally, acute toxicity is

also frequently tested on the early developmental stages of

sensitive aquatic insects (Kreutzweiser et al., 2008; Beketov

et al., 2013; Camp & Buchwalter, 2016). Many crustacean

species are widely employed in both freshwater and marine

ecotoxicology [American Public Health Association (APHA),

American Water Works Association (AWWA), World

Economic Forum (WEF), 1995-Standard Methods for the

Examination of Water and Wastewater; Pane et al., 2012],

as they are strongly affected by environmental stressors such as

pollution by light, nutrients, and toxins (Wacker &

Harzsch, 2021). Among the crustaceans, Cladocera,

Copepoda, and Amphipoda are the most extensively used

taxa in bioassays.

Cladocera (Diplostraca), commonly known as water ﬂeas,

are small crustaceans widely used as bioassay organisms

when evaluating the impact of toxic substances (Sarma &

Nandini, 2006). Cladocera reproduce by cyclic parthenogen-

esis when conditions are suitable. When conditions worsen,

males and resting eggs are produced. Ernest Warren intro-

duced Daphnia magna as a model organism for toxicity stud-

ies, laying the groundwork for the ﬁeld of ecotoxicology

(Warren, 1900). Since then, D. magna has become the most

widely used crustacean in ecotoxicity tests and is consid-

ered the standard bioassay organism by many academic insti-

tutions and governmental organisations (Siciliano et al., 2015).

Various chemicals have been examined on cladocerans,

including heavy metals (Sadeq & Beckerman, 2019), pesticides

(Toumi et al., 2015), nanomaterials (Ellis et al., 2021a), micro-

plastics (MPs) (Frydkjær, Iversen & Roslev, 2017), and natural

toxic substances such as the cyanobacterial toxin microcystin

(Herrera, Echeverri & Ferrao-Filho, 2015). In early life stages,

Daphnia is sensitive to virulence traits and is therefore used to

study interaction with bacterial pathogens (Ebert, 2008). The

genome of D. pulex was sequenced in 2007 by the Daphnia

Genomics Consortium (DGC), revealing that it has the highest

similarity to human genes among arthropods (http://

wﬂeabase.org/). The use of Daphnia (Fig. 2A) in genetic screen-

ing could facilitate an understanding of the intricate control of

genes and of the cellularand molecular processes thatrespond

to environmental challenges (Siciliano et al., 2015). Since D.

magna naturally occurs only in temperate regions, toxicity test-

ing with this species conducted in tropical regions has drawn

criticism, as D. magna experiences low reproduction and high

mortality rates at tropical temperatures (Mark & Solbé, 1998).

Copepoda form the largest crustacean group with over

13,000 known species (Longhurst, 1985). Most copepod species

Fig. 2. (A) Daphnia sp. (B) Gammarus fossarum.(C–H) Whole zooplankton community crude-oil acute toxicity (LC50) test using

experimental microcosms. (C) Experimental setup. (D–H) Zooplankton species exposed to crude oil in concentrations of

50–1000 ppm (v/v) for 48 h. (D) Sapphirina sp. (E) Oithona nana. (F) Oithona plumifera. (G) Pseudoevadne tergestina. (H) Cirripedia nauplius.

Black scale bar =100 μm; red scale bar =5 mm.

A broad-taxa approach as an important concept 7

are omnivorous, feeding on a range of macroinvertebrates,

protozoa, algae, and bacteria. Copepods go through six nau-

pliar phases and six copepodite stages throughout develop-

ment. Reproduction is sexual, and most species have resting

stages that permit survival during periods of poor environ-

mental conditions. These varied traits of copepods make

them excellent model organisms for ecotoxicological studies

(Kulkarni et al., 2013). The calanoid copepod Acartia tonsa

and the harpacticoid species Amphiascus tenuiremis and Nitocra

spinipes have been recommended by the International Organi-

sation for Standardisation (ISO,2007)andOECD(2007b)for

the evaluation of chronic and acute lethal toxicity of marine

contaminants such as herbicides (Noack et al., 2016), sediment

organic contaminants (Macken et al., 2008), discharge waters

(Sonmez, Sivri & Dokmeci, 2016) and oestrogens (Andersen,

Halling-Sørensen & Kusk, 1999). Other studies have stressed

the importance of using local species to ensure the ecological

relevance of such tests (Butler et al., 2020). Marine copepods

have also been utilised as model organisms for the acute and

chronic testing of crude oil and chemical dispersants (Almeda

et al., 2013; Fig. 2C–H), with the regulation of their cyto-

chrome P450 (CYP) genes being one of the best-known tests

(Han et al., 2017).

Amphipods are ubiquitous crustaceans that include nearly

10,000 marine and freshwater species. They cover a wide

trophic range as herbivores, detritivores, and predators,

while providing an important food source for many organ-

isms higher in the food chain. Amphipods are very sensitive

to a wide range of pollutants. Experiments with amphipods

include lethal concentration 50% (LC50) and effect concen-

tration 50% (EC50) assays for the testing of chemicals

(Alonso, De Lange & Peeters, 2010). Amphipods are also

used in the Polychaeta/Amphipoda (P/A) index, which mea-

sures the differential sensitivity of these two taxonomic

groups (Dauvin, 2018). The genus Gammarus (Fig. 2B)is

widely distributed in freshwater and marine ecosystems and

has been extensively studied for its responses to various

stressors and pollutants (Barnard, 1983). Biomarker analyses

on Gammarus spp. have been used to assess the effects of anti-

oxidant responses, behaviour, cellular damage, defence

mechanisms, energy reserves, endocrine responses, iono/

osmoregulation mechanisms, and lysosomal responses

(Kunz, Kienle & Gerhardt, 2010; Sroda & Cossu-Leguille,

2011; Arce Funck et al., 2013; Gismondi & Thomé, 2014;

Trapp et al., 2014; Mehennaoui et al., 2016; Gouveia et

al., 2018; Batista et al., 2021). In addition, multigenerational

studies (Geffard et al., 2010; Vigneron et al., 2019; Cribiu et

al., 2020) have led to a more ecologically relevant under-

standing of the impact of toxicants on ecosystems (Minguez

et al., 2015). Recent advances in transcriptomics (Caputo et

al., 2020) and proteomics (Trapp et al., 2014,2016; Gouveia

et al., 2019) have enabled the identiﬁcation of new

biomarkers, including reference genes for quantitative PCR

(qPCR) data normalisation (Mehennaoui et al., 2018), and

microbiome (Gouveia et al., 2020). These studies have con-

tributed to a better understanding of the physiological and

molecular responses of non-model species to contaminants

and the pathways underlying detoxiﬁcation (Armstrong

et al., 2019).

Other crustaceans commonly employed in ecotoxicological

studies and environmental biomonitoring include decapods

(Reynolds & Souty-Grosset, 2011), barnacles (Da Silva, Ridd

& Klumpp, 2009), brine shrimps (Hnamte, Kaviyarasu &

Siddhardha, 2020), notostracans (Lahr, 1997), and isopods

(Reboleira et al., 2013). The use of decapods in toxicological

tests is likely to be amended following recent observations of

their ability to feel pain and distress (Passantino, Elwood &

Coluccio, 2021). As an alternative to ecotoxicological testing,

the development of in silico models in crustaceans appears to

be a promising method for predicting chemical toxicity,

which can offer a practical and trustworthy tool for evaluat-

ing environmental risk and ranking chemicals for testing

(Cao et al., 2018; Varsou et al., 2021).

(2) Mollusca

Mollusca are the second largest phylum in the Kingdom

Animalia (Fig. 1) and include seven clades: Aplacophora,

Monoplacophora, Polyplacophora, Bivalvia, Gastropoda,

Cephalopoda, and Scaphopoda. Molluscs are essential

components of the food chain and play an important eco-

logical role in structuring benthic communities due to their

ubiquitous distribution (Fortunato, 2015). As well as being

highly sensitive to pollution exposure (Mouthon & Charvet,

1999), molluscs show a variety of responses to toxic contam-

inants (Grabarkiewicz & Davis, 2008), have a rapid growth

index, short life cycle, wide distribution, different lifestyles,

bioaccumulation capability for a wide range of pollutants,

various life stages with different sensitivity, and can be main-

tained under laboratory conditions (EPA, 2003). Bivalves

and gastropods are recommended for standardised tests in

water and sediment quality assessment as bioindicators

(Table 1).

Most bivalves live at the water–sediment interface, exhibit

burrowing behaviours, and are ﬁlter feeders (McLeod,

Luoma & Luthy, 2008). These features lead them to accumu-

late various chemical pollutants including metals (Rzymski

et al., 2014; Shi et al., 2016; Yuan et al., 2020), PCBs (Dodoo,

Essumang & Jonathan, 2013; Milun et al., 2020), PAHs (Yap,

Shahbazi & Zakaria, 2012; Yoshimine & Carreira, 2012),

organochlorine pesticides (Tong et al., 2019), per- and poly-

ﬂuoroalkyl substances (Cui et al., 2021), and pharmaceuticals

released into the environment (Gomez et al., 2021). Mytilus

edulis (Fig. 3A; Rosenberg & Loo, 1983), Pecten spp. (Fig. 3B;

Metian et al., 2007,2008), Crassostrea gigas (Fig. 3C), and C.

virginica (Perrino & Ruez, 2019) are the recommended species

for the American Society for Testing and Materials (ASTM)

bioconcentration standard test (ASTM, 1994) designed to

assess the ability of an aquatic species to accumulate test

materials directly from the water. C. virginica is the standard

species for two other EPA test guidelines: an acute toxicity

test (OPPTS 850.1025; EPA, 1996) and a bioaccumulation

test (OCSPP 850.1710; EPA, 2016g). The ASTM standard

guide E2122-01 (ASTM, 2001) describes in situ bioassay

8Amalia Rosner and others

using caged bivalves as allowing more precise results about

the impact of pollutants in natural environments. Another

asset of bivalves is the differential susceptibility of their vari-

ous life stages to pollutants, and this is used by two standard

guides, ASTM E724-98 (ASTM, 1998) and OCSPP

850.1055 (EPA, 2016e). The procedures outlined in ASTM

E724-98 (ASTM, 1998) and ASTM E2455-05 (ASTM,

2005) describe how to obtain laboratory data regarding the

acute effects of test materials (individual chemicals; mixtures

in different environmental matrices) on embryos and larvae.

Gastropoda are the largest clade of molluscs (Frýda, 2021)

and in some ecosystems, they can represent 20–60% of the

total quantity and biomass of macroinvertebrates (Anderson

& Smith, 2000). Due to their sensitivity to a broad variety of

organic and inorganic chemicals, gastropods are widely used

as effective indicators of both water and sediment quality

(Tallarico et al., 2014; Leomanni et al., 2015,2016)inenviron-

mental risk assessment (OECD, 2010). Freshwater gastropods

have been utilised for laboratory studies on the effects of endo-

crine-disrupting chemicals (EDCs) on reproduction (OECD,

2010). In particular, the snails Potamopyrgus antipodarum and

Limnaea stagnalis were recommended as the standard species

for reproduction assays by the OECD (2016a,b), with this rec-

ommendation validated within the Conceptual Framework

for Endocrine Disrupters (OECD, 2018).

Besides ecotoxicological tests based on whole-organism-

related endpoints, additional molecular and cellular

biomarkers have been developed in molluscs. These include:

hepatopancreas metallothionein concentration for metals

(Lionetto et al., 2001; Lionetto, Caricato & Giordano, 2021),

oxidative stress biomarkers (depletion of intracellular

reduced glutathione; Lima et al., 2007), alteration of the

activity of antioxidant enzymes [e.g. glutathione peroxidase

(GPX), superoxide dismutase (SOD), and catalase (CAT);

Jena, Verlecar & Chainy, 2009], alterations of the lysosomal

system (Martínez-Gomez, Bignel & Lowe, 2015), induction

of lipid membrane peroxidation, cholinesterase inhibition

as a biomarker of neurotoxicity (Leomanni et al., 2015) and

vitellogenin induction as an EDC biomarker (Tran et al.,

2019). In addition, genotoxicity has been assessed using the

micronucleus assay applied in both laboratory and ﬁeld

experiments to bivalves (Bolognesi & Fenech, 2012).

In recent years, research for alternatives to in vivo

approaches has led to the development of in vitro tools such

as primary cell cultures from various organs (Galloway &

Depledge, 2001; Nogueira et al., 2013; Yoshino, Bickham &

Bayne, 2013; Daugavet & Blinova, 2015) and larval stages

(Van der Merwe et al., 2010), allowing the use of cell cultures

as models for environmental contaminant testing, the analy-

sis of cellular responses to pollutants, and investigation of the

Fig. 3. (A–C) Mollusca. (A) Mytilus edulis. (B) Pecten maximus. (C) Crassostrea gigas.(D–F) Annelida. (D) Polychaeta, Eunicidae.

(E) Polychaeta, Opheliidae. (F) Leech, Hirudo verbana.(G–K) Nematoda. (G) Vasostoma sp., head. (H) Vasostoma sp., tail in female.

(I) Vasostoma sp. (J) Thershelingia sp. (K) Dorylaimopsis sp. am, amphid; bc, buccal cavity; cs, cephalic setae; mo, mouth opening; rp,

reproductive pore; ut, uterus. Black scale bar =1 cm; red scale bar =100 μm.

A broad-taxa approach as an important concept 9

underlying mechanisms (Rinkevich, 1999,2005a). Further-

more, adult stem cells have been identiﬁed (Vogt, 2012;

Jemaà et al., 2014; Deryckere & Seuntjens, 2018; Cherif-

Feildel et al., 2019; Rodriguez et al., 2020) increasing interest

in developing new tools that may lead to ecotoxicological

applications (Rosner et al., 2021).

(3) Annelida

Annelida is a phylum of over 25,000 species of segmented

worms that are found worldwide in various environments

(Fig. 1; WoRMS, 2022; Glasby & Timm, 2008; Aguado

et al., 2014; Glasby, Erséus & Martin, 2021). They can repro-

duce both sexually and asexually and exhibit high regenera-

tion potential, including the restoration of germ cells, cell

repair, regrowth of body parts, and whole-body regenera-

tion (Hyman, 1940; Berrill, 1952; Herlant-Meewis, 1964;

Bely, 2006,2014; Dannenberg & Seaver, 2018;Zattara,

2020; Kostyuchenko & Kozin, 2021).

Polychaetes (Fig. 3D, E) are highly diverse marine worms

that are abundant in ocean sediments and also live in fresh-

water and shallow brackish waters (Verdonschot, 2015;

Jørgensen & Jensen, 1978; Niederlehner et al., 1984). They

have soft bodies, a characteristic segmented shape, and

occupy a wide range of habitats, including those in extreme

conditions (Glasby, 1999; Verdonschot, 2015; Magalh˜

aes et

al., 2021). They play an important ecological role in benthic

ecosystems through scavenging, ﬁlter-feeding, and predation

(Brusca & Brusca, 1990; Hickman & Roberts, 1994). They

are a crucial food source for organisms such as ﬁsh and wad-

ing birds (Verdonschot, 2015). Platynereis dumerilii is a well-

known polychaete species with several life phases and can

survive in polluted and acidic environments (see online

Supporting Information, Fig. S1; Fischer & Dorresteijn, 2004;

Fischer, Henrich & Arendt, 2010;Luceyet al., 2015;

Schenkelaars & Gazave, 2021). It is a dioecious, semelpa-

rous animal that can be bred efﬁciently in a laboratory

(Kuehn et al., 2019), producing numerous embryos for exper-

imentation and culture populations.

Polychaetes are commonly used in marine toxicity tests to

assess the impact of pollutants and toxic materials on sedi-

ments (Pocklington & Wells, 1992; Scaps, 2002; Bat, 2005;

Dean, 2008). More than 48 species of polychaetes are used

in standard toxicity tests (ASTM, 2000,2007), with the six

species used most frequently being Neanthes arenaceodentata,

Nereis diversicolor,Nereis virens,Laeonereis acuta,Arenicola marina,

and Capitella teleta (Forbes, Andreassen & Christensen, 2001;

Christensen, Banta & Andersen, 2002). They are used for

both acute and chronic toxicity tests (Reish & Gerlinger,

1997) and to evaluate the effects of new and emerging

pollutants on reproductive, larval development, and beha-

vioural endpoints (Lewis & Watson, 2012). Polychaetes

can accumulate harmful organic compounds in their tis-

sues and are considered good indicators of bioaccumula-

tion (Caldwell et al., 2005;Janssenet al., 2010; Jørgensen

et al., 2005;Langstonet al., 2005). The possible toxic

impacts of multi-walled carbon nanotubes (MWCNTs)

(Baughman, Zakhidov & de Heer, 2002) and MPs on

polychaetes has been studied, revealing neurotoxicity, activa-

tion of antioxidant defences, and alterations in energy-related

biochemical processes (Du et al., 2013;Hidalgo-Ruzet al.,

2012;Andrady,2015). Exposure to MWCNTs and MPs

reduces the regenerative capacity of some polychaete species

(Leung & Chan, 2018). Despite their ability to accumulate

harmful materials, some polychaete species are considered

resistant to pollutants due to their positive responses to organic

enrichment. This results in the proliferation of numerous

opportunistic polychaete species and an increase in their abun-

dance after pollution events. By contrast, amphipods are more

sensitive tostress and have a higher death rate. To track signif-

icant alterations in benthic ecosystems caused by oil spills,

urban sewage outfalls, and organic matter enrichment, 23

countries use P/A ratio-based indices such as benthic opportu-

nistic polychaetes amphipods index (BOPA) and benthic

opportunistic annelida amphipods index (BO2A) in estuarine

and coastal environments (Dauvin, 2018).

Nearly 1100 species of freshwater oligochaetes exist

(Martin et al., 2008). About half of these are widely distributed

worldwide (Timm & Martin, 2015) while the rest have limited

distributions (Wang & Liang, 1997; Timm & Martin, 2015;

Verdonschot, 2015). Oligochaetes are commonly used in eco-

toxicological studies due to their abundance, cosmopoli-

tanism, and ability to accumulate toxic substances such as

metals (Helling, Reinecke & Reinecke, 2000; Corbi, Guil-

herme & Regiane, 2015; Gomes et al., 2017). The most com-

mon species used in toxicological studies are Pristina leidyi,

Branchiura sowerbyi,Lumbriculus variegatus,Tubifex tubifex, and

Allonais inaequalis (Lobo et al., 2016; Hurley, Jamie &

James, 2017; Neto et al., 2019, Felipe et al., 2020). L. variegatus,

T. tubifex and A. inaequalis are the most widely used species for

evaluating toxic effects caused by MPs and MWCNTs

(Castro et al., 2020; Scopetani et al., 2020; Silva et al., 2021).

Transmission electron microscopy showed that MWCNTs

do not penetrate cell membranes, and that toxicity caused

by ingesting them might result from metals solubilised from

the nanotubes (Mwangi et al., 2012). Ingestion of MPs did

not appear to affect survival and reproductive rate, but

this does not rule out the possibility of risks to biodiversity

(Koelmans et al., 2014; Barboza & Gimenez, 2015).

Leeches (Hirudinea) are a group of annelids with a variety

of life-history strategies and reproductive behaviours that are

primarily found in freshwater habitats, but also occur in

other habitats (Apakupakul, Siddall & Burreson, 1999; Sket

& Trontelj, 2008). H. medicinalis and H. verbana (Fig. 3F) are

perhaps the best-known species (Trontelj, Sotler & Verovnik,

2004;Trontelj&Utevsky,2005)andareusedinmedicineand

research due to their ease of maintenance and well-charac-

terised physiology and behaviour (Sawyer, 1986;Grimaldi,

Tettamanti & de Eguileor, 2018;Baranziniet al., 2020). Studies

using the alkaline comet test on H. verbana haemocytes found

that exposure to MWCNTs causes stress and potential risks to

public health (Tice et al., 2000; Mihaljevicet al., 2009). In vitro

treatments of leech phagocyte cultures with MWCNTs

decreased cell proliferation, increased apoptotic events, and

10 Amalia Rosner and others

induced the production of amyloid ﬁbrils and reactive

oxygen species (ROS), which are indicators of toxicity

induced by environmental pollutants (Girardello et al.,

2015a,b,2017; Torres & Fadeel, 2013). Thus, H. medicinalis

and H. verbana are ideal species to conduct rapid in vivo and

in vitro environmental tests with innovative methods that uti-

lise molecular-based endpoints at the sub-cellular or cellular

level (Bodoet al., 2020).

(4) Nematoda

Nematodes (Fig. 3G–K) are common in aquatic habitats,

mainly inhabiting sediments as free-living organisms

(Hogue, 1982;Heip,Vincx&Vranken,1985;Decho&

Fleeger, 1988;Eckman&Thistle,1988; Hodda, 1990;

Hugot, Baujard & Morandi, 2001; Van Gaever et al.,

2006; Mokievsky, Udalov & Azovsky, 2007; Gallucci, Moens

& Fonseca, 2009). Nematode distribution depends on the

physico-chemical properties of the sediments, climate,

oxygen and food sources (Lee et al., 1977; Bell, Watzin &

Coull, 1978; Montagna et al., 1983; Decho & Fleeger, 1988;

Blanchard, 1990; Rice & Lambshead, 1994; Brown et al.,

2001; Steyaert et al., 2003; Fonseca & Soltwedel, 2007; Lee

& Riveros, 2012; Braeckman et al., 2013). In intertidal areas,

some nematode species are also sensitive to the water content of

the sediment (Jansson, 1968;Steyaertet al., 2001). Nematodes

are important in bioturbation, bioirrigation and trophic pro-

cesses, and feed on a variety of resources, from dissolved organic

matter to multicellular organisms, including other nema-

todes (Montagna, 1984; Moens, Verbeek & Vincx, 1999).

Most of them have digestive and nervous systems and are

dioecious. Despite being common, only 14% of estimated

free-living nematode species in marine habitats are currently

classiﬁed (Appeltans et al., 2012). Additional information on

nematode species is available online (Nemaplex, 2023).

Nematodes, as important benthic constituents and food

sources for many predators, play signiﬁcant roles in the tro-

phic transfer of pollution from sediments, with some species

more susceptible to environmental stressors (Hägerbäumer

et al., 2015). In consequence, nematodes are emerging as

bioindicators for freshwater (Hägerbäumer et al., 2016) and

marine pollution (Ridal & Ingels, 2021), in single-species

and community composition tests. Community composition

tests require expertise in identifying genera and species,

although this can be circumvented by using molecular tools

(Avoet al., 2017; Carta & Li, 2018; Knot et al., 2020) and

automated image recognition apparatus (e.g. FlowCAM;

Kitahashi et al., 2018), both of which have been shown to

be credible alternatives.

Most ecological research on nematodes as bioindicators in

the marine environment has focused on their responses to

heavy metals, hydrocarbons, and organic enrichment (Ridal

& Ingels, 2021). Studies have demonstrated species-speciﬁc

susceptibility to different metals and changes in the diversity

and abundance of some species in response to heavy metal

pollution (Bastami et al., 2017). Nematode genera have

shown differential tolerance to oil spills and PAHs, with some

demonstrating tolerance (e.g. Sabateria,Dorylaimopsis) and

others increased mortality (e.g. Encheliidae; Allouche et

al., 2020a). Different nematode species have also shown var-

iable susceptibility to crude oil or diesel pollution (Stark

et al., 2017; Monteiro et al., 2018). Studies of organic and/

or sewage-enriched pollution have shown inconsistent nema-

tode abundances (Bertocci et al., 2019; Sahraeian et al., 2020),

highlighting the need for additional pollution evaluation

metrics such as an index of trophic diversity based on nema-

tode trophic guild compositions (Kandratavicius et al., 2018).

Aquacultural pollution has also been found to affect nema-

tode assemblage compositions, with a shift from long-lived,

slow-growing genera to short-lived genera (Lacoste et al.,

2020). These experiments have demonstrated that some

genera are speciﬁcally tolerant to pollutants (e.g. Sabatieria

and Daptonema) while others are sensitive to pollutants

(e.g. Pomponema and Halalaimus)(Ridal&Ingels,2021).

Nematode-based biomarkers have also been used to eval-

uate the effects of MNPs, demonstrating lethality in

adults, changes in brood size and embryo number, and spe-

cies-speciﬁc effects on population growth (Lei et al., 2018;

Mueller et al., 2020). Biochemical biomarkers have also been

successfully tested on nematodes, showing changes in enzy-

matic activities following exposure to various pollutants

(Allouche et al., 2020b; Hedﬁet al., 2021).

Single-organism tests complement community-level

assessments of the impact of pollution. They allow a faster

evaluation of the impact of pollution under controlled condi-

tions, enabling the establishment of concentration–response

relationships and the identiﬁcation of pollutant mechanisms

of action. Aquatic nematodes are challenging to cultivate

and therefore only a few freshwater and marine species are

used as models for pollution assessment (Moens & Vincx,

1998). To overcome this shortage, Caenorhabditis elegans, pri-

marily a soil inhabitant (Zullini, 1988), is used as a standar-

dised model in freshwater (Carresse et al., 2021) and soil

quality control (ISO 10872:2020). C. elegans has been tested

with various pollutants, including heavy metals, PAHs,

pesticides, endocrine disruptors, bacterial toxins, and nano-

materials (Hägerbäumer et al., 2016). Litoditis marina has been

proposed as a marine model due to its sensitivity to crude oil

and diesel pollution, pesticide pollution, and sodium dodecyl

sulfate (SDS) detergent pollution. Studies have used a wide

range of parameters to measure response to pollution,

including mortality assay, LC50, fecundity, egg deposition

time, abundance of eggs, embryonic and post-embryonic

development time, and adult sex ratio (Monteiro et al.,

2018; Oliveira et al., 2020; Francolino et al., 2021).

The low costs associated with nematode sampling should

encourage their inclusion in environmental pollution assess-

ment tests. However, the main obstacles to the large-scale

use of nematodes as ecological indicators include: (i) the lack

of useful cultivation protocols for individual species; (ii) the

scarcity of literature on individual nematode species; (iii)

the low number of taxonomists able to identify species; (iv)

the paucity of metabarcoding conducted on marine and

freshwater nematodes (excluding C. elegans); and (v) the

A broad-taxa approach as an important concept 11

absence of standardised effective metrics for each pollutant

type or pollutant assembly. Some of these difﬁculties might

be surmounted by the development of automated image rec-

ognition and improved DNA barcoding analysis techniques

for the rapid identiﬁcation of species.

(5) Echinodermata

Echinoderms are marine invertebrates widely distributed

across all seas, including highly anthropogenically modiﬁed

areas. They are important representatives of many marine

ecosystems and frequently constitute the majority of the

benthic macrofauna. More than 7400 living species are

divided into ﬁve extant clades: Asteroidea (starﬁsh; Fig. 4A),

Holothuroidea (sea cucumbers; Fig. 4B), Echinoidea (sea

urchins and sand dollars; Fig. 4C), Crinoidea (sea lilies and

feather stars; Fig. 4F), and Ophiuroidea (brittle stars and bas-

ket stars; Fig. 4H). Feeding strategies depend on taxon and

species and can include ﬁlter-feeding, carnivory, scavenging,

grazing, and omnivory. Crinoids are generally ﬁlter-feeders;

ophiuroids are carnivores, scavengers, and ﬁlter-feeders;

asteroids are predators; echinoids are grazers and omnivores;

and holothurians are scavengers and ﬁlter-feeders. Members

of this phylum share some features including pentaradial

symmetry in the adult stage, a water vascular system, a cal-

cium carbonate dermaskeleton, mutable connective tissue

and remarkable regeneration capabilities that can also be

used in reproduction (Wessel, 2018). In general, echinoderms

(excluding crinoids) produce feeding larvae (Fig. 4D), while

some asteroids, echinoids, and ophiuroids are direct devel-

opers (McEdward & Miner, 2001). They live in close contact

with substrates and sediments, and possess highly developed

and permeable external epithelia, normally used for respira-

tion and excretion. Due to these latter features, they are easily

exposed to various environmental pollutants (Sugni et al.,

2007). As deuterostomes, they share a rather close phylogenetic

relationship with vertebrates, suggesting the existence of shared

mechanisms of responses to environmental contamination.

Both sea urchins (Echinoidea) and sea cucumbers

(Holothuroidea) have high commercial value and are exten-

sively exploited (Kelly, 2005; Cirino et al., 2017;Huet al.,

2021; Rubilar & Cardozo, 2021). Sea urchins are considered

ideal models for marine (eco)toxicological tests (Goldstone et

al., 2006; Gharred et al., 2016) as their embryos can sense

adverse effects related to a wide range of environmental

stressors, including metals and plastics (Pinsino et al., 2017;

Messinetti et al., 2018; Morroni et al., 2018; Bergami et al.,

2019; Oliviero et al., 2019), polluted sediments and ocean

acidiﬁcation (Pagano et al., 2017; Dorey et al., 2018;

Bonaventura et al., 2021; Caetano, Pereira & Envangelista, 2021).

Accordingly, assessing the embryonic and larval development

of sea urchins under stress conditions is useful in monitoring

and risk assessment programs (EPA, 2002;Environment

Canada, 2011;Sartoriet al., 2017;ASTM,2021). Chronic

or sub-chronic short-term embryotoxicity tests for the

analysis of environmental quality have been used since

the 1960s, due to the accessibility and reliability of sea

urchin embryos and the speed with which these tests can

be conducted. The guidelines cover procedures for per-

forming short-term (48–96 h) laboratory assays on sea

urchin embryos using a variety of sea urchin species, such

as Arbacia punctulata and Strongylocentrotus droebachiensis from

the East Coast of the USA, Strongylocentrotus purpuratus,

S. droebachiensis,andDendraster excentricus from the West

Coast of the USA, and Paracentrotus lividus (Fig. 4C)from

the Mediterranean. The ASTM (2021) guide also includes

procedures for the use of other echinoids (e.g. sand dollars).

Modiﬁcations to the standard procedures can be applied.

For example, the sea urchin embryotoxicity test, considered

to be a valuable tool for assessing the quality of sediments in

harbour areas, may result in ﬂawed outcomes when based

exclusively on normal versus abnormal embryos. To overcome

this risk, several Integrative Toxicity Indices (ITIs) have been

developed and validated (Morroni et al., 2016; Bonaventura

et al., 2021). Among these, the ITI 4.0 index discriminates

strictly between developmental delay and morphological

defects from fertilised eggs to the gastrula stage (Bonaventura

et al., 2021). Notably, some sea urchin species are suffering

from increasing anthropogenic pressures in coastal environ-

ments, including intensive exploitation. As a result, the popu-

lations of species such as the European sea urchin P. lividus

(distributed from the northeastern Atlantic to the coasts of

the Mediterranean Sea) have recently collapsed, and these

species are becoming extremely rare in some regions

(Yeruham et al., 2015). In addition, the depletion of reproduc-

tive adults renders the procurement of an acceptable number

of wild mature animals, able to release gametes of good qual-

ity, increasingly difﬁcult (Cirino et al., 2017). However, in

recent years, new research opportunities have emerged, such as

the use of the sea urchin for environmental immunotoxicology,

and a successful culture of P. lividus adult immune cells has

been established (Pinsino & Alijagic, 2019). This may provide

the basis for the design of new approaches for monitoring the

quality of the environment and estimating the hazards repre-

sented by test materials. Besides sea urchin embryos, larvae

(Fig. 4D) and cultured immune cells (Fig. 4E), adult sea

urchins (e.g. P. lividus) have also been used successfully in eco-

toxicological testing and environmental monitoring studies,

thus further highlighting the suitability of these organisms

as diversiﬁed model species for this type of research (Sugni

et al., 2007; Rouane-Hacene et al., 2018; Parolini et al., 2020).

Other non-conventional echinoderms have been successfully

used in research, particularly in assays focusing on regeneration

as an alternative and complementary perspective to embryo-

toxicity tests (Sugni et al., 2007). Regeneration is an intrinsic

part of the life history of echinoderms. Alterations to regener-

ation can strongly affect animal performance, including sur-

vival and ﬁtness, and thus, potentially, have an immediate

impact on the entire population. Among the different model

species, the crinoid Antedon mediterranea (Fig. 4F) is a good can-

didate for environmental testing and monitoring. Arm regen-

eration (Fig. 4G) is an extremely sensitive endpoint as it is

impacted by exposure to endocrine disruptors in environmen-

tally relevant quantities, which lead to alterations in skeletogenic

12 Amalia Rosner and others

Fig. 4. Representative echinoderm experimental models used in ecotoxicological research. (A) The starﬁsh Asterias rubens; scale

bar =2 cm. (B) The sea cucumber Holothuria tubulosa on a muddy substrate; scale bar =2.5 cm. (C) The sea-urchin Paracentrotus

lividus feeding on Posidonia oceanica; scale bar =1 cm. (D) A pluteus larvae of P. lividus; scale bar =25 μm. (E) Culture of P. lividus

immune cells; scale bar =30 μm. (F) The crinoid Antedon mediterranea; scale bar =2 cm. (G) Arm regeneration (arrow) of

A. mediterranea 2 weeks post-amputation; scale bar =1 mm. (H) The brittle star Ophiactis virens undergoing regeneration (arrow)

after asexual reproduction (ﬁssion); scale bar =1.5 mm.

A broad-taxa approach as an important concept 13

processes, cell recruitment pathways, and proliferation

(Candia Carnevali et al., 2001; Sugni et al., 2007,2008,2010).

Brittle stars can also serve as optimal marine sentinels for envi-

ronmental monitoring. Burrowing and deposit/ﬁlter feeder

species such as Amphiura spp., Microphiopholis gracillima,orthecos-

mopolitan Amphipholis squamata, can easily accumulate contami-

nants (D’Andrea, Stancyk & Chandler, 1996; Gunnarsson &

Sköld, 1999; Barboza, Martins & Lana, 2015) and eventually

display altered physiology, for example, a reduction in natural

bioluminescence (Deheyn, Jangoux & Warnau, 2000). Notably,

evidence of ingestion of plastic materials by brittle stars was ﬁrst

reported over 40 years ago (Courtene-Jones et al., 2019). Similar

to A. mediterranea, arm regeneration in ophiuroids (Fig. 4H)isa

valuable property in the assessment of environmental contami-

nation: in the epibenthic carnivorous Ophioderma brevispina,

the organotin compounds bis(tri-n-butyltin)oxide (TBT) and

bis(triphenyltin)oxide (TPT) reduced arm regrowth at concen-

trations as low as 0.1 μg/L, possibly as a result of neurotoxic

effects (Walsh et al., 1986). Lastly, sea cucumbers (holothuroids)

have emerged as bioindicators of environmental contamination

(Marrugo-Negrete et al., 2021). These benthic scavengers and

burrowing organisms represent excellent tools for monitoring

contaminants accumulated in sediments. Furthermore, their

non-selective feeding strategy causes the ingestion of any avail-

able item on the substratum, thus leading to a diffuse presence

of particles of anthropogenic origin in their digestive tracts

(e.g. MPs; Iwalaye, Moodley & Roberston-Andersson, 2020).

(6) Standardised models –overview

Very few species among the above-mentioned taxa have

been used for (eco)toxicological tests. However, many of the

species used in standardised protocols possess exceptional

characteristics such as high sensitivity to various chemicals,

ubiquitous presence in aquatic ecosystems, ease of laboratory

maintenance, and the capacity to measure numerous physio-

logical parameters. These features drive their exploitation in

the ﬁeld of (eco)toxicology. In recent years, new species have

been used to provide additional data about the ecotoxicity of

pollutants in aquatic ecosystems. Additionally, efforts are

being made to develop alternative in vitro and in silico models.

The development of in vitro tools such as primary cell cultures

and cell lines from various organs allows the use of cell cul-

tures as models to analyse the underlying mechanisms of tox-

icity. From this perspective, organisms that are not currently

used as standard models are of great interest for the develop-

ment of new tools.

IV. NON-STANDARD MODELS

(1) Porifera

Sponges (Porifera) are cosmopolitan sessile organisms

distributed in fresh and marine waters across the globe and

are key invertebrates in marine benthic ecosystems (Fig. 5A, B).

TheyareamongsttheoldestofthemodernMetazoa,

located at the basal position of the phylogenetic tree

(Wörheide et al., 2012; Redmond & McLysaght, 2021).

Phylum Porifera is divided into four clades: (i)Calcarea

(calcareous sponges with calcium carbonate skeletal spic-

ules); (ii) Demospongiae (demosponges, mostly with

organic or silicon dioxide skeletons; a few species without

skeletons), the largest group containing more than 76%

of all living sponge species; (iii) Homoscleromorpha (the

only class of sponges with a true basal lamina); and (iv)

Hexactinellida (glass sponges, with syncytial structure

and silicon dioxide spicules; not used as bioindicators

due to their rarity). Porifera are diblastic organisms with-

out organs, and many of their cells are capable of de-

and trans-differentiation into other cell types. The number

of cell types in sponges can exceed a dozen, and the diver-

sity of morphogenesis during development is comparable

to that of bilaterians (Simpson, 1984;Ereskovsky,2010).

The outer layer of the sponge, composed of exo-pinaco-

cytes, is pierced by the channels of the aquiferous system.

Theaquiferoussystemconsistsofchannelsandchambers,

the latter lined by choanocytes, which are ﬂagellate cells.

The beating of choanocyte ﬂagella produces a ﬂow of

water through the aquiferous system, and the choanocytes

remove food particles from the water for consumption.

The space between the outer layer (exo-pinacocytes) and

the wall of the aquiferous system (a layer of choanocytes

and endopinacocytes) is ﬁlled with mesohyl,

an extracellular matrix containing populations of resident

cells, e.g. amoebocytes/archaeocytes, sclerocytes, lopho-

cytes, and spherulous cells (Simpson, 1984). Sponges con-

tribute greatly to ecosystem functioning as bio-ﬁltrators:

they ﬁlter more than 900 times the volume of their body

perhour(Ludeman,Reidenbach&Leys,2017), and effec-

tively trap particles smaller than 10 μminsize(Pile,Pat-

terson & Witman, 1996;Comaet al., 2001;Yahelet

al., 2007). They receive dissolved organic material from

the water column and make it available to higher trophic

levels as detritus (de Goeij et al., 2013;Rixet al., 2016,

2017,2018). Sponges are rich in symbiotic bacteria, which

contribute to the overall recycling of organic material, poly-

phosphate production and the storage and supply of fresh

photosynthate (Taylor et al., 2007;Colman,2015). Addi-

tionally, sponges are critical components of ecosystems,

providing habitats for other animals and food sources for

other species. As is the case for other ﬁlter-feeders, sponge

cells exhibit physiological responses to pollutants present

in the water.

Roveta et al.(

2021) reviewed 50 biomonitoring studies per-

formed on sponges. Most of the work relied on the ability of

sponges to accumulate chemical elements from the water in

their tissues. Heavy metal content of sponges collected from

contaminated sites differed signiﬁcantly from that in sponges

collected from uncontaminated locations (Patel, Balani &

Patel, 1985; Hansen, Weeks & Depledge, 1995; Araújo et

al., 2003; Perez et al., 2005; Rao et al., 2006; Cebrian, Uriz

& Turon, 2007; Batista et al., 2014). Demosponges with silica

spicules are of particular interest, because during spiculogenesis,

14 Amalia Rosner and others

Fig. 5. Non-standard models. (A, B) Sponges. (A) Halichondria panicea. (B) Aplysina cavernicola.(C–F) Cnidarians. (C) Stylophora pistillata.

(D) Pocillopora damicornis. (E) Nubbins (small fragments) of Stylophora pistillata portraying horizontal spread on the substrate. (F) Coral

nursery. (G) Nematostella vectensis.(H–K) Flatworms. (H) Schmidtea mediterranea. (I) Dugesia japonica. (J) Dugesia tigrine. (K) Macrostomum

lignano.

A broad-taxa approach as an important concept 15

the sclerocytes accumulate not only silicon but also heavy metals

in their spicules (Truzzi et al., 2008; Annibaldi et

al., 2011; Illuminati et al., 2016). Other physiological changes

that may be observed following exposure to sublethal

concentrations of pollutants include changes in sponge cell

behaviour (Cebrian et al., 2003,2006), metabolism (Saby et

al., 2009; Selvin et al., 2009), production of ROS, expression

of stress genes (Müller et al., 1994,1995,1996) and increased

apoptosis (Batel et al., 1993). For example, in the case of

induction of genotoxicity by exposure to heavy metals

(Akpiri, Konya & Hodges, 2017), changes in the expression

of metallothionein (Berthet et al., 2005), post-translational

modiﬁcation of tubulin (Ledda et al., 2013), and expression

of apoptosis-associated prosurvival factor (Luthringer et al.,

2011) have been reported. Other biomarkers currently used

in sponges include the altered expression or activity of heat

shock proteins (HSPs) (e.g. the effect of copper on Crambe

crambe; Agell et al., 2009), glutathione (GSH) and glutathione

S-transferases (GST) (e.g. the effects of industrial, urban, and

harbour pollution on Sarcotragus spinosulus; Khati et al., 2018).

These lines of evidence support the use of sponges in retroac-

tive assessments of water pollution (i.e. they can be viewed as

providing an ‘ecotoxicological memory’), particularly in

long-living species. Suggested models for toxicological tests

include ubiquitous species of Demospongiae like Halichondria

panicea (Fig. 5A), which is well studied in many respects, Aply-

sina cavernicola (Fig. 5B), and Spongia ofﬁcinalis, which is found in

the Mediterranean Sea, a focal region for aquatic pollution

studies.

At the cellular level, the states of cells like choanocytes,

which are in direct contact with the ﬁltered water, can be

used as biomarkers. Along with archaeocytes, which are

amoebocytes residing in the mesohyl (Funayama, 2013), cho-

anocytes are part of the stem cell system of sponges and

undergo constant renewal due to high rates of proliferation

and the shedding of old cells into the lumen of choanocyte

chambers (De Goeij et al., 2009). Under constant stress

conditions, the pool of both stem cells in general, and cho-

anocytes in particular, can change (e.g. in cell distribution, cell

cycle phases, population size); this trait can be easily assessed

by ﬂow cytometry, as described for Suberites domuncula and

Haliclona oculata (Sipkema et al., 2004;Schipperset al., 2011).

In addition, the sexual reproduction, asexual reproduction

(by budding, gemmule formation) and regeneration (the

development of a new animal from a body fragment and

whole-body regeneration from cell aggregates) of sponges

could be of interest in the assessment of environmental stress

factors. The sexual reproduction of sponges leads to the for-

mation of free-ﬂoating larvae that, following settling,

undergo metamorphosis into juvenile sponges (Ueda et

al., 2016). Exposure of the larvae of C. crambe and Scopalina

lophyropoda to heavy metals and PAHs inhibits their settling.

Copper ions act synergistically with PAHs, although

treatment with Cu

alone does not inhibit larval settling

(Cebrian & Uriz, 2007a). The model of larval settlement

and metamorphosis is potentially attractive and may be more

sensitive than the model for adult sponges, but the molecular

mechanisms that enable competence acquisition and meta-

morphosis are poorly understood (Conaco et al., 2012).

Additionally, the extensive asexual reproduction and regen-

eration capabilities of sponges, even from adult body frag-

ments (Baldacconi et al., 2010; Çelik et al., 2011), has

enabled the production of clonal animals providing repro-

ducible responses to contamination (Osinga, Tramper &

Wijffels, 1999; Schippers et al., 2012) that can be used for pol-

lution monitoring. Furthermore, primmorphs, artiﬁcial

models generated from sponges belonging to the taxa

Demospongiae, Calcarea, and Homoscleromorpha (Lavrov

& Kosevich, 2014; Akpiri, Konya & Hodges, 2020), also have

potential as in vitro tools for toxicological tests. Primmorphs

are obtained from sponge tissues dissociated into cells by

mechanical or chemical methods. These cells form aggre-

gates and dedifferentiates, and the outer layer of the aggre-

gated cells forms a covering layer, resulting in the formation

of a radially symmetrical primmorph. The cells within the

primmorph then differentiate and small fragments that

attach to the substrate form new sponges by extending their

structures (Lavrov & Kosevich, 2014,2016; Ereskovsky

et al., 2021). Differentially expressed genes, and some of

the mechanisms underlying the dissociation–reaggregation

phenomena in the sponge Oscarella lobularis have been

revealed (Vernale et al., 2021). Heavy metals inﬂuence the

behaviour of cells in the reaggregation process (Cebrian &

Uriz, 2007b,c). Further studies of the effects of pollutants

on the aggregation and development of primmorphs will

allow the creation of effective and suitable methods for eval-

uating biological reactions to pollution. Additional break-

throughs in terms of in vitro tools include the development

of cell cultures. Primary cultures of various species

have been initiated (Rinkevich, Blisko & Ilan, 1998a;

Pomponi, 2006; Urban-Gedamke et al., 2021) and devel-

oping cells have been cryopreserved (Munroe et al., 2018),

but many attempts to obtain cell lines have failed (Grasela

et al., 2012). Conkling et al.(2019) reported rapid cell division

in primary cultures established from nine sponge species, dem-

onstrating a cultured cell life span of 21–35 days. Such

improvements may open additional avenues for the broad-

scale use of sponge cell cultures in in vitro pollution assessment.

(2) Cnidaria

Cnidarians, especially corals (Fig. 5C, D), have been successfully

used in ecotoxicological experiments (Fig. 1;Rovetaet al., 2021)

and their potential for applications in biomonitoring has

been repeatedly highlighted (Rainbow, 2002). This potential

is supported by the very simple tubular body morphology of

cnidarians, which are diploblastic organisms consisting

of two tissue layers, the outer epidermis and the inner

gastrodermis, which lines the gastrovascular cavity. These

layers are separated by an extracellular matrix, the mesoglea,

containing some cells in Scyphozoa and Anthozoa. Both the

epidermal and gastrodermal layers are in constant contact

with the environment. Cnidarians can reproduce both

sexually and asexually. Asexual reproduction, which

16 Amalia Rosner and others

characterises many cnidarian species, ensures a supply of

high numbers of genotypic replicates with low variations in

physiological/biochemical parameters, which is an impor-

tant asset in performing reliable tests (Shaﬁr, Van Rijn & Rin-

kevich, 2001,2003,2006). Other advantages of cnidarians for

ecotoxicological testing include their wide distribution in almost

all freshwater, brackish and marine ecosystems, their pres-

ence in both temperate and tropical zones (Howe, Reichelt-

Brushett & Clark, 2012), and the number of cnidarian species

that deposit hard skeletons. However, there is a relative pau-

city of ecotoxicological studies exploiting cnidarians, as

shown in studies using the comet assay to examine aquatic

genotoxicity (Svanfeldt et al., 2014). Only seven cnidarian

species were included in a recent review on the comet assay

in model invertebrates and unicellular organisms (Gajski et

al., 2019), which covered almost 300 publications and hun-

dreds of species: the hydrozoan Hydra magnipapillata, the sea

anemones Anthopleura elegantissima,Actinia equina and Bunodo-

soma cangicum, and the corals Stylophora pistillata (Fig. 5C), Ser-

iatopora hystrix and Montastraea franksi. Testing on cnidarians

typically involves a small number of model animals for which

laboratory husbandry is available, with difﬁculties emerging

when animals collected from the wild are used, as these pro-

cedures are inherently destructive (Summer, Riechelt-Brush-

ett & Howe, 2019).

Hydra is the most widely used hydrozoan genus in monitoring

and ecotoxicological studies. Hydra polyps are effective for tox-

icity testing since they are easily adapted for mass culture in

the laboratory (Loomis, 1953), forming large numbers of

individuals that normally reproduce asexually by budding.

Hydra is a reliable indicator of pollution due to its single polyp

structure, its basic anatomy, and the possibility to use genet-

ically identical or similar individuals in the same or separate

tests (Beach & Pascoe, 1998): this ensures reproducible results

with a low coefﬁcient of variation (Quinn, Gagné & Blaise,

2012). The Hydra genome has been completely sequenced

and the full range of next-generation tools is available

(Chapman et al., 2010). During the last three decades, several

Hydra species [primarily H. attenuata (=H. vulgaris) and H.

magnipapillata] have been used in toxicology studies with

numerous compounds, including acute, sub-chronic, chronic,

reproductive, developmental, carcinogenic, and genetic toxins

(Quinn et al., 2012; Patwardhan & Ghaskadbi, 2013;Zeeshan

et al., 2017; Murugadas et al., 2019;Ceraet al., 2020). Many of

these studies used the Hydra assay (Johnson & Gabel, 1992). In

comparison, ecotoxicological research on jellyﬁsh species is

sparse (Faimali et al., 2014;Ohderaet al., 2018). While gener-

ally considered to be robust towards anthropogenic stressors

(e.g. Richardson et al., 2009), it has long been known that jelly-

ﬁsh accumulate toxic metals from the environment in high

concentrations and are therefore a potential source of metal

transfer in food chains (Romeo & Gnassia-Barelli, 1992)and

may themselves be sensitive to environmental toxicity. The

size of medusae can be a limitation in studies employing a

large number of specimens, although ecotoxicological assays

on ephyras have been carried out successfully, primarily on

the common species Aurelia aurita (e.g. Faimali et al., 2014;

Costa et al., 2015; Olguín-Jacobson et al., 2020), a proposed

model organism in ecotoxicology due to its high sensitivity to

pollutants (Olguín-Jacobson et al., 2020). In addition, some

recent studies have explored the impact of chemicals on the

sessile stage, the polyps (Olguín-Jacobson et al., 2020;Pinteus

et al., 2020), a protocol that has simpliﬁed the use of scypho-

zoan species in ecotoxicological assays.

Members of the Anthozoa (including sea anemones and

corals) are unique tools for ecotoxicological studies since they

may live for prolonged periods of hundreds of years, accumu-

lating an ecotoxicological memory, and some, like scleracti-

nian corals, may assimilate environmental records into their

skeletons (Shah, 2021). A search of Web of Science identiﬁed

3045 papers published in the last decade that contained the

key words ‘coral +(ecotoxicology or toxicology or pollu-

tion)’. One of the most diversiﬁed ecosystems on Earth is

found in coral reefs that provide numerous ecosystem ser-

vices, including a means of living for hundreds of millions

of people (UN Environment Programme, 2023). Their mon-

itoring requires the development of a suite of sensitive,

standardised ecotoxicological tests for assessing short- and

long-term anthropogenic impacts on corals (Branton, 2018).

However, coral ecotoxicology at the laboratory level (Fig. 5E)is

methodologically restricted, as coral husbandry is not easy. This

is primarily due to the numbers of organisms required for

adequate replication, as collecting corals from the ﬁeld is

inherently destructive (Vijayavel & Richmond, 2012). To

overcome this difﬁculty, stocks of coral colonies (with varying

numbers of coral genotypes for each species) can be devel-

oped and maintained in underwater coral nurseries

(Fig. 5F;Levyet al., 2010; Rinkevich, 2015,2021; de la Cruz

et al., 2015), an approach with new market opportunities

(Rinkevich, 2015). Numerous healthy ramets can be pro-

duced from each coral genet, opening new avenues for eco-

toxicological research.

A range of laboratory-based ecotoxicological assays have

been developed for corals, including histopathology, produc-

tivity levels, calciﬁcation rates, reproductive effort, and state

of symbionts (i.e. bleaching events and/or photosynthetic

activity), yet there is a need for the development of more

deﬁned bioassays (Branton, 2018). One such assay is the nub-

bins assay (Shaﬁret al., 2001,2003,2006; Fig. 5E), based on

the repeated use of numerous very small fragments from

selected coral genotypes. This assay provides a cheap, stan-

dardised and low-variation approach, and limits the ecologi-

cal impact of harvesting large numbers of corals for research.

This protocol has been employed to evaluate the toxicity of

organic carbon, heavy metals, household detergents, MPs,

oil and oil dispersants, ultraviolet (UV) ﬁlters and more to

reef corals (Ferrier-Pagès et al., 2005; Kuntz et al., 2005;

Shaﬁr, Van Rijn & Rinkevich, 2007;Shaﬁr, Halperin &

Rinkevich, 2014;Chenet al., 2012; Vijayavel & Richmond,

2012;Svanfeldtet al., 2014; Corinaldesi et al., 2018;He

et al., 2019; Aminot et al., 2020;Mitchelmoreet al., 2021;Xiao

et al., 2021). In addition, toxicity tests conducted on ﬁeld-

collected coral material, including planulae, gametes, and

fragments, have provided important results (reviewed in

A broad-taxa approach as an important concept 17

Howe et al., 2012;Loya&Rinkevich,1980) and additi-

onal methodologies, like vital staining (Shefy, Shashar &

Rinkevich, 2021), have recently been added.

Sea anemones are also good candidates for routine

ecotoxicological tests. The absence of a calcium carbonate skele-

ton is a major feature of scleractinians, and the availability of

symbiotic and aposymbiotic sea anemones has been noted

(Howe et al., 2012). Sea anemones can be easily maintained in

laboratory settings and in large cultures, and research has shown

that they are susceptible to a variety of pollutants (Anjos et

al., 2017;Howeet al., 2012,2017;Trenﬁeld et al., 2017; Ianna

et al., 2020;Vitaleet al., 2020;Rosneret al., 2023). The sea anem-

ones Aiptasia pulchella (Exaiptasia pallida), Anemonia viridis and A.

equina (Howe et al., 2012;Trenﬁeld et al., 2017;Vitaleet

al., 2020) have been suggested as the best model taxa for

research. Following the genomic sequencing of Nematostella vecten-

sis (Fig. 5G), ecotoxicology studies have revealed transcriptome

responses to stress and pollutants (Goldstone, 2008;Elranet

al., 2014;Tinoet al., 2014;Tarrantet al., 2018). The use of in vitro

approaches (Rabinowitz, Moiseeva & Rinkevich, 2016)mayfur-

ther enhance ecotoxicological tests on cells derived from this sea

anemone and other related species. Examples of toxicological

effects on N. vectensis are shown in Fig. 6, including impacts

on development and regeneration (Klein et al., 2021).

(3) Platyhelminthes

Platyhelminthes, commonly known as ﬂatworms, are a

phylum of bilaterian acoelomate organisms. They contain

distinct brain, digestive, and excretory systems, but lack

circulatory, skeletal, or respiratory organs (Hyman, 1951).

Platyhelminthes can be free-living, commensal, or parasitic.

The phylum is subdivided into several clades, including

Tricladida. This taxon includes both marine and freshwater

species, some of which have been utilised in ecotoxicology over

the last decade. The wide range of environments in which they

live makes them ideal models to test ecosystem health. Among

the Platyhelminthes, the term ‘planarian’is used to describe

the free-living ﬂatworms belonging to Tricladida, which are

the best studied models from this phylum. These animals

reproduce through sexual and asexual (animal ﬁssion) strate-

gies (Vila-Farré & Rink, 2018). The ecosystem functions of

ﬂatworms have recently been reviewed (Majdi, Kreuzinger-

Janik & Traunspurger, 2016; Vila-Farré & Rink, 2018)and

we refer the reader to these reviews for an extended discussion.

Many species of planarians have an immense capacity for

regeneration, with small pieces of a single animal being able

to generate a multitude of progeny (Ivankovic et al., 2019).

Their regeneration capacity depends on the presence of

ASCs called neoblasts, which can represent >30% of cells

in a single adult individual (Rink, 2013). Several well-studied

models are available, including Schmidtea mediterranea (Fig. 5H).

A complete genome, various transcriptomes and a culture of

neoblasts are available for this species (Grohme et al., 2018;

Wu & Li, 2018;Leiet al., 2023). Its ability to regenerate in

the presence of various chemical compounds has been used in

ecotoxicology. Studies have also considered behavioural

endpoints, such as mobility (Deochand, Costello & Deoc-

hand, 2018; Pestana & Ofoegbu, 2021). In all experiments,

a culturing methodology, following stringent standardised

protocols, has been indispensable to the screening process

(e.g. Wu & Li, 2018). This is rarely the case for other species.

In the absence of standardisation, the effects of speciﬁc che-

micals are (and must be) evaluated in different populations,

as populations from different geographical areas can show

differences in sensitivity (Indeherberg, Van Straalen &

Schockaert, 1999). More economical and efﬁcient technol-

ogies have been developed recently to analyse the effects of

molecules on the development and behaviour of model

systems. Such technologies can be applied to a variety of

planarians, particularly Dugesia japonica (Fig. 5I), S. mediterranea,

and Girardia tigrina. These methods allow cross-species compar-

isons, indicating the best species to be utilised for each chemi-

cal test or the optimal physical attributes to be studied

(Hagstrom et al., 2015; Hagstrom, Cochet-Escartin & Collins, 2016;

Ireland et al., 2020). Interestingly, some studies have found

that, under certain conditions, planarians such as D. japonica

possess comparable sensitivity to established models used in

mammalian toxicology (Hagstrom et al., 2019).

Most ecotoxicological studies use the morphology and

behaviour of planarians as their endpoints due to the well-

characterised morphology of many species and the availability

of tests that allow the tracking of a few well-known

behavioural traits. These include locomotion, thermotaxis,

negative phototaxis, and the capacity to ‘scrunch’(i.e. display

a muscle-driven oscillatory escape gait) in reaction to toxic

heat (Cochet-Escartin, Mickolajczk & Collins, 2015). The neu-

ropharmacological effects of many chemicals have been

assessed using planarians (Pagan, Rowlands & Urban, 2006;

Yuan, Zhao & Zhang, 2012; Stevens et al., 2015). Such studies

focused on speciﬁc behavioural patterns following exposure to

drugs that act on neural transmission (e.g. Buttarelli, Pellicano

&Pontieri,2008;Rawlset al., 2008; Sacavage et al., 2008;

Dziedowiec et al., 2018) and the effects of different drugs on

the natural behaviour and regeneration of planarian species.

For example, compounds such as carbamazepine, used to treat

epilepsy, or ﬂuoxetine, used to treat depression, have revealed

effects on nutrition and reproduction of S. mediterranea

(Ofoegbu et al., 2019). Compounds used for the treatment of

Alzheimer’s disease (e.g. donepezil, tacrine, galantamine, and

rivastigmine) were studied in the related species, Dugesia tigrina

(Fig. 5J; Bezerra da Silva et al., 2016). The behaviour of planar-

ians can now be tracked on a large scale using emerging new

technologies such as automatic tracking systems (Zhang et

al., 2019a). Such platforms enable the systematic analysis of

large numbers of compounds and the tracking of complex sets

of parameters. Using large-scale screening platforms also

allows the testing of multi-target mixtures with therapeutic

potential that are derived from natural marine sources (e.g.

Henry & Wlodkowic, 2019; Zhang et al., 2019a).

Although the potential for automatisation is clear, most

toxicological research on planarians has focused on a

small number of substances and employed more tradi-

tional methodologies. These approaches have been used

18 Amalia Rosner and others

to assess the impact of insecticides, pesticides, metals, and

neurotoxic compounds (Simao et al., 2020;Dornelas

et al., 2021;Silvaet al., 2022).

Few studies have tracked the effects of chemicals on the

molecular and cellular composition of ﬂatworms (Plusquin

et al., 2016). These studies rely on ﬂatworms because

Fig. 6. Toxicological tests applied to Nematostella vectensis as a model organism. (A) Gene expression changes in mature N. vectensis after

4 days exposure to polluted sea water collected from Haifa port and Herzliya marina. Relative gene expression comparisons were

made using quantitative polymerase chain reaction (qPCR) between animals exposed to polluted sea water diluted with double-

distilled water (DDW; N=6 for Haifa and N=6 for Herzliya) in a 1:2 ratio, and animals grown in clean sea water samples from

Tel Shikmona beach, also diluted with DDW in a 1:2 ratio (N=6). The expression changes are presented as fold changes on the

log

scale. The tested genes represent N. vectensis orthologues of well-known oil pollution biomarkers retrieved from the N. vectensis

genome database (Putnam et al., 2007). Transcript id is speciﬁed in parentheses; Asterisks are used to distinguish between

paralogous genes. CCS, copper chaperone for superoxide dismutase (227361); GPX, glutathione peroxidase (90698); GST,

glutathione S-transferase (GST*113255; GST** 86756); SOD, superoxide dismutase (SOD*94316; SOD** 234825; SOD***

165732). Error bars represent standard deviations (SD). (B–E) N. vectensis embryos following electrophoresis with pTimer-1 Vector

(Takara) under the control of the CCS gene promoter and exposed to the accommodated fraction of a medium containing 30 ppm

crude oil. The time following electroporation is indicated. The embryos were alive at the time the photographs were taken.

A broad-taxa approach as an important concept 19

they are particularly well suited for investigating how

environmental stressors regulate stem cells since it is simple

to observe stem cell dynamics (Stevens et al., 2018). Plusquin

et al.(

2016) analysed the neoblast dynamics of the ﬂatworm

Macrostomum lignano (Fig. 5K) in the presence of environmen-

tally relevant metals, using a combination of morphological,

gene expression and immunochemical methodologies, allow-

ing for detailed characterisation of the effects of metals on the

proliferation of stem cells. In a similar study, the exposure of

S. mediterranea neoblasts to the carcinogen methyl methanesul-

fonate (MMS), resulted in a reduction in neoblast prolifera-

tion rate, which was attributed to induction of DNA

damage (Plusquin et al., 2012).

In addition to existing studies on the general effects of

pollutants on behaviour and regeneration, other studies have

examined their speciﬁc effects on cellular components, such

as DNA (e.g. chromosomal assays, nuclear DNA fragmen-

tation) or enzymatic activities (e.g. CAT or HSPs;

reviewed by Knakievicz, 2014). Environmental effects on

the population level were tested by tracing reproductive

ﬁtness, such as the number of offspring or changes in sex

ratios (Miyashita et al., 2011)whileanimpactonthemicro-

biota was demonstated by Bijnens et al.(

2021)onS.

mediterranea.

(4) Tunicata

Tunicates are ﬁlter-feeding marine invertebrates that are

found in oceans and seas with salinities over 2.5%. They

are classiﬁed as a sister group of vertebrates (Delsuc

et al., 2006,2008) and are named after their ‘tunic’, which

is the outer layer that encloses the animal body (Deck,

Hay & Revel, 1967; Welsch, 1984; Van Daele et al., 1992;

Burighel & Cloney, 1997;DiBella,Carbone&DeLeo,2005;

Xu et al., 2008; Hirose, 2009). Tunicates are subdivided into

sessile ascidians, pelagic thaliaceans, and larvaceans (Stach,

Braband & Podsiadlowski, 2010). Ascidians are the best-stud-

ied group of tunicates and encompass around 2300 species.

They are considered reference organisms in investigations

of the developmental biology, regeneration, allorecognition,

and immunobiology of invertebrate chordates (Satoh, 1994,

2016;Burighel&Cloney,1997; Stoner, Rinkevich &

Weissman, 1999;Corbo,DiGregorio&Levine,2001;

Khalturin et al., 2003;Rinkevich,2004; Jeffery, 2015;

Franchi & Ballarin, 2017; Ferrario et al., 2020; Gordon,

Manni & Shenkar, 2019; Gordon et al., 2021; Ballarin et al.,

2021). Most ascidians have a biphasic life history with two

distinct body plans: a swimming tadpole larva and a sessile

adult. The tadpole larva is considered a prototype of the

chordate ancestor (Fig. 7A), with which it shares features such

as a notochord, dorsal neural tube, pharynx provided with gill

slits, and a muscular tail (Satoh, 1994;Burighel&Cloney,

1997). Studies have found ascidians to be reliable model organ-

isms for ecotoxicological bioassays due to their production of

eggs almost all year round, rapid development, simplicity of

the larval stage (Fig. 7A), ease of management in the laboratory,

and their small sequenced genome (Tosti & Gallo, 2012; Gallo

&Tosti,2015;Metriet al., 2019). Ascidians include both solitary

and colonial species.

(a)Solitary ascidians

Solitary ascidian species have become increasingly popular

for toxicological studies. Ciona intestinalis is a well-studied

ascidian species (Mansueto et al., 1993; Bellas, Beiras &

Vazquez, 2003) that has been shown to include at least two

cryptic species, Ciona robusta (Fig. 7B) and C. intestinalis

(Suzuki, Nishikawa & Bird, 2005; Caputi et al., 2007; Nydam

& Harrison, 2007; Zhan, Macisaac & Cristescu, 2010;

Brunetti et al., 2015). We refer to both species herein as Ciona

spp. Additional models being investigated include Halocynthia

roretzi,Ciona savignyi,Microcosmus exasperatus,Phallusia fumigata,

and Phallusia mammillata (Pennati et al., 2006; Choi et al.,

2014; Cahill et al., 2016a; Gomes et al., 2019; Anderson &

Shenkar, 2021). The solitary ascidian phylogenetic position,

a fully sequenced genome and available genomic tools make

them attractive models for studying the mode of action

(MOA) of toxic compounds (Dehal et al., 2002; Stolﬁ&

Christiaen, 2012).

The larvae have a simple structure consisting of only six

tissue types (Fig. 7A), making it easy to assess phenotypic

alterations during development and to discriminate between

speciﬁc and non-speciﬁc toxicity of substances (Katz, 1983;

Nicol & Meinertzhagen, 1991; Meinertzhagen, Lemaire

&Okamura,2004;Jianget al., 2005;Horieet al., 2008;

Hudson, 2016).Thejuvenilesaretransparent,allowing

easy observation of morphological changes caused by

stressors, and have been used to evaluate the impact of pol-

lutants such as MPs, tributyltin, bisphenol A, drugs, and

oil dispersants on survival and morphology (Mansueto,

Cangialosi & Faqi, 2011;Mizotaniet al., 2015; Messinetti

et al., 2019;Elisoet al., 2020b). The use of software tools

such as Toxicosis (Gazo et al., 2021) allows for the high-

content analysis of larval phenotypes and the evaluation

of embryonic malformations through scoring morphomet-

ric endpoints. Other commonly used endpoints include the

percentage of normal hatched larvae and the progression

of metamorphosis to adults (Fig. 7C–F). Behavioural phe-

notyping, such as swimming activity, also has the potential

for identifying effects of toxic substances on neuro-beha-

vioural performance (Zega, Thorndyke & Brown, 2006;

Rudolf et al., 2019). Comparing embryotoxicity data for

chemicals in Ciona spp., bivalves (Mytilus spp.) and sea

urchins (Paracentrotus spp.) demonstrates that Ciona spp.

can support classical tests (Table 2).

Adult ascidians are abundant in both contaminated and

pristine environments. They are important ﬁlter-feeders in

many benthic ecosystems and can ﬁlter dozens of litres of

water per day, retaining submicron-sized particles and

accumulating low concentrations of toxicants from the

watercolumntowhichthey may show sensitivity

(Draughon, Scarpa & Hartmann, 2010;Jacobi,Yahel&

Shenkar, 2018;Tzafriri-Miloet al., 2019; Vered et al., 2019).

Therefore, adults are useful for bioaccumulation studies

20 Amalia Rosner and others

of speciﬁc tissues and organs and for evaluating how ingested

toxicants may affect reproduction and development. In recent

years, adult solitary ascidians, including invasive species,

have been used as bioindicators to monitor anthropogenic

stressors such as heavy metals, MPs, phthalate acid esters,

and pharmaceutically active compounds (Vered et al., 2019;

Navon et al., 2020). Biomonitoring methods and indices,

including both chemical and physiological analyses, have

been used to quantify the distribution of chemicals in tissues

and organs (Tzafriri-Milo et al., 2019). Increasing evidence

suggests that stressful conditions stimulate the production of

ROS, leading to an inability to detoxify pollutants or repair

induced damage (Kaloyianni et al., 2009;Tomanek,2014;

Canesi, 2015; Puppel, Kapusta & Kuczynska, 2015; Zeeshan

et al., 2016). Antioxidant responses have been used as

markers of stress conditions related to pollutants (Shida

et al., 2003; Franchi et al., 2014; Drago et al., 2021) in the

digestive system (the ﬁrst organ to contact pollutants) and cir-

culating haemocytes (the major detoxiﬁcation organ) in

ascidians.

(b)Colonial ascidians

Colonial ascidians are the only chordates capable of asexual

reproduction (Manni et al., 2007) and have been widely

used in studies investigating asexual reproduction (Manni

&Burighel,2006;Manniet al., 2014,2019;Rosner

et al., 2014;Gaspariniet al., 2015;Kowarskyet al., 2021),

regeneration (Fig. 8A–F; Rinkevich, Shlemberg & Fishelson,

1995b;Rinkevichet al., 2007a;Voskoboyniket al., 2007;

•

Fig. 7. Ascidians. (A) Diagram of a typical larva. Bv, brain vesicle; gd, gastrodermis; ep, epidermis; mc, mesenchyme; ms, muscle; nc,

nerve chord; nt, notochord; oc, ocellus; ot, otolith; vg, visceral ganglion. (B) A typical solitary ascidian Ciona robusta.(C–F) Solitary

ascidian life stages used in (eco)toxicological tests.

A broad-taxa approach as an important concept 21

Table 2. Comparison of embryonic sensitivity towards inorganic and organic compounds among three marine invertebrates: Ciona spp., Paracentrotus spp. and Mytilus spp.

Compounds EC50*Ciona

spp. (20 h)

EC50*Paracentrotus

spp. (48 h)

EC50*Mytilus

spp. (48 h) References

Copper 0.72 μM 1.8 μM 0.05 μM His et al.(

1999); Bellas et al.(2001);

Fernandez & Beiras (2001); Gharred

et al.(

2016)

Mercury 0.27 μM 0.04 μM 0.61 μM

Cadmium 7.46 μM 3.1 μM 19.6 μM

Tributyltin 7.1 μg/L (0.022 μM) 0.309 μg/L (0.009 μM) 0.377 μg/L (0.0012 μM) Bellas et al.(

2005)

Lindane 4412 μg/L (15.2 μM) >91,000 μg/L (>313.5 μM) 1992 μg/L (6.8 μM) Bellas et al.(

2005); Beiras & Bellas (2008)

Chlorpyrifos 5666 μg/L (15.7 μM) 300 μg/L (0.83 μM) 154 μg/L (0.44 μM)

Sodium dodecyl sulphate 5145 μg/L (17.8 μM) 4100 μg/L (14.18 μM) 2353 μg/L (8.2 μM)

Naphthalene 1.9 μg/L (15 μM) 4.72 μg/L (37.3 μM) 6.55 μg/L (51.7 μM) Bellas et al.(

2008)

Phenanthrene >2400 nM >2400 nM 809 nM

Pyrene >640 nM >640 nM >640 nM

Fluoranthene >1250 nM >1250 nM >1250 nM

Chlorothalonil 123 nM 25 nM 33 nM Bellas (2006)

Sea-Nine 211 372 nM 43 nM 38 nM

Dichloﬂuanid 846 nM 1881 nM 244 nM

Tolylﬂuanid 625 nM 1165 nM 213 nM

Irgarol 1051 8346 nM 15871 nM 6076 nM

Bisphenol A 168 μg/L (0.74 μM) 710 μg/L (5.7 μM) 3.68 μg/L (0.016 μM) Özlem & Hatice (2008); Matsushima

et al.(

2013); Fabbri et al.(2014)

Paclitaxel 3 μM (MTC**)10μM–Semenova et al.(

2006); Mizotani et al.

(2015)

PS-NH

*** 7.52 μg/mL 2.61 μg/mL 0.142 μg/mL Della Torre et al.(2014); Balbi et al.

(2017); Eliso et al.(2020a,2023)

The data demonstrate that Ciona spp. can support classical embryonic tests previously performed with species already incorporated in standardised protocols.

EC50*, half maximal effective concentration; MTC**, minimum toxic concentration; PS-NH2***, polystyrene nanoparticles amino-modiﬁed.

22 Amalia Rosner and others

° °°

(Figure 8 legend continues on next page.)

A broad-taxa approach as an important concept 23

Brown et al., 2009; Rosner, Kravchenko & Rinkevich, 2019),

stem cells (Rinkevich et al., 2010;Voskoboyniket al., 2008;

Rosner et al., 2013) and allorecognition (Sabbadin, 1962;

Karakashian & Milkman, 1967;Oka,1970;Mukai&

Watanabe, 1974;Koyama&Watanabe,1982;Saito&

Watanabe, 1982; Taneda & Watanabe, 1982a,b; Rinkevich

& Weissman, 1992; Ballarin, Cima & Sabbadin, 1995;

Ballarin et al., 2002; Rinkevich, Porat & Goren, 1995a;

Rinkevich, Tartakover & Gershon, 1998; Cima, Sabbadin

& Ballarin, 2004; Rinkevich, 2005b;Voskoboyniket al.,

2013; Taketa & De Tomaso, 2015;Franchi&Ballarin,2017).

Very few colonial ascidians have been used as sentinel organ-

isms in toxicological assays and most data refer to the species

Botryllus schlosseri (Fig. 8G). Toxicological tests on B. schlosseri

larvae have primarily focused on antifouling paints.

Results suggest that antifoulants interfere with larval adhe-

sion and metamorphosis, cause developmental delays and

increase mortality (Cima & Ballarin, 2004; Cima, Burighel

& Ballarin, 2006).

The effects of exposure to xenobiotics on adult colonial

ascidians have mainly been studied on haemocytes. At high

concentrations, xenobiotics can change many parameters,

from phagocytosis capability and membrane permeability

to increasing cell mortality by inducing apoptosis. At lower

concentrations, they can alter cell shape by changing cyto-

solic levels of Ca

, hence affecting the cytoskeleton. Such

endpoints have been used to screen various antifouling com-

pounds and compare their toxicity (Cima et al., 1995,

1997,1998a,2002; Cima, Spinazi & Ballarin, 1998b;

Cima, Bragadin & Ballarin, 2008; Cima & Ballarin, 1999,

2000,2004,2012,2015;Meninet al., 2008; Matozzo &

Ballarin, 2011;Matozzo,Franchi&Ballarin,2014;Cima

&Varello,2020)ortoevaluatetheimpactofcadmium

(Franchi & Ballarin, 2013).

Responses of whole colonies to pollutants were also tested.

The response of B. schlosseri depends on pollutant concentra-

tion and exposure duration, ranging from changes in tran-

scription of genes involved in glutathione synthesis and

antioxidant responses, detectable only in haemocytes

(Franchi, Ballin & Ballarin, 2017), to phenotypic modiﬁcations

such as disconnected zooid oral siphons, reduced circulation,

and darkened ampullae, or even colony death (Gregorin et

al., 2021). Pseudistoma crucigaster colonies transplanted for

6 months to polluted areas of the Catalan coast with high con-

centrations of dissolved copper exhibited negative effects on

growth (Agell et al., 2004). Other experiments have shown an

increase in the activity of enzymes like SOD and CAT in

Botryllus colonies transplanted to sites exposed to high anthro-

pogenic impact (Tasselli et al., 2017). Alterations in GPX gene

expression due to exposure to crude oil and environmental

pollutants were also detected (Fig. 8G).

To date, the use of colonial ascidians in ecotoxicology has

been limited to a few species, as reported above. Important

features of colonial species that have been neglected, such

as their asexual reproduction and their huge regenerative

ability could be exploited as reliable biomarkers in environ-

mental monitoring.

(c)Appendicularia

Appendicularia, free swimming tunicates, are another group

of tunicates proposed as bioindicators for monitoring the

quality of estuarine areas. The density of Oikopleura longicauda,

Oikopleura dioica,Oikopleura fusiformis, and Fritillaria haplostoma

populations has been used to evaluate water quality in Rio

de Janeiro state (de Carvalho, Bonecker & Nassar, 2016).

(5) Non-standard models –overview

Many hitherto underused species (poriferans, cnidarians,

planarians, and colonial ascidians) have exceptional charac-

teristics such as enormous regenerative ability, and the pos-

session of populations of pluripotent ASCs, capable of

differentiation into both soma and germ lineages (Rinkevich

et al., 2022; Rosner et al., 2021; Ballarin et al., 2022). These

features can be exploited in the ﬁeld of (eco)toxicology in

many ways: (i) production of many ramets from a speciﬁc

genet, all with identical genetic information and epigenomes,

(Figure legend continued from previous page.)

Fig. 8. (A–F) Various time points during regeneration of a Botryllus schlosseri colony after removal of all buds from the colony

(budectomy). (A) A colony immediately after budectomy. (B) Morphological changes occurring in the budectomised colony upon

entering stage D (takeover stage). These changes include the destruction of zooids, dilation of blood vessels, and increased

pigmentation of ampullae. (C) Formation of new zooids. (D, E) Healing of the colony through normal blastogenic cycles. (F) The

recovered colony. am, ampulla; bv, blood vessel; pb, primary bud; tu, tunic; zo, zooid. (G) Changes in B. schlosseri glutathione

peroxidase (GPX) messenger RNA (mRNA) expression following the submersion of colonies (N=3 for each test) in crude oil-

polluted sea water samples and environmental seawater samples. Each colony was subcloned into several ramets. A ramet was

submersed in tested seawater samples while the matched control was submersed in clean sea water originating from Tel-Shikmona

(near Haifa). GPX expression was measured using relative quantitative polymerase chain reaction (qPCR) analysis and is

expressed as fold changes at the log

scale. Some of the tested samples consisted of clean sea water (from Tel-Shikmona) spiked

with 20 or 40 parts per thousand (ppt) crude oil while others contained only sea water without any additional additives. Shafdan is

near Tel Aviv, where regional treated sewage was discharged into the Mediterranean Sea until the end of 1996. The Haifa port

sample was collected from a highly polluted region inside Haifa port and the ‘open sea’sample was taken from open sea outside

Haifa port. Each experiment was performed with three different genets. Statistical analysis was performed by the paired t-test

method; statistically signiﬁcant upregulation of GPX mRNA following treatment (P≤0.005) is marked by an asterisk (A. Rosner,

unpublished data).

24 Amalia Rosner and others

for testing various endpoints under controlled laboratory and

ﬁeld experiments; (ii) the possibility of assessing the impact of

various pollutants on regeneration (Best et al., 1981; Best &

Morita, 1982) using new models like artiﬁcial embryos

(Johnson et al., 1982; Johnson & Gabel, 1983), the nubbins

assay (Shaﬁret al., 2003) and the use of primmorphs (Akpiri

et al., 2020) that include endpoints such as the inhibition of

regeneration, regeneration time and formation of teratomas;

(iii) evaluation of the direct effects of pollutants on stem cell

populations, and the use of these systems to design effective

interpretations of in vitro outcomes in order to understand

whole-body impact better; and (iv) investigation of the epige-

netically mediated inheritance of the impacts of pollution

by unexposed descendants (Ellis, Kissane & Lynch, 2021b;

Rosner et al., 2021).

V. NEW OPTIONS FOR BIO-MONITORING

USING NON-STANDARD MODELS FOLLOWING

THE DEVELOPMENT OF INNOVATIVE

METHODS

Advances in omics and bioinformatics methodologies over

recent decades have expanded the use of non-model organ-

isms (Pai et al., 2018; Amil-Ruiz et al., 2021), and enabled fast

and effective studies on the impact of environmental pollu-

tion at different organisational levels, from the cell to the

population. Omics approaches cover genomics for studies

at the DNA landscape level, transcriptomics for studies on

RNA repertoires, metabolomics for studies on metabolites

and epigenomics to study epigenetic modiﬁcation.

Transcriptomic analyses performed on aquatic inverte-

brates exposed to various pollutants (Srivastava et al., 2010;

Elran et al., 2014; Riesgo et al., 2014; Ereskovsky et al.,

2017; Kenny et al., 2018,2020; Zhang et al., 2019b; Luter

et al., 2020; DeLeo et al., 2021; Rubin et al., 2021) have shed

light on species-speciﬁc differences in response to environ-

mental pollution. Tests based on the mode of regulation

and expression of genes of the aryl hydrocarbon receptor sig-

nalling pathway (Hahn, Karchner & Merson, 2017) and

xenobiotic-metabolising enzymes have become prominent

examples of these approaches, serving as reliable pollution

biomarkers in various organisms, including non-model

organisms (Zhou et al., 2020). Proteome proﬁles

have also been used as pollutant-speciﬁcbiomarkers

(Diz & Calvete, 2016;Gouveiaet al., 2018). For example,

a differential expression of proteins has been observed in

solitary ascidians (Kuplik, Novak & Shenkar, 2019)and

corals (Tisthammer et al., 2021) exposed to pollution.

Metabolomic analysis revealed that microbiome-derived

bioactive metabolites contribute signiﬁcantly to the overall

organism metabolites, with changes observed in the meta-

bolome of the amphipod crustacean Hyalella azteca exposed

to the anti-inﬂammatory drug Diclofenac (Fu et al., 2021).

In the sponge S. ofﬁcinalis, exposure to anthropogenic

pollutants containing the synthetic surfactant coconut

diethanolamide (C11 DEA) resulted in the discovery of

new metabolites, while increased levels of metabolites were

found in the coral S. pistillata following exposure to polyethyl-

ene MPs (Bauvais et al., 2017;Lanctôtet al., 2020). Site-associ-

ated differences in metabolome were also observed in the

ascidian Ciona intestinalis (Utermann et al., 2020).

Pollution can also modify the epigenetic signature of

organisms, and this can persist for several generations, even

after pollutant removal. Such alterations can change the sen-

sitivity of organisms to other chemicals, resulting in hormesis

and resistance (Vaiserman, 2011; Roberts & Gavery, 2012;

Oziolor, De Schamphelaere & Matson, 2016; Calabrese &

Mattson, 2017; Ellis et al., 2021b). However, some of these

techniques rely on sequenced genomes, therefore, to bypass

such obstacles, new techniques have been developed [such

as reduced-representation bisulﬁte sequencing (RRBS-Seq),

epigenotyping by restriction-site associated DNA sequencing

(EpiRADseq), bisulﬁte-based restriction-site associated DNA

sequencing (BsRADseq), and epi-genotyping by sequencing

(epiGBS(; Schield et al., 2016; Trucchi et al., 2016; Van Gurp

et al., 2016]. Although invertebrate epigenetic research is in

its infancy, epigenetic changes can be a valuable source of

information for the development of innovative tools for

monitoring the effects of pollution (Brander, Biales & Connon,

2017). In a recent comprehensive review on the impact of pol-

lution on invertebrate epigenomes, ˇ

Srut (2021) highlighted

various exposure scenarios, epigenetic endpoints and methods

for detecting epigenetic impact. However, much work still

needs to be done before such tools can be used in models to

predict the impact of speciﬁc pollutants. Alterations in the epi-

genetic proﬁle may also be linked to changes in the micro-

biome and changes to organism resistance to environmental

factors (Barno et al., 2021).Themicrobiomecanalsobe

directly affected by pollution, ultimately leading to

adverse effects in the host (Lederberg & Mccray, 2001;

Haiser & Turnbaugh, 2013; Claus, Guillon & Ellero-

Simatos, 2016; Adamovsky et al., 2018). Studies of the

impact of pollution on the microbiomes of non-model

aquatic hosts include tests on the Manila clam Ruditapes

philippinarum (Bernardini et al., 2021), some sponge species

(Yang et al., 2011; Pita et al., 2013; Turon et al., 2019), reef-

building corals of the genus Acropora (Littman et al., 2009;

Ziegler et al., 2018,2019), the planarian S. mediterranea

(Bijnens et al., 2021) and ascidians (Cahill et al., 2016b; Evans

et al., 2018; Goddard-Dwyer, Lopez-Legentil & Erwin, 2021).

These results suggest high potential for microbiomes as tools

to test the impact of pollution; however, the standardisation

of protocols is required to avoid methodological bias

(Evariste et al., 2019).

Environmental pollution can result in changes to population

composition, including the loss of native species and the

emergence of invasive species. Pollution can also impact phe-

notypic plasticity, which may adversely affect the accuracy of

traditional morphology-based taxonomic classiﬁcations of

organisms (Abdelhady et al., 2018). DNA barcoding is a

A broad-taxa approach as an important concept 25

genetic-based method for species classiﬁcation that utilises

highly conserved regions ﬂanking species-speciﬁc sequences,

which are ampliﬁed by polymerase chain reaction (PCR)

and sequenced (Hebert & Gregory, 2005). One commonly

used gene for this purpose in many animals is the mito-

chondrial cytochrome oxidase subunit 1 (CO1) gene.

The Barcode of Life Data System (BOLD), a global data-

base established in 2005, stores species-speciﬁcinforma-

tion obtained using this approach from all over the

world,alongwithLinnaeanclassiﬁcation, photographs

andcollectionsites(Ratnasingham&Hebert,2007).

BOLD facilitates the identiﬁcation of cryptic and invasive

species (Douek et al., 2020,2021;Galilet al., 2021)andwas

also used to identify phenotypic plasticity induced by pol-

lution (Weigand et al., 2011,2019). DNA metabarcoding

(Pawlowski et al., 2018), which applies the same principles

as DNA barcoding, is a cost-effective tool for observing

biodiversity alterations across environments. This approach

involves using PCR amplicons from a variety of species

in environmental samples and then subjecting them to

high-throughput sequencing and bioinformatic analyses.

Alternatively, Droplet Digital PCR (ddPCR) can be

employed for water samples (Wood et al., 2018; Hernandez

et al., 2020). Standardised methods for collecting environ-

mental samples, such as from river streams or sediments,

are crucial for the reliability of such methods (Emilson et al.,

2017; Holman et al., 2019).

In addition to the use of genetic material extracted from

organisms, environmental DNA (eDNA) and environmental

RNA (eRNA) analyses of water or sediment samples are

promising approaches for detecting genetic material from

various sources, including microscopic organisms, physical

damage to animals and reproductive products (Wood

et al., 2020). eDNA- and eRNA-based methods offer beneﬁts

over conventional methods, such as non-invasive sampling,

lower costs and improved detection sensitivity (Bohmann

et al., 2014; Rees et al., 2014; Valentini et al., 2016). eDNA

is already used to record aquatic organism distribution

(Lamy et al., 2021), while eRNA experiments are at a proof-

of-concept stage, enabling the detection of alternative gene

spliced transcripts or non-coding RNA species via an RNA

sequencing (RNA-seq) technique.

Quantitative trait loci (QTLs) are used to evaluate the

impact of pollution on species diversity by linking pollution resis-

tance with speciﬁc genomic regions/loci (gene–environment) or

gene–gene epistatic interactions. These approaches involve

crosses between resistant phenotypes and the comparison of

genetic polymorphisms of exposed/unexposed individuals.

Comprehensive methods like pooling-based sequencing

(pool-seq) sequencing and population resequencing are

replacing traditional methods like ampliﬁed fragment

length polymorphism (AFLP), microsatellites, single nucleo-

tide polymorphisms, and restriction site-associated DNA

sequencing (RADseq) to detect the responses of multiple

regions to multiple selective stressors and have successfully

been applied to ﬁsh populations. The coral species A. millepora

and Pocillopora damicornis have also successfully been used to

correlate environmental stressors with speciﬁc QTLs for

thermal stress, water clarity, eutrophication and high-

temperature stress conditions (Lundgren et al., 2013; Jin

et al., 2020).

These are just a few examples of the new methods that are

continually emerging and can be applied to non-model

aquatic invertebrates. Consequently, they eliminate bound-

aries in ecotoxicological studies, allowing broader ecotoxico-

logical research that considers the diversity of the animals,

the representation of their natural habitats and their role in

the food chain.

VI. INTEGRATION OF DATA FOR REUSE IN

(ECO)TOXICOLOGY AND ENVIRONMENTAL

RISK ASSESSMENT

Environmental risk assessment (ERA) is the process of

predicting the risk of adverse effects on the environment

caused by pollutants through successive steps of hazard iden-

tiﬁcation, hazard characterisation, exposure characterisation

and risk assessment. The main outputs of ERA are risk

management and communication plans (Blasco & DelValls,

2008). Recent reports (including from the World Health

Organisation) have demonstrated that the integration of

health, safety and environmental (HSE) risk assessment

approaches is needed to protect human health and the envi-

ronment, a so-called One Health approach (Aguirre et al.,

2016). Integration improves the efﬁciency and quality of

decisions related to human health and ecological risk assess-

ments (Suter et al., 2005). In addition, the integration of

HSE risk assessment and risk-mitigation measures requires

the integration of scientiﬁc data, information and knowledge.

Integrated approaches in toxicity testing and assessment

(IATA) are already included in OECD testing recommenda-

tions. IATA is a practical, scientiﬁc approach to the analysis

of chemical danger relying on an integrated study of the

available data along with the development of new data

(experimentally and/or computationally; OECD, 2021).

The IATA approach of integration of knowledge, informa-

tion, and data may enable more comprehensive safety proﬁl-

ing (Rivetti et al., 2020). The ﬁrst step in ERA is hazard

assessment, but it is unrealistic to identify the hazards pre-

sented by all pollutants released into the environment.

Therefore, the reuse and integration of existing data, infor-

mation and knowledge represents a way forward to a more

holistic approach to ERA (Bennekou, 2019). Data, informa-

tion and knowledge integration in (eco)toxicology is facing

many challenges, which are not only related to informatics

and technical issues, but to an even greater degree to the

assessment of the weight of test outcomes and their overall

relevance (Neagu & Richarz, 2019). For example, available

studies vary in study design, methodology, and in the level

of detail reported. Regulators must therefore evaluate each

individual study for reliability in accordance with

good research practice (Schwab et al., 2022). Adherence to

26 Amalia Rosner and others

metadata content standards, i.e. an agreed list of common

metadata items and the standardisation of terminology and

deﬁnitions for these items is a prerequisite for quality data

sharing, reuse and integration. In addition to metadata stan-

dards, data reporting templates can also play an important

role in ensuring quality data sharing, reuse and integration.

Data reporting templates provide a standardised format for

reporting data, including metadata, and can help ensure that

important information is not omitted, and that data are

reported consistently across different data sets. At present, over

50 harmonised data entry templates for various types of studies

have been elaborated in the nanomaterial safety community,

including the physicochemical characterisation of materials,

hazard assessments (cell viability, genotoxicity, environmental

organism dose–response tests, omics) and the reportingof data

from exposure and release studies. The templates can be used

in other research domains and have already been extended

and adapted for MPs and advanced materials research. The

harmonised templates aim to make data presentation, interla-

boratory comparisons and meta-analyses more reliable and to

streamline the evaluation and regulation process. Data entry

templates are created collaboratively with the active involve-

ment of data providers. Once the layout and content are

agreed, the templates are integrated into the Template Wiz-

ard, which is an online tool allowing the sharing and down-

loading of dynamically customisable templates. The tool is

designed to be user-friendly and attractive to data providers,

as well as to improve awareness and reuse of existing templates

in new projects and when adding new (or extended) endpoints.

The online template validator allows self-evaluation of the

template and transformation by the open-source parser into

a machine-readable format (e.g. json or rdf) compliant with

the FAIR (ﬁndable, accessible, interoperable, reusable) princi-

ples (Jeliazkova et al., 2021).

The integration of data and knowledge is outlined by the

FAIR data principles (Wilkinson et al., 2016). This set of prin-

ciples is focused on ensuring that research objects are reus-

able, and will be reused, and thus become as valuable as

possible. The FAIR principles put speciﬁc emphasis on

enhancing the ability of machines automatically to ﬁnd

and use the data, in addition to supporting its reuse by

individuals. The idea of being machine-actionable applies

in two contexts –ﬁrst, when referring to the contextual

metadata surrounding a digital object (‘what is it?’), and second,

when referring to the content of the digital object itself (‘how do

I process it/integrate it?’). This means that domain experts

should also have a basic understanding of how to organise,

document, store and share data, to ensure they are properly

managed and can be understood and (re)used in the future.

VII. CERTAINTIES AND UNCERTAINTIES IN

ASSESSING AQUATIC ECOTOXICOLOGY

Species sensitivity distribution (SSD) models have

been established as a key tool for the ERA of chemicals

(Posthuma, Suter & Traas, 2002). SSDs consist of a statistical

approach to predict the potential biological impact of a

chemical in nature. SSDs simulate the range of sensitivity of

various species to a variety of chemical concentrations. These

tools are used to estimate the potentially affected fraction

(PAF) of species that will be harmed by exposure, and to

establish threshold concentrations. When using SSDs, it is

assumed that the species toxicity data represent a random

sample from a statistical distribution that is typical for a com-

munity or ecosystem. The precision of SSDs in predicting

environmental toxicity impact will rise as more data become

accessible for a variety of species. SSDs have been useful tools

for risk assessment purposes for decades, despite their numer-

ous limitations (Belanger & Carr, 2019).

De facto testing of the effects of chemicals is only possible on

a restricted number of species. Ecotoxicologists, therefore,

face the major problem of translating the measurements

acquired from the tested species into predictions of effects

on the wider range of species in aquatic ecosystems. A broad

comparison of the toxic concentrations of various classes of

compounds for some aquatic invertebrates has highlighted

that species belonging to highly diverse phyla, such as arthro-

pods and molluscs, exhibit a wide range of sensitivities

(Rosner et al., 2021). For example, a study on arthropods

comparing Deleatidium spp. and D. magna exposed to heavy

metals showed similar sensitivities to Cr

and Cu

whereas a signiﬁcantly higher sensitivity was observed in

Daphnia exposed to Cd

and Zn

(Hickey & Vickers, 1992).

Differences in sensitivity can be observed even among cryptic

species. For example, comparisons of two cryptic species of

the polychaete annelid Capitella found that Capitella sp. I pos-

sessed a greater ability to biotransform ﬂuoranthene than

Capitella sp. S (Selck, Palmqvist & Forbes, 2003; Li, Bisgaard

& Forbes, 2004). The observation of such differences in

invertebrates, as opposed to vertebrates, could be explained

by the huge molecular diversity (divergence in protein

sequences, for example) arising during their evolutionary tra-

jectories. This molecular diversity could be directly linked to

disparities in inherent sensitivities to chemical compounds

dependent on speciﬁc monoamine oxidase (e.g. inhibition

of an enzyme or receptor by xenobiotics via their attachment

to speciﬁc sites of action; Chaumot et al., 2014) and is further

illustrated by the high sensitivity of arthropods to acetylcho-

linesterase (AChE)-inhibiting insecticides compared to

rotifers, molluscs, and annelids (Van Wijngaarden et al.,

2005; Bally et al., 2016). This wide molecular diversity also

results in unexpected xenobiotic toxicity in speciﬁc groups

of invertebrates; for example, the biocide TBT is responsible

for the abnormal development of the genital tract, with mas-

culinisation of females, in about 100 species of marine gastro-

pods (Migula, 2005).

To overcome the limitations of SSDs, trait-based

approaches have been proposed as complementary tools for

ERA. These approaches allow the deciphering of the mech-

anisms behind the effects of exposure and prediction of the

responses of species to chemicals with the same MOA. For

example, the SSD for various MOAs highlighted groups of

A broad-taxa approach as an important concept 27

species with different sensitivity levels. Arthropods were

classiﬁed as more sensitive than average, followed by nema-

todes, molluscs and annelids. However, bryozoans, cnidar-

ians and ﬂatworms were never of above-average sensitivity

(Van den Berg et al., 2019). It is interesting to note that species

used routinely for ecotoxicological tests, e.g. Daphnia, are not

the most sensitive species (Van den Berg et al., 2019).

Another way to improve the accuracy of ecotoxicological

tests on aquatic organisms consists of designing adverse out-

come pathways (AOPs; OECD, 2017). AOPs serve as the

basis for mechanistically driven toxicological approaches

using a variety of toxicological data including details of

chemical interactions with a speciﬁc biomolecule, commonly

called the molecular initiating event (Ankley et al., 2010).

Toxicokinetic (TK) pathways together with toxicodynamics

(TD) are also key elements in the design of AOPs, as

TK pathways determine the concentration of chemicals in

an organism by considering adsorption–distribution–

metabolism–excretion (ADME) traits (McCarty & Mackay,

1993; Nyman, Schirmer & Ashauer, 2012), while TD

describes the interaction between the chemicals and the tar-

get(s) (McCarty & Mackay, 1993;Nymanet al., 2012). The

OECD actively supports AOP development to standardise

testing methods to assess substance toxicity. However, to date,

AOPs in aquatic organisms have not been fully developed.

They range from putative to partially characterised forms,

even in cases where toxicant effects are highly conserved across

species, such as those of organophosphates due tothe presence

of conserved AChE receptors (Brockmeier et al., 2017).

Indeed, for other xenobiotics, such as heavy metals, interspe-

cies variation has been observed due to microevolutionary

processes leading to the acquisition of resistance mechanisms

(see review by Posthuma & Van Straalen, 1993; Chandrangsu,

Rensing & Helmann, 2017). Modiﬁcation in the expression of

metallothioneins has been shown to cause a change in the

resistance capacity of adapted species. For instance, genotypes

with a speciﬁc metallothionein promoter are detected most

frequently in cadmium-tolerant populations of the springtail

Orchesella cincta in comparison to other O. cincta populations

(Costa et al., 2012). This adaptation is also observed in the

springtail Folsomia candida, which exhibits constitutive metal-

lothionein expression. Additional examples of adaptation have

been observed in natural populations of oligochaetes, mol-

luscs, crustaceans (Isopoda), myriapods, arachnids, aptery-

gotes and insects (Migula, 2005). Nevertheless, it is important

to remember that these adaptations are species dependent.

For instance, the isopod Porcellio scaber, considered an accumu-

lator of metals, has a smaller body size and starts to reproduce

earlier in polluted sites than in unpolluted sites, and shows dif-

ferent responses to O. cincta, which is a very rapid eliminator of

metals (Drobne, 1997;Migula,2005). In addition to these spe-

cies’adaptations to environmental stresses, increasing evi-

dence is accumulating on the potential contribution of the

host-associated microbiome to adaptation to environmental

stress. A prominent example is the change in the mammalian

gut microbiome in laboratory animals that results in altered

concentrations of inorganic arsenic metabolites in urine and

in increased sensitivity to arsenic toxicity (Lu et al., 2013;

Chi et al., 2019). Speciation of organoarsenic species in aquatic

organisms also highlights the importance of the microbiome

(Langdon et al., 2002; Rahman, Hasegawa & Lim, 2012;

Liebeke et al., 2013). The degradation of organic chemicals,

such as pesticides, by the host-associated microbiome has also

been demonstrated (Daisley et al., 2018;Fernandezet al., 2019;

Wang et al., 2020). In Hydra, an increased tolerance to copper

has been associated with the presence of microalgal symbionts

(Karntanut & Pascoe, 2005), while in metal-rich hydrothermal

vent environments, symbiotic bacteria on mussels (Bathymodiolus;

Hardivillier et al., 2004) protect their host by transforming

absorbed metal ions into metal particles. Additionally, micro-

bial symbionts of aquatic invertebrates frequently provide their

hosts with crucial micronutrients (Stock et al., 2021). As an

example, the settling of poriferan, cnidarian and mollusc

larvae requires bacteria. Chromatiales supply the amino

acid L-arginine to Amphimedon queenslandica to allow larvae

to settle successfully and metamorphose. Overall, even if

the role of the underlying mechanisms of microbiomes in

pollution adaptation are not always evident, case studies

suggest that associated microbiota can play a relevant role

(White & Torres, 2009).

VIII. DISCUSSION

This review summarises many (eco)toxicological studies on

chemicals affecting the aquatic environment, with a particu-

lar focus on several major aquatic invertebrate phyla, includ-

ing those represented by non-model organisms. It is clear that

interspecies and even intraspecies differences in sensitivity

and responses to toxicants do occur (Hendriks et al., 2013).

The key issue remains the heterogeneity of the species and

the number of endpoints required to enable the design of reli-

able predictive toxicity platforms, including tools for the esti-

mation of ecological risks and decision-making processes.

Currently, standardisation by the main international

organisations is available for only a few species representative

of a limited number of invertebrate phyla: Arthropoda,

Mollusca, and Annelida (Table 1). Sea urchins and sand dol-

lars are the only echinoderms for which there are standar-

dised protocols, while C. elegans, the nematode model

species recommended for freshwater toxicity studies, is not

even typically an inhabitant of fresh water. In some cases,

the selected model organisms include invasive species such as

Corbicula ﬂuminea [ENV/JM/MONO(2019)11]. By contrast,

many ecosystem engineers and keystone species like sponges

and corals are not represented in the standardised protocols,

and some pollution-related properties like hormesis, micro-

biome changes and population diversity properties are studied

de facto using varied protocols. This is the current status of the

ﬁeld, although a growing body of information has been accu-

mulated on corals, sponges and ascidians. Moreover, for pre-

dictive models like SSD, recommendations regarding the

minimum number of species to be tested exceed this: eight

28 Amalia Rosner and others

for the US EPA and 5–8 for the European Union (for SSD;

TenBrook et al., 2009). It is therefore logical to extend testing

to additional taxa. However, as demonstrated in Fig. 1,most

of the aquatic (eco)toxicological research performed in the last

decade still used vertebrates (54.2%), and the most studied

invertebrates were arthropods (12.5%) and molluscs (19%).

Performing an additional search of Web of Science (see Q3 and

Q4 in Table S1 for search terms) for the genera listed in

Table 1demonstrated that 58% and 32% of all tests per-

formed with arthropods and molluscs, respectively, used only

this small subset of model species. Clearly, only a limited and

biased subset of taxa still make up a substantial part of toxicity

testing.

The available results from non-model animals from

Porifera, Cnidaria, Platyhelminthes and Tunicata encourage

the use of species in these phyla as reliable model organisms

for acute and chronic tests (Fig. 9), with similar endpoints to

those used for testing the impact of chemicals on standard

models (Table 2) or with additional new tests. Some tests

based on non-standard species are relatively well established

and generally accepted (although non-validated), such as H.

attenuata reproduction, survival, and development tests used

as indicators of teratogenic effects [ENV/MC/CHEM(98)

19 (Johnson et al., 1982; Johnson & Gabel, 1983)]. A draw-

back of the lack of standardisation is the creation of multi-

ple laboratory-speciﬁc protocols, which affects the

outcomes and limits or complicates the inclusion of the

resulting data into predictive models. In other cases, cer-

tain types of tests may be performed with unsuitable

models, because standardisation does not exist for the best

animal model. Additionally, major regulatory frame-

works, including the Canadian Environmental Protection

Act, the US Toxic Substances Control Act, and the ECHA

REACH initiative, all encourage increased reliance on in

silico approaches, with the latter being based on the acute

toxicity of pollutants to a few species only, such as Daphnia

and ﬁsh (Zhou et al., 2021). The Danish QSAR Database

(Danish QD; DTU, 2018), the virtual models for property

evaluation of chemicals within a global architecture

(VEGA; Benfenati, Manganaro & Gini, 2013), the Kashinhou

tool for ecotoxicity (KATE; Furuhama et al., 2010), the toxicity

estimation software tool (TEST; EPA, 2016h), the QSAR tool-

box developed by the OECD (OECD, 2014), and the

ecological structure activity relationships (ECOSAR;

Mayo-Bean et al., 2012), are several of the in silico technol-

ogies created for ERA and utilised to support chemical

regulation.

The preservation of aquatic biodiversity is now widely

recognised as an important conservation goal, which requires

the use of effective toxicology and ecotoxicology tools as well

as modern safety assessment technologies. Hazard identiﬁca-

tion and prompt responses are necessary for pollutants such

as waste, micro- and nanopollution (including nanoplastics),

endocrine disruptors, and persistent chemicals, as they can

have harmful effects. In parallel, newly emerging supporting

technologies are becoming increasingly affordable and

accessible. These include various omics-based applications,

microbiomics, metabarcoding (Pawlowski et al., 2018),

Fig. 9. Summary of the broad-taxa approach proposed herein for environmental assessment and ecotoxicological studies.

A broad-taxa approach as an important concept 29

automated species identiﬁcation technologies (Gorsky et al.,

2010; Le Bourg et al., 2015; Kalaﬁ, Town & Dillon, 2018;

Wäldchen & Mäder, 2018, First et al., 2021), in vitro technol-

ogy (Rosner et al., 2021), bioinformatics, big data processing

techniques and increased computing power. Omics technol-

ogies will facilitate studies on non-model organisms; in vitro

technology will enable the simultaneous analysis of samples

from many species using a large number of tests; while tech-

nologies like DNA barcoding (Weigand et al., 2019; Paz &

Rinkevich, 2021) and automated species identiﬁcation will

reduce reliance on professional taxonomists, facilitating the

processing of large amounts of data originating from (eco)

toxicological tests performed on a variety of aquatic species.

By increasing the number and diversity of species used in tox-

icity assessment studies and incorporating these advances,

predictive models can adopt a more precise ‘broad-taxa

approach’(Fig. 9). Transformation in research approaches

is evidenced by the recent increase in tests using non-model

species performed using newly emerging techniques (Fig. 1),

especially with coral and sponges. It is important for the sci-

entiﬁc community to take a more proactive role in integrat-

ing large volumes of experimental data into formats that

are user-friendly and readily accessible, further inﬂuencing

the ways (eco)toxicity studies are performed under interna-

tional regulations, such as the EU Chemicals Strategy for

Sustainability (https://www.chemistryworld.com/news/eu-

commits-to-overhaul-of-chemicals-legislation/4012615.article)

and the EU Green Deal environmental programme (https://

ec.europa.eu/info/strategy/priorities-2019-2024/european-

green-deal_fr). Integrated approaches to test and assess are

already recommended by the OECD via IATA, but our goal

should be holistic. The term ‘holism’describes the necessity

for knowledge governance between various knowledge

sources and decision-making levels, represented by scientists,

experts, citizens and laypeople, as well as administrative and

political decision-makers (Giebels et al., 2020). This necessi-

tates problem-focused collaboration between multiple scien-

tiﬁcﬁelds and non-scientiﬁc actor groups in order to produce

transdisciplinary knowledge.

Rigorous reporting of research results is crucial for the

scientiﬁc community to advance. A minimum annotation

checklist for reporting research results needs to be extended

beyond omics research and should be applied to other ﬁelds,

including environmental sciences. The application of artiﬁ-

cial intelligence has opened up new and exciting possibilities

in a holistic and integrative way, to study the complexity of

biological systems (Johnson et al., 2021).

IX. CONCLUSIONS

(1) The preservation of the aquatic environment and its

biodiversity necessitates the application of state-of-the-art

toxicological and ecotoxicological tools and the development

of accurate prediction tools.

(2) The ﬁeld of (eco)toxicology is experiencing the emergence

of new methods and technologies that can be applied to all

living organisms, including in vivo,in vitro,andin silico

methods, which may eventually lead to a reduction in ani-

mal experimentation.

(3) Accurate evaluation of the impact of toxicants, and the

prediction of their mode of action requires the use of a wide

range of animal models that encompass both interspecies and

intraspecies variations, as well as the normal geographic

distribution and habitat of each model.

(4) A broad range of endpoints, spanning multiple levels of

organisation (from subcellular to population level) and

including aquatic non-model invertebrates, is necessary to

gain a comprehensive understanding of pollution impacts.

(5) Standardisation and predictive models are needed to

compare data obtained by different groups and on different

animal models, further leading to a comprehensive global

approach to aquatic toxicology.

X. ACKNOWLEDGEMENTS

A. R. was supported by the Israeli Ministry of Energy

(contracts no-215-17-025 and 214-17-013); T. G-H. was

supported by the Israeli Ministry of Energy (grant no. 219-

17-015); K. M. was partly supported by the Luxembourg

National Research Fund and the French National Research

Agency (ANR) in the framework of the Fond National de la

Recherche (FNR)/INTER/ANR research programme

(contract no. INTER/ANR/15/11209808/ECOTREE); I.

L. and S. C. were partly supported by the European Union’s

Horizon 2020 Research and Innovation Programme under

grant agreement no. 814425 (Grant No. 814425: RiskGONE

–Risk Governance of Nanotechnology). We thank Shai Shaﬁr

for the Daphnia photo; Elad N. Rachmilovitz for the coral

pictures; Alexander V. Ereskovsky for the sponge photos, and

Ximena Velasquez-Dubinsky for the zooplankton photos.

We thank the European Cooperation in Science & Technol-

ogy programme (EU COST), grant title: ‘Stem cells of

marine/aquatic invertebrates: from basic research to innova-

tive applications’(Action 16203 MARISTEM) for providing

the platform for this joint work. We also thank Lindsey Stokes

for editing the English.

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XII. SUPPORTING INFORMATION

Additional supporting information may be found online in

the Supporting Information section at the end of the article.

Fig. S1. The lunar-controlled life cycle of Platynereis dumerilii.

After fertilisation, the zygote starts segmentation, giving rise

to a nectochaete larva (72 h) (stomodeum and prototroch

highlighted in green). After the ﬁrst metamorphosis, the juve-

nile worm forms and starts to feed. After 8–10 days of ben-

thic feeding, the small worm undergoes cephalic

metamorphosis to become an atoke worm inside its tube,

which then grows continuously until sexual maturation. After

sexual maturation (female shown in yellow; male in red), the

epitoke worm leaves its tube and swims into the water col-

umn, eventually to take part in mass spawning coordinated

by the lunar cycle. Drawing modiﬁed after Schenkelaars &

Gazave (2021) and Fischer & Dorresteijn (2004)byK.M.

Karahan and A. Karahan.

Table S1. List of search terms used in Web of Science. Q1 was

used to identify all publications on marine and freshwater

species in the domains of ecotoxicology, toxicology, and pol-

lution in the last 10 years (see green numbers in Fig. 1). Q2

was used to identify all publications on marine and freshwa-

ter species based on omics technology in the domains of eco-

toxicology, toxicology, and pollution in the last 10 years

(see red numbers in Fig. 1). Q3 and Q4 are the search terms

used to assess the proportion of studies that focus only on the

subset of taxa listed in Table 1as common model taxa used in

standardised regulatory tests.

(Received 31 August 2022; revised 23 August 2023; accepted 28 August 2023)

46 Amalia Rosner and others

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Jun 2022
PLOS COMPUT BIOL

Common Environmental Pollutants Negatively Affect Development and Regeneration in the Sea Anemone Nematostella vectensis Holobiont

Article

Full-text available

Dec 2021

The anthozoan sea anemone Nematostella vectensis belongs to the phylum of cnidarians which also includes jellyfish and corals. Nematostella are native to United States East Coast marsh lands, where they constantly adapt to changes in salinity, temperature, oxygen concentration and pH. Its natural ability to continually acclimate to changing environments coupled with its genetic tractability render Nematostella a powerful model organism in which to study the effects of common pollutants on the natural development of these animals. Potassium nitrate, commonly used in fertilizers, and Phthalates, a component of plastics are frequent environmental stressors found in coastal and marsh waters. Here we present data showing how early exposure to these pollutants lead to dramatic defects in development of the embryos and eventual mortality possibly due to defects in feeding ability. Additionally, we examined the microbiome of the animals and identified shifts in the microbial community that correlated with the type of water that was used to grow the animals, and with their exposure to pollutants.

3-D Culture of Marine Sponge Cells for Production of Bioactive Compounds

Article

Full-text available

Oct 2021
MAR DRUGS

Production of sponge-derived bioactive compounds in vitro has been proposed as an alternative to wild harvest, aquaculture, and chemical synthesis to meet the demands of clinical drug development and manufacture. Until recently, this was not possible because there were no marine invertebrate cell lines. Recent breakthroughs in the development of sponge cell lines and rapid cell division in improved nutrient media now make this approach a viable option. We hypothesized that three-dimensional (3-D) cell cultures would better represent how sponges function in nature, including the production of bioactive compounds. We successfully cultured sponge cells in 3-D matrices using FibraCel® disks, thin hydrogel layers, and gel microdroplets (GMDs). For in vitro production of bioactive compounds, the use of GMDs is recommended. Nutrients and sponge products rapidly diffuse into and out of the 3-D matrix, the GMDs may be scaled up in spinner flasks, and cells and/or secreted products can be easily recovered. Research on scale-up and production is in progress in our laboratory.

Effects of benzo(a)pyrene on meiobenthic assemblage and biochemical biomarkers in an Oncholaimus campylocercoides (Nematoda) microcosm

Article

Full-text available

Oct 2021
ENVIRON SCI POLLUT R

A microcosm experiment was carried out to determine how benzo(a)pyrene (BaP) may affect marine meiofauna community, with a main emphasis on nematode structure and functional traits. Three increasing concentrations of BaP (i.e. 100, 200 and 300 ng/l, respectively) were used for 30 days. The results revealed a gradual decrease in the abundance of all meiobenthic groups (i.e. nematodes, copepods, amphipods, polychaetes and oligochaetes), except for isopods. Starting at concentrations of 200 and 300 ng/l BaP, respectively, significant changes were observed at community level. At taxonomic level, the nematode communities were dominated at the start of the experiment and also after being exposed or not to BaP by Odontophora villoti, explicable through its high ecologic ubiquity and the presence of well-developed chemosensory organs (i.e. amphids), which potentially increased the avoidance reaction following exposure to this hydrocarbon. Moreover, changes in the activity of several biochemical biomarkers (i.e. catalase ‘CAT’, gluthatione S-transferase ‘GST’, and ethoxyresorufin-O-deethylase ‘EROD’) were observed in the nematode species Oncholaimus campylocercoides, paralleled by significant decreases in CAT activity for non-gravid females compared to controls at concentrations of 25 ng/l BaP and associated with significant increase in GST and EROD activities for both types of individuals.

Using Caenorhabditis Elegans As An Environmental Indicator for Impaired Urbanized Watersheds

Preprint

Full-text available

Sep 2021

Background Third Fork Creek is a historically impaired urban stream that flows through the city of Durham, North Carolina. Caenorhabditis elegans (C. elegans) are non-parasitic, soil and aquatic dwelling nematodes that have been used frequently as a biological and ecotoxicity model. We hypothesize that exposure to Third Fork Creek surface water will inhibit the reproduction and chemotaxis of C. elegans. Using our ring assay model, nematodes were enticed to cross the impaired water samples to reach a bacterial food source which allowed observation of chemotaxis. The total number of nematodes found in the bacterial food source and the middle of the plate with the impaired water source was recorded for three days. Results Our findings suggest a reduction in chemotaxis and reproduction on day three in nematodes exposed to Third Fork Creek water samples when compared to the control (pvalue<0.05). These exploratory data provide meaningful insight to the quality of Third Fork Creek located near a Historically Black University. Conclusions Further studies are necessary to elucidate the concentrations of the water contaminants and implications for human health. The relevance of this study lies within the model C. elegans, that has been used in a plethora of human diseases and exposure research but can be utilized as an environmental indicator of water quality impairment.

Do bio-insecticides affect only insect species? Behavior, regeneration, and sexual reproduction of a non-target freshwater planarian

Article

Full-text available

Feb 2022
ENVIRON SCI POLLUT R

Bio-insecticides have been increasingly used worldwide as ecofriendly alternatives to pesticides, but data on their effects in non-target freshwater organisms is still scarce and limited to insects. The aim of this study was to determine the lethal and sub-lethal effects of the bio-insecticides Bac Control (based on Bacillus thuringiensis kurstaki—Btk) and Boveril (based on Beauveria bassiana—Bb) on regeneration, behavioral, and reproductive endpoints of the freshwater planarian Girardia tigrina. The estimated LC50–48h were > 800 mg a.i./L for Btk and 60.74 mg a.i./L for Bb. In addition, exposure to Btk significantly decreased locomotion and feeding activities of planarians (lowest observed effect concentration (LOEC) of 12.5 mg a.i./L Btk) and fecundity rate (LOEC = 3.12 mg a.i./L Btk), whereas exposure to Bb significantly delayed regeneration (LOEC = 0.75 mg a.i./L Bb) and decreased fecundity rate (1.5 mg a.i./L Bb) of planarians. Thus, both bio-insecticides induced deleterious sub-lethal effects on a non-insect freshwater invertebrate species. However, only Bb-based formulation affected the survival, fecundity rate, and regeneration at concentrations below the maximum predicted environmental concentration (PEC = 247 mg/L). Thus, care should be taken when using such formulations as alternatives to chemical insecticides near aquatic ecosystems.

Host under epigenetic control: A novel perspective on the interaction between microorganisms and corals

Article

Full-text available

Aug 2021

Coral reefs have been challenged by the current rate and severity of environmental change that might outpace their ability to adapt and survive. Current research focuses on understanding how microbial communities and epigenetic changes separately affect phenotypes and gene expression of corals. Here, we provide the hypothesis that coral-associated microorganisms may directly or indirectly affect the coral's phenotypic response through the modulation of its epigenome. Homologs of ankyrin-repeat protein A and internalin B, which indirectly cause histone modifications in humans, as well as Rv1988 histone methyltransferase, and the DNA methyltransferases Rv2966c, Mhy1, Mhy2, and Mhy3 found in coral-associated bacteria indicate that there are potential host epigenome-modifying proteins in the coral microbiome. With the ideas presented here, we suggest that microbiome manipulation may be a means to alter a coral's epigenome, which could aid the current efforts to protect coral reefs.

Application of transcriptome profiling to inquire into the mechanism of nanoplastics toxicity during Ciona robusta embryogenesis

Article

Dec 2022

The growing concern on nanoplastics (<1 μm) impact on marine life has stimulated a significant amount of studies aiming to address ecotoxicity and disclose their mechanisms of action. Here, we applied an integrative approach to develop an Adverse Outcome Pathway (AOP) upon acute exposure to amino-modified polystyrene nanoparticles (PS-NH2 NPs, 50 nm), as proxy for nanoplastics, during the embryogenesis of the chordate Ciona robusta. Genes related to glutathione metabolism, immune defense, nervous system, transport by aquaporins and energy metabolism were affected by either concentration tested of 10 or 15 μg mL-1 of PS-NH2. Transcriptomic data and in vivo experiments were assembled into two putative AOPs, identifying as key events the adhesion of PS-NH2 as (molecular) initiating event, followed by oxidative stress, changes in transcription of specific genes, morphological defects, increase in reactive oxygen species level, impaired swimming behavior. As final adverse outcomes, altered larval development, reduced metamorphosis and inhibition of hatching were identified. Our study attempts to define AOPs for PS-NH2 without excluding that chemicals leaching from them might also have a potential role in the observed outcome. Overall data provide new insights into the mechanism of action of PS-NH2 NPs during chordate embryogenesis and offer further keys for a better knowledge of nanoplastics impact on early stages of marine life.

Biological Reviews - 2023 - Rosner

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