Elsevier

Ecological Indicators

Volume 69, October 2016, Pages 35-49
Ecological Indicators

A review of toxicity testing protocols and endpoints with Artemia spp.

https://doi.org/10.1016/j.ecolind.2016.04.017Get rights and content

Highlights

  • We presented an overview of toxicity testing methods with Artemia spp.

  • No international standards for hatching, swimming, immobilization and mortality.

  • Cysts hatching conditions before testing, varied greatly.

  • Exposure conditions and data presentation limited their comparison.

  • Need to standardize the use of Artemia spp. as a reference biological model.

Abstract

Artemia spp. is an historically popular biological model still requiring an official internationally based standardization. Several endpoints are currently available. Short-term acute endpoints include biomarker (acetylcholinesterase; heat stress proteins; lipid peroxidation; thiobarbituric acid reactive substances; thioredoxin reductase; glutathione-peroxidase; glutathione S-transferase; glutathione reductase; aldehyde dehydrogenase; and adenylpyrophosphatase and Fluotox), hatching (dry biomass, morphological disorders and size), behavioral (swimming speed and path length), teratogenicity (growth), and immobilization (meaning mortality after 5–30 s observation). Long-term chronic tests focus on growth, reproduction and survival or mortality after 7–28 d exposure from larval to adulthood stage. We analyzed each test looking at its endpoint, toxicant and experimental design including replicates, exposure time, number of exposed cysts or organisms and their relative life stage, exposure conditions during hatching and testing (salinity, pH, light intensity, aeration dilution media, and food supply), type of testing chambers, and quality assurance and quality control criteria. Similarities and differences between the identified approaches were highlighted. Results evidenced that hatching 24 h short-term and 14 d long-term mortality are the most promising Artemia spp. protocols that should go forward with international standardization.

Introduction

The adoption and implementation of the European legislation about the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) (EC, 2006) required several additional ecotoxicity data promoting the decrease of vertebrates used in toxicity testing encouraging alternative strategies with invertebrates, plants as well as organ, tissue, and cell cultures (Dvorak et al., 2012). During the last 50 years, various invertebrates were assessed to investigate their sensitivity to many physical and chemical agents for their possible use as pre-screening or screening models. Internationally, Artemia spp. brine shrimps (Crustacea, Branchiopoda, Anostraca), commonly known also as sea monkeys, are one of the most frequently used species for toxicity testing (Van Steertegem and Persoone, 1993a, Van Steertegem and Persoone, 1993b).

Artemia spp. is a major taxon in many hypersaline biotypes throughout the world feeding primarily on phytoplankton and being an important primary consumer (Persoone and Sorgeloos, 1980; Vanhaecke et al., 1987; Triantaphyllidis et al., 1998). They are of economical importance being used in aquaculture and in aquariology. They also act as an efficiency and productivity stimulator for salt production in solar salt works (Jones et al., 1981, Migliore et al., 1997, Treece, 2000).

The main advantages of using brine shrimps in toxicity testing are: (i) rapidity (i.e. 28–72 h from hatching to the first endpoint); (ii) cost-effectiveness;(iii) the availability of nauplii hatched from commercial durable cysts (eggs) (i.e. homogeneity of the population, availability all year-round without the necessity of culturing) (Nunes et al., 2006a, Manfra et al., 2012). Other advantages are: (i) good knowledge of its biology and ecology; (ii) easy manipulation and maintenance under laboratory conditions; (iii) small body size allowing accommodation in small beakers or microplates; (iv) high adaptability to various testing conditions (Nunes et al., 2006a; Kokkali et al., 2011). Conversely, several criticisms about Artemia spp. sensitivity were presented by a learning-by-doing approach (Libralato et al., 2010a, Libralato et al., 2010b, Libralato, 2014). For example, the cysts’ production can reflect the occurrence of genetic variation caused by crustaceans’ geographical origin that is rarely known (Migliore et al., 1997), although certified cysts are usually utilized in toxicity testing. Their origin can have consequences on the growth, survival and reproduction of Artemia spp. specimens considering especially salinity and temperature (Vanhaecke and Sorgeloos, 1989; Triantaphyllidis et al., 1995).

Artemia spp. nauplii were used to test the toxicity of a wide range of chemicals such as arsenic (As) (Brix et al., 2003), cadmium (Cd) (Kissa et al., 1984, Hadjispyrou et al., 2001, Sarabia et al., 1998a, Sarabia et al., 2002, Sarabia et al., 2006, Brix et al., 2006; Leis et al., 2014), chromium (Cr) (Hadjispyrou et al., 2001; Leis et al., 2014), cobalt (Kissa et al., 1984), copper (Cu) (Browne, 1980, Jorgensen and Jensen, 1977, Brix et al., 2006), mercury (Hg) (Sarabia et al., 1998b; Leis et al., 2014), nickel (Kissa et al., 1984), tin (Sn) (Hadjispyrou et al., 2001), zinc (Zn) (Brix et al., 2006, Garaventa et al., 2010), potassium permanganate, potassium dichromate, and silver nitrate (Boone and Baas-Becking, 1931, Vanhaecke et al., 1980), antibiotic drugs (Migliore et al., 1993a, Migliore et al., 1993b, 1997), engineered nanomaterials (Libralato, 2014, Minetto et al., 2014, Corsi et al., 2014, Callegaro et al., 2015), nano-sized polystyrene (Bergami et al., 2016), asbestos (Stewart and Schurr, 1980), phenolic compounds (Guerra, 2001), ethanolamines (Libralato et al., 2010a) and trace elements (Petrucci et al., 1995), triazine herbicides, insecticides, pesticides (Kuwabara et al., 1980, Varó et al., 1997, Varó et al., 2002), acrylonitrile (Tong et al., 1996), carbammates (Barahona and Sánchez-Fortún, 1999), phthalates, antifouling agents (Grosch, 1980, Persoone and Castritsi-Catharios, 1989a, Persoone and Castritsi-Catharios, 1989b, Okamura et al., 2000, Castritsi-Catharios et al., 2007, Castritsi-Catharios et al., 2013, Castritsi-Catharios et al., 2014, Koutsaftis and Aoyama, 2007), pharmaceuticals (Xu et al., 2015), anticorrosive agents (Tornambè et al., 2012; Manfra et al., 2015a, Manfra et al., 2016), oil (Trieff, 1980) and oil dispersants (Zillioux et al., 1973, Savorelli et al., 2007), various plant extracts (Cáceres et al., 1998), toxins (Granade et al., 1976, Medlyn, 1980, Vezie et al., 1996, Beattie et al., 2003) and environmental matrices such as wood leachates (Libralato et al., 2007), wastewater (Krishnakurmar et al., 2007, Libralato et al., 2010b), seawaters (Manfra et al., 2011) and marine discharges (Manfra et al., 2010).

Currently, various toxicity tests with Artemia spp. are available including short-term and long-term methods. Short-term toxicity tests are more frequently used, some long-term protocols have been developed in the last 10 years, but none of them is an internationally standardised method like International Standard Organization (ISO), American Society for Testing and Materials (ASTM) or Organization for Economic Co-operation and Development (OECD). Methods for testing immobilization/mortality were standardised only in Italy by the Italian Agency for Environmental Protection and Italian Institute for Water Research (APAT IRSA-CNR) and Italian Agency for Standardization in the Chemical sector (Unichim). Despite the frequent and widespread use of Artemia spp. in toxicity testing, the harmonization of protocols followed by international standardization activities is still lacking, and intercalibration exercises are urgently necessary (Libralato, 2014).

The aim of this review paper is to collect, organize, select and discuss the existing knowledge about Artemia spp. methods for toxicity testing including both short- and long-term bioassays and organism hatching and maintenance conditions providing tips for protocols definition, implementation and standardization.

Section snippets

Hatching of cysts

Artemia spp. cyst hatching conditions can vary greatly as reported in Table S1 (n = 42). This can result in a different evaluation of cysts/nauplii sensitivity, although the first factor that can affect organism sensitivity is the geographical origin of cysts. Other species are commercially available, but their sensitivity must be evaluated on Vanhaecke et al. (1980) a case-by-case basis if no certification or traceability is available (Guzzella, 1997).

The second key point is to start the

Toxicity tests

We decided to cluster toxicity tests with Artemia spp. as short-term acute and long-term chronic bioassays (Fig. 1) considering the life span of Artemia spp. varying between 2–4 months depending on salinity, temperature and species-specific characteristics (Browne et al., 1991). Acute toxicity tests assess the effects based on relatively high exposure concentrations (i.e. mg/L) for no more than 96 h. Toxicity is generally expressed as lethal concentration causing the death of 50% of the group of

Discussion

After more than five decades of use in ecotoxicology, Artemia spp. demonstrated its ability mainly as pre-screening of toxic agents (Dvorak et al., 2012), thus Artemia spp. endpoints seem to respond to the market need of toxicity testing tools, even though no internationally standardised toxicity testing protocols currently exist according to OECD and ISO.

Among the short-term toxicity tests, biomarkers and teratogenicity are the less popular endpoints with just few papers and citations probably

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