Intercalibration of ecotoxicity testing protocols with Artemia franciscana
Introduction
In recent years, many countries’ environmental legislation has introduced the use of bioassays as additionally recommended analyses assessing the status of aquatic environments (i.e., water and sediment) and characterizing the ecotoxicity of chemicals (Chapman, 2007). Toxicity tests using a wide variety of taxa have been developed and new methodologies are in progress (Gorbi et al., 2012). In 2014, three national standard toxicity test methods were published: (a) two protocols (acute and chronic mortality) with the copepod Acartia tonsa Dana (Gorbi et al., 2012); and (b) one protocol (acute swimming behavior) with the cirripede Balanus amphitrite L. (Piazza et al., 2012).
Artemia spp. (Crustacea, Anostraca) is among the most commonly used live food sources in aquaculture; it has a key role in the food chain energy flow in the marine environment and is frequently utilized as a saltwater biological model in ecotoxicology (Migliore et al., 1997). Its use is well documented in the scientific literature of the past 30 years (Nunes et al., 2006, Libralato et al., 2007, Libralato, 2014). The main advantage of the species in this context is that nauplii can be hatched from commercial available durable cysts (eggs), allowing homogeneity of the population and its continuous year-round use, without animal breeding in the laboratory, as required for almost all the species used in ecotoxicity tests (Manfra et al., 2012). Other advantages are: (a) good knowledge of its biology and ecology; (b) easy manipulation and maintenance under laboratory conditions; (c) small body size that allows accommodation in small beakers or plates; (d) adaptability to a wide range of salinities and temperatures (USEPA, 2002).
Some criticisms about Artemia sensitivity have been presented in the context of a learning-by-doing approach (Libralato et al., 2010, Libralato, 2014). For example, the cysts’ production may reflect the occurrence of genetic variation caused by their geographical origin that is rarely known (Migliore et al., 1997), although certified cysts are usually utilized in toxicity testing.
Various toxicity test protocols are available in the literature and are easily applicable in a minimally equipped laboratory by personnel with little experience. Short-term toxicity test protocols (≤96-h) are the most common. They include various endpoints: (a) survival (Persoone et al., 1993, Guzzella, 1997, Artoxkit, 2014); (b) hatching and growth (Migliore et al., 1997, Sarabia et al., 2008); and (c) behavioral (i.e., swimming) (Garaventa et al., 2010, Gambardella et al., 2014). Some long-term protocols have also been studied in the last 10 years, showing that survival is the most sensitive endpoint compared to growth and reproduction (Brix et al., 2003, Brix et al., 2004, Savorelli et al., 2007, Manfra et al., 2012, UNICHIM, 2012). These long-term procedures employ the larval developmental stages of Artemia spp. (Instar II–III), which are the most sensitive stages in the entire life cycle (Gorbi et al., 2012).
The existence of several approaches but the absence of standardized methods (ISO, ASTM or OECD) with Artemia spp. is a crucial gap that should be filled as soon as possible to make Artemia spp. an official standard biological model in ecotoxicology and nanoecotoxicology (Libralato, 2014). The availability of standardized protocols is a great concern for all test species because the procedures should be reproducible in different laboratories and from different operators. In particular, Artemia spp. is widely used in several countries as a result of the above-described advantages and the rich storehouse of information existing in the literature for this species.
Despite the frequent and widespread use of Artemia spp. in toxicity testing, the harmonization of protocols followed by standardization activities is still lacking, and round-robins are urgently necessary (Libralato, 2014). To date, only the results from the 24-h toxicity test intercalibration exercises have been published, providing data on copper sulfate as a reference toxicant (Persoone et al., 1993).
To remedy this lack of information, the aim of this paper is to present the Italian intercalibration outcomes of three toxicity-testing protocols with Artemia franciscana: (a) the static acute mortality test (24-h); (b) the static acute hatching test (48-h); and (c) the static-renewal long-term test (14-d).
Attention was mainly focused on these methods because: (a) the 24-h mortality test is routinely used for toxicity screening, such as in the case of industrial pollution monitoring or within specific national regulatory requirements; (b) the 48-h hatching procedure is easy to perform, sensitive in a short-time-lag framework and performed on a huge number of individuals (Migliore et al., 1997); (c) the 14-d mortality test presents the same starting conditions (hatched nauplii as testing organisms) of the 24-h protocol, being already recognized by UNICHIM (UNI associated agency, the Italian organization devoted to standardization and unification of methods) and illustrated in a peer reviewed scientific video journal (Manfra et al., 2012). As a preliminary step, a reference laboratory assessed the repeatability of the suggested protocols. Afterwards, seventeen laboratories (three Institutes from the National Research Center, three Universities, one private laboratory and 10 Regional Environmental Protection Agencies) were involved in evaluating the reproducibility of protocols; these laboratories conducted intercomparison exercises on two reference toxicants.
Section snippets
Intercalibration design and set-up
National intercomparison exercises took place between 2006 and 2009 with 11 participating laboratories. These laboratories were designated Lab 1, Lab 2, Lab 3, Lab 4, Lab 5, Lab 6, Lab 7, Lab 8, Lab 9, Lab 10 and Lab 11.
All participating laboratories were supplied with: (a) certified cysts; (b) reference toxicants; (c) algal cultures (as brine shrimp feeding only for the 14-d test); (d) detailed protocols (also for microalgae culturing) and (e) specific training courses organized by the
Acute mortality method
All LC50 values generated by participants were considered valid because the mortalities of control tests were <10% (Guzzella, 1997, Artoxkit, 2014). The values of mean LC50, standard deviation (SD), coefficient of variation (CV) and repeatability (r) of the reference laboratory are reported in Table 2. The CV value and the differences between results obtained in two different repetitions (not greater than r value) indicated acceptable intra-laboratory test–reliability. In Fig. 1A, the mean LC50
Discussion
The intercomparison exercises with A. franciscana involved eleven, seven and nine participants for the acute mortality test, the acute hatching test and the long-term mortality test, respectively. Most of the participants in the intercalibration activities already knew the acute mortality test protocol. This method did not require specific technical expertise but simply required that the operator pay attention when transferring the hatched larvae from the Petri dish to the multi-well plate. The
Conclusions
Intercalibration exercises are highly essential steps in standardization processes. This study focused on a series of round-robins to validate and standardize the acute mortality test, the acute hatching test and the long-term mortality test. The outcomes from the inter-laboratory exercises suggested a good repeatability and reproducibility of all protocols. This favorable result was demonstrated by intralaboratory coefficients of variation <30% and interlaboratory coefficients of variation
Acknowledgements
The interlaboratory test was partially funded by the Italian Ministry of the Environment and Territory and Sea Protection. The sponsor did not participate in the study design, the collection, analysis or interpretation of data, the writing of the report or the decision to submit the article for publication. The authors are grateful to Andrea Tornambè for his support in the development and production of the hatching data, and the authors especially thank the staff of all laboratories that
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