**4. Prospects for development of toxicity assessment with** *Artemia* **spp.**

To rapidly figure out the deleterious effects brought about by environmental toxicants, acute toxicity assessment with *Artemia* spp. is of paramount importance as it shows a decent ability in pre-screening of toxic substances [10] and, thus, will be further developed in the future.

Despite the widespread application of this bioassay, there is currently no internationally standardized method. Hence, intercalibration exercises as well as international standardization activities are rather necessary [71]. Among the three frequently used endpoints involving acute mortality, acute cyst hatchability, as well as behavioral response, acute mortality was intercalibrated based on the available standards [40, 69, 72], while acute hatchability was intercalibrated at the Italian level [69]. To make *Artemia* spp. an international standard model in ecotoxicity testing calls for joint efforts engaging all relevant stakeholders including the government, NGOs, researchers, industry, consumer associations, and others.

Swimming speed as the most popular behavioral endpoint promises to be of great potential. This is because results can be obtained via easy video camera analysis at ease and also because the swimming speed is of great ecological significance as the behavior alteration means an integral whole body response that can connect the physiological and ecological features of an organism with its environment [73]. Nevertheless, to better employ this endpoint, the interaction of *Artemia* spp. with contaminants, particularly the mechanisms of response to toxic effect, needs to be illuminated.

One is to believe that owing to the advantages of using *Artemia* spp. as the biological model described in the previous section of this paper, besides toxic testing application itself, application into other environmentally related fields such as applied biology might also be put into practice. For example, from a bio-conservation point of view, the unique biological characteristics of brine shrimp *Artemia* make it a model organism to evaluate management policies for the protection of aquatic

*Assessment and Management of Radioactive and Electronic Wastes*

benefit the offshore fishery industry.

ecotoxicological data in an extensive manner.

**3.3 Acute behavioral test (swimming speed)**

**3.2 Acute cyst hatching test**

**Figure 4.** *Dose-response curve.*

Another example in relation to the *Artemia* acute toxicity test [50, 51] is for the purpose of prevention and reduction of red tides. The red tide induced by algae is quite disastrous and may pose a threat to inshore fishery. The poisonous *Chattonella marina* that produces reactive oxygen species (ROS) [52] and hemolytic toxins [53] is one kind of red tide-related algae and has caused massive fish death and a considerable amount of economic loss in many places around the world. The "Brine Shrimp Lethality" study in this regard can help reveal the toxic characteristics of *Chattonella marina*, offer some valuable red tide prevention evidences, and further

Analogous to the acute mortality test, acute cyst hatching testing, which observes the retarded emergence of nauplii from cysts [54] or the morphological disorders and size of hatched nauplii [55] when exposed to toxic agents, is another frequently used assay for toxicity assessment. The hatching toxicity test lasting between 24 and 96 h in static conditions was investigated to assess the effect of environmentally deleterious agents such as heavy metals [54, 56, 57], organic compounds [58, 59], antibiotic drugs [60], and others. As temperature profoundly influences the hatching percentage of cysts [61] and significantly affects the chemicals' effect [62], it is a variable of great interest to be considered while carrying out the hatching test, and the use of a full temperature range might help increase the

Regarding the acute behavioral test, motion behavior changes in response to pollutant exposure have been investigated for a range of aquatic organisms [63–67]. In particular, swimming speed as a sublethal behavioral endpoint can be detected by employing a video camera tracking system developed by Faimali et al. [63],

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resources [74]. *Artemia* is such a versatile creature that it is a paradigmatic model having not only scientific research values but also the ability to satisfy human needs, owing to its unique life traits including a well-developed adaptability to high salinity conditions as well as easy handling under laboratory conditions, which have been successfully applied to marine fish farming that uses *Artemia* nauplii as food for fish larvae. However, the booming marine fish farming activity worldwide is likely to give rise to some risks in terms of the high genetic divergence between different *Artemia* species. Exploitation of new *Artemia* cyst harvesting sites and introduction of an exotic species linked to traits relevant to aquaculture can drive other local genotypes to extinction. Risk assessment and evaluation of management decisions in exploited resources, for instance, the availability of genetic information as well as molecular tools for follow-up gene pool monitoring, therefore, become quite necessary in order to maintain biodiversity. Gene banks established from cysts collected from various sites guarantee population persistence while proceeding with management affairs. Taking into account the simple constitution of hypersaline habitats, the evaluation of population/species persistence with *Artemia* can be modeled in laboratories and further extrapolated to other species, offering some of the aspects regarding rational aquatic resource utilization and, more importantly, biodiversity preservation.

### **5. Conclusions**

After more than five decades of use in ecotoxicology, *Artemia* spp. have demonstrated its suitability for use in pre-screening of toxic agents [10]; thus, it seems that *Artemia* sp. endpoints may be used as a toxicity testing method to meet market demand, even though there are no internationally standardized toxicity testing protocols at present according to the ISO and OECD.

Biomarkers and teratogenicity are the less popular endpoints used in short-term toxicity tests because of their limited sensitivity. However, behavioral endpoints, especially swimming inhibition, seem to have a wider application potential in the future with the development of computer technology. Both continuous and intermittent observations of single or groups of living organisms can be studied by image and video analysis. Hatching rate and acute mortality are the most commonly used endpoints in the standardization process at a different level. Usually, hatching rate (48 h static test) was intercalibrated at the Italian level [69], while acute mortality (24 h static test) was intercalibrated based on the available standard [40] at the Italian [69] as well as the European level [72]. Both provided data on CuSO4 as a reference toxicant. Among the long-term toxicity tests, the 14-d static renewal mortality test was intercalibrated at the Italian level [69] with SDS according to the UNICHIM protocol (2012).

Further concentrated efforts are necessary to make *Artemia* sp. an official internationally recognized standard biological model in ecotoxicology evaluation. It involves (I) a national member (who then contacts the ISO) upon a request by an industry sector or group for a standard; (II) scope, main definitions, and contents of standards which are scientifically assessed by experts in relevant fields; and (III) multi-stakeholder discussion and reviewing process including experts from related industries, consumer associations, academic institutions, nongovernmental organizations, and governments.

### **Acknowledgements**

The authors of this study express their gratitude to the National Natural Science Foundation of China (No. 31600257), Public Projects of Zhejiang Province

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**Author details**

Yin Lu\* and Jie Yu

Hangzhou, China

provided the original work is properly cited.

\*Address all correspondence to: luyin\_zjsru@aliyun.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

College of Biology and Environmental Engineering, Zhejiang Shuren University,

*A Well-Established Method for the Rapid Assessment of Toxicity Using* Artemia *spp. Model*

(No. 2016C32022), Academic Climbing Project for Young and Middle-Aged Leads in Universities of Zhejiang Province (pd2013339), and Project of Zhejiang Provincial Department of Education (Y201738582) for their financial supports of this study.

*DOI: http://dx.doi.org/10.5772/intechopen.85730*

*A Well-Established Method for the Rapid Assessment of Toxicity Using* Artemia *spp. Model DOI: http://dx.doi.org/10.5772/intechopen.85730*

(No. 2016C32022), Academic Climbing Project for Young and Middle-Aged Leads in Universities of Zhejiang Province (pd2013339), and Project of Zhejiang Provincial Department of Education (Y201738582) for their financial supports of this study.
