**3.1 Acute mortality test**

*Assessment and Management of Radioactive and Electronic Wastes*

*The life cycle and different stages of Artemia as a salty survivor.*

the brine shrimp *Artemia* (individuals, populations, and species) subject to critical life conditions imposed by salty lakes, as schemed in **Figures 2** and **3**, may be summarized as follows: (1) Females are able to cope with the forthcoming environmental conditions by switching the type of offspring to produce either cysts under stressful conditions or free-swimming nauplii under stable conditions, and (2) cysts are

*The reproduction of Artemia brine shrimp (individuals, populations, and species) subject to critical* 

**16**

**Figure 3.**

*life conditions imposed by salty lakes.*

**Figure 2.**

Acute mortality is one of the most commonly used endpoints for toxicity testing, though there is no standardized protocol based on OECD and ISO regulations. Since the establishment of the *Artemia* Reference Center (ARC-test) and the issuance of the first short-term acute mortality (24 h static test) protocol with *Artemia* larvae [35–38], extensive toxicity assessment research using this bioassay has been carried out via calculating the median effectiveness concentration on mortality (24 h LC50). Besides observation of lethal endpoints for *Artemia* exposed to reference toxicants including CuSO4, K2Cr2O7, and SDS [39, 40], many are related with toxicity monitoring of environmental pollutants such as heavy metals, pesticides, oil drilling fluids, organic compounds of ecotoxicological concern, and others [41–44]. Indeed, in the wake of various environmental issues challenging humans and living surroundings, the importance of toxicity assessment using *Artemia* has been gradually recognized and more frequently employed. The following are two examples in recent years.

The "Brine Shrimp Lethality" study is one of the biological assays to determine the safe exposure limit of naturally occurring agents extracted from plants before being used as pesticides for crops and for other botanical protections [16]. Crop protection is one of the important food safety-related issues and is thus vital to human populations worldwide. As crop protection nowadays rely heavily on synthetic pesticides [45], the massive use of these pesticides for the purpose of killing pests and preventing diseases in plants has inevitably led to several side effects such as pest resistance resulting in the use of increased application rates [46], harm to nontarget organisms, and environmental contaminations with the potential influence on the food chain [47] that might cause pesticide poisoning of humans directly. Botanically derived natural products therefore have attracted attention among phytochemists. "Brine Shrimp Lethality," a rapid general bioassay, offers a unique advantage in the standardization and quality control of those bioactive compounds that are usually undetectable using traditional physical analytical methods. The objective of carrying out the biological assay focuses on establishing a cause-effect relationship (**Figure 4**) between exposure to a hazardous substance and an appeared effect expressed by dose-response curve to determine a safe exposure limit [48]. The threshold level as well as the toxicity features obtained from the dose-response curves can help determine the safe levels of chemicals in botanical extracts and chemical exposure [49]. The threshold information (ThD0.0) measured in mg/kg/day and based on the assumption that human beings are as sensitive as the tested animals; in this case the brine shrimp *Artemia* sp. is of paramount importance in generalizing animal data to humans and interpolating what might be considered a safe human dose for a given chemical.

**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 benefit the offshore fishery industry.

#### **3.2 Acute cyst hatching test**

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 ecotoxicological data in an extensive manner.

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

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],

**19**

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

also known as the Swimming Speed Alteration (SSA) recording system, which has already been used on the brine shrimp, *Artemia* [68]. Moreover, the research results of Garaventa et al. [68] and Manfra et al. [69] showed that swimming speed was more sensitive than mortality and had a sensitivity similar to and sometimes higher than that of the hatching rate endpoint. Therefore, it is a well-defined behavioral response and an adaptable endpoint that can be used for ecotoxicity testing. For instance, Manfra et al. [69] recorded the swimming speed alteration of *Artemia* exposed to diethylene glycol (DEG), an organic substance ecotoxicological concern, and observed a decline in the swimming speed under the toxicant concentration of 40–160 g/L after 24 h exposure and 10–160 g/L after 48 h exposure. Another example is related with marine pollution such as oil spilling, oil mining, and oily water discharge that can greatly threaten human health as contaminants can be accumulated in the human body through the food chain. In this regard, *Artemia* spp., as one of the toxicity-monitoring species, is of great importance in the evaluation of the health of the marine ecosystem. Pan [70] investigated the swimming speed and motion angle alteration of *Artemia* exposed to diesel oil. For comparison purposes, when experiments were carried out under normal conditions, namely, seawater, the swimming speed of *Artemia* increased by 51%, from 2.47 mm/s at the start time to 3.72 mm/s after 12 h exposure on average, and in a similar trend, the motion angle of *Artemia* increased from 25 to 37°. In contrast, when subject to diesel oil, the swimming speed of *Artemia* decreased by 40%, from 2.37 mm/s at the start time to 1.42 mm/s after 12 h exposure on average, and in a similar trend, the

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

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 govern-

ment, 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

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

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

motion angle of *Artemia* decreased from 30 to 21°.

be further developed in the future.

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

also known as the Swimming Speed Alteration (SSA) recording system, which has already been used on the brine shrimp, *Artemia* [68]. Moreover, the research results of Garaventa et al. [68] and Manfra et al. [69] showed that swimming speed was more sensitive than mortality and had a sensitivity similar to and sometimes higher than that of the hatching rate endpoint. Therefore, it is a well-defined behavioral response and an adaptable endpoint that can be used for ecotoxicity testing. For instance, Manfra et al. [69] recorded the swimming speed alteration of *Artemia* exposed to diethylene glycol (DEG), an organic substance ecotoxicological concern, and observed a decline in the swimming speed under the toxicant concentration of 40–160 g/L after 24 h exposure and 10–160 g/L after 48 h exposure. Another example is related with marine pollution such as oil spilling, oil mining, and oily water discharge that can greatly threaten human health as contaminants can be accumulated in the human body through the food chain. In this regard, *Artemia* spp., as one of the toxicity-monitoring species, is of great importance in the evaluation of the health of the marine ecosystem. Pan [70] investigated the swimming speed and motion angle alteration of *Artemia* exposed to diesel oil. For comparison purposes, when experiments were carried out under normal conditions, namely, seawater, the swimming speed of *Artemia* increased by 51%, from 2.47 mm/s at the start time to 3.72 mm/s after 12 h exposure on average, and in a similar trend, the motion angle of *Artemia* increased from 25 to 37°. In contrast, when subject to diesel oil, the swimming speed of *Artemia* decreased by 40%, from 2.37 mm/s at the start time to 1.42 mm/s after 12 h exposure on average, and in a similar trend, the motion angle of *Artemia* decreased from 30 to 21°.
