**2. Toxicity assessment with** *Artemia* **spp. and its advantages**

The predominant EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) legislation with the aim of sound management of the ecoenvironment and protection of human societies promoted the decrease in the use of vertebrates and encouraged the use of invertebrates and plants, as well as organ, tissue, and cell cultures, as alternative study materials for toxicity and ecotoxicity testing [10]. Among various invertebrates screened and assessed to investigate their sensitivity to several physical and chemical substances, brine shrimps, *Artemia* spp., which are extremely sensitive to toxicity, stand out as one of the most frequently used species for toxicity testing [11] and are recognized and listed by the US Environmental Protection Agency [12] as the model organism for toxicity testing and emission monitoring.

*Artemia* sp. is a crustacean adapted to harsh conditions such as those in hypersaline lakes [13], living mainly on phytoplankton [14, 15]. It is closely related to other zooplankton such as copepods and daphnia (**Figure 1**) [16]. Normally, it is routinely employed as a test organism for ecotoxicological studies. The molecular, cellular, and physiological states of *Artemia* spp. change dramatically when they are under contamination stress [17]. At present, a variety of toxicity tests with *Artemia* spp. have been carried out covering both short-term acute and long-term chronic methods (**Table 1**), with the former being the more frequently used. Acute toxicity tests, which are highlighted in this paper, mainly assess the effect exposure to relatively high concentrations (at a mg/L level) for no more than 4 days (96 h). Toxicity under normal conditions is expressed as the lethal concentration causing the death of half of the tested animals (LC50) and is also manifested in impeded hatching and swimming behavior. Chronic toxicity tests mainly have to do with the long-term exposure to relatively low concentrations (at a μg/L level) ranging from a few weeks up to the entire life cycle of *Artemia* spp. [18].

**15**

marine discharges [29].

**Table 1.**

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

Hatching Dry biomass

Swimming Speed

Immobilization Mortality

Reproduction Mating

Immobilization Mortality *PS: AChE = acetylcholinesterase; HSP = heat stress proteins; LP = lipid peroxidation; TBARS = thiobarbituric acid reactive substances; TRed = thioredoxin reductase; GPx = glutathione peroxidase; GST = glutathione S-transferase;* 

*GRed = glutathione reductase; ALDH = aldehyde dehydrogenase; and ATPases = adenyltriphosphatase*

Long-term Growth Body size

*Summary of Artemia short- and long-term toxicity tests [19].*

HSP Fluotox

Size

Teratogenicity

Path length

Weight

LP, TBARS, and TRed GRed, GPx, and GST ALDH and ATPases

Morphological disorder

Morphological disorder

Reproductive rate Offspring

**Test type Method Parameter index**

Short-term Biomarker AChE

Considering the environmental aspect, *Artemia* spp. nauplii were employed to assess the toxicity of various hazardous metal substances such as As, Cr, Sn, etc. [19–22]; organic compounds including pharmaceuticals, agrichemicals, etc. [23–26]; and environmental media such as wastewater [27], seawater [28], and

The principal advantages of using *Artemia* spp. in toxicity testing are as follows:

(1) rapidity in hatching, (2) cost-efficiency, and (3) commercial availability of nauplii hatched from durable cysts, which dispenses with the need for self-culturing [30, 31]. Moreover, other significant factors that have been taken into consideration include good cognition of its biological and ecological features, small size allowing for easy laboratory operation, as well as its well-developed adaptability to diversified testing conditions [30, 32]. It is noteworthy that the complex adaptive response evolved by *Artemia* to live through and thrive in critical conditions not only explains why it is a favorable candidate for toxicity testing but to some extent also offers insights with regard to biological and environmental perspectives, which in turn might contribute to toxicity testing itself and eventually the well-being of human populations. With that being said, the response mechanism developed by *Artemia* to deal with harsh conditions [13] is worth mentioning (see **Figures 2** and **3**). The harsh living condition is exemplified in hypersaline lakes (salty lakes) where *Artemia* is often the only macroplanktonic inhabitant [13]. The survival and reproduction of

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

**Figure 1.** *An adult of Artemia spp.: male (left) and female (right).*


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

*PS: AChE = acetylcholinesterase; HSP = heat stress proteins; LP = lipid peroxidation; TBARS = thiobarbituric acid reactive substances; TRed = thioredoxin reductase; GPx = glutathione peroxidase; GST = glutathione S-transferase; GRed = glutathione reductase; ALDH = aldehyde dehydrogenase; and ATPases = adenyltriphosphatase*

#### **Table 1.**

*Assessment and Management of Radioactive and Electronic Wastes*

is a remarkable similarity in cellular structure, signaling processes, anatomy, and physiology, particularly in the early stages of development [4–8]. Current estimates show that more than 90% of the human open reading frames are homologous to those in the genes of this fish [9]. Thus, investigations using this model system can

The predominant EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) legislation with the aim of sound management of the ecoenvironment and protection of human societies promoted the decrease in the use of vertebrates and encouraged the use of invertebrates and plants, as well as organ, tissue, and cell cultures, as alternative study materials for toxicity and ecotoxicity testing [10]. Among various invertebrates screened and assessed to investigate their sensitivity to several physical and chemical substances, brine shrimps, *Artemia* spp., which are extremely sensitive to toxicity, stand out as one of the most frequently used species for toxicity testing [11] and are recognized and listed by the US Environmental Protection Agency [12] as the model organism for toxicity testing and emission monitoring.

*Artemia* sp. is a crustacean adapted to harsh conditions such as those in hypersaline lakes [13], living mainly on phytoplankton [14, 15]. It is closely related to other zooplankton such as copepods and daphnia (**Figure 1**) [16]. Normally, it is routinely employed as a test organism for ecotoxicological studies. The molecular, cellular, and physiological states of *Artemia* spp. change dramatically when they are under contamination stress [17]. At present, a variety of toxicity tests with *Artemia* spp. have been carried out covering both short-term acute and long-term chronic methods (**Table 1**), with the former being the more frequently used. Acute toxicity tests, which are highlighted in this paper, mainly assess the effect exposure to relatively high concentrations (at a mg/L level) for no more than 4 days (96 h). Toxicity under normal conditions is expressed as the lethal concentration causing the death of half of the tested animals (LC50) and is also manifested in impeded hatching and swimming behavior. Chronic toxicity tests mainly have to do with the long-term exposure to relatively low concentrations (at a μg/L level) ranging from a few weeks up to the

reveal subtle interactions that are likely to be conserved across species.

**2. Toxicity assessment with** *Artemia* **spp. and its advantages**

**14**

**Figure 1.**

entire life cycle of *Artemia* spp. [18].

*An adult of Artemia spp.: male (left) and female (right).*

*Summary of Artemia short- and long-term toxicity tests [19].*

Considering the environmental aspect, *Artemia* spp. nauplii were employed to assess the toxicity of various hazardous metal substances such as As, Cr, Sn, etc. [19–22]; organic compounds including pharmaceuticals, agrichemicals, etc. [23–26]; and environmental media such as wastewater [27], seawater [28], and marine discharges [29].

The principal advantages of using *Artemia* spp. in toxicity testing are as follows: (1) rapidity in hatching, (2) cost-efficiency, and (3) commercial availability of nauplii hatched from durable cysts, which dispenses with the need for self-culturing [30, 31]. Moreover, other significant factors that have been taken into consideration include good cognition of its biological and ecological features, small size allowing for easy laboratory operation, as well as its well-developed adaptability to diversified testing conditions [30, 32]. It is noteworthy that the complex adaptive response evolved by *Artemia* to live through and thrive in critical conditions not only explains why it is a favorable candidate for toxicity testing but to some extent also offers insights with regard to biological and environmental perspectives, which in turn might contribute to toxicity testing itself and eventually the well-being of human populations. With that being said, the response mechanism developed by *Artemia* to deal with harsh conditions [13] is worth mentioning (see **Figures 2** and **3**). The harsh living condition is exemplified in hypersaline lakes (salty lakes) where *Artemia* is often the only macroplanktonic inhabitant [13]. The survival and reproduction of

#### **Figure 2.**

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

#### **Figure 3.**

*The reproduction of Artemia brine shrimp (individuals, populations, and species) subject to critical life conditions imposed by salty lakes.*

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

**17**

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

**3. Application status of the toxicity assessment with** *Artemia* **spp.**

Ecotoxicological studies employing *Artemia* spp. as testing species have been extensively performed, and among the endpoints that were mainly investigated, acute mortality, acute cyst hatchability, as well as behavioral response, as a result of

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

the most environmental stress-resistant among all animal life history forms, while motile stages are the best osmoregulators in the animal kingdom [33]. Cysts are gene banks that store a genetic memory of historical population conditions. They play a role aiding in the dispersal of *Artemia* and serve as reservoirs of genetic variability

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

[34] and the source of evolutionary change and resilience.

their relatively high sensitivity, are commonly used.

considered a safe human dose for a given chemical.

**3.1 Acute mortality test**

the most environmental stress-resistant among all animal life history forms, while motile stages are the best osmoregulators in the animal kingdom [33]. Cysts are gene banks that store a genetic memory of historical population conditions. They play a role aiding in the dispersal of *Artemia* and serve as reservoirs of genetic variability [34] and the source of evolutionary change and resilience.
