Lethality Bioassay Using Artemia salina L.

Summary

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

Abstract

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Introduction

Natural products from plants, animals, or microorganisms have been a growing area of interest over the years in the development of new bioactive molecules because of their varied range of biological and pharmacological activities¹. However, the associated side effects, drug resistance, or inadequate specificity of the agents, especially when used as anticancer drugs, represent the major factors that can lead to ineffective treatment^1,2.

Over the last few decades, several plant-derived cytotoxic agents have been discovered, some of them used as anticancer agents^1,2,3. In this context, paclitaxel is reported as one of the best-known and most active chemotherapeutic drugs of natural origin^3,4. Currently, it is estimated that more than 35% of all medicines on the market are derived from or are inspired by natural products⁵. The potential high toxicity of these compounds requires consideration during all of the study phases, since different types of contaminants or even metabolic components of the plant itself can cause toxic effects. For this reason, pharmacological and toxicological profiles should be undertaken in the preliminarily phase, to assess the biological activity and safety of new potential plant-based treatments. To evaluate the toxicity of new bioactive samples, invertebrate animals can be considered as the best models to study. They demand minimal ethical requirements and allow preliminary in vitro assays, to prioritize the most promising products for the next round of testing in vertebrates^1,6.

Commonly known as brine shrimp, A. salina is a small halophilic invertebrate belonging to the genus Artemia (family Artemiidae, order Anostraca, subphylum Crustacea; Figure 1). In marine and aquatic saline ecosystems, brine shrimps play an important nutritional role as they feed on microalgae and are constituents of the zooplankton used to feed fish. Moreover, their larvae (known as nauplii) are widely used in the assessment of general toxicity during preliminary studies^1,3,7.

Artemia spp. are widely used in lethality studies and are also a convenient starting point for toxicity assessments, by tracking the toxicity of potentially bioactive compounds based on their ability to kill nauplii grown in the laboratory^1,8. For this reason, the use of A. salina gained attraction in general toxicity studies, because it is a very efficient and easy-to-use method, compared to other tests on animal models⁹.

Owing to their simple anatomy, tiny size and short life cycle, a vast number of invertebrates can be studied in a single experiment. As such, they combine genetic amenability and low-cost compatibility with large-scale screenings¹. In this context, the use of brine shrimp in a general toxicity assay shows several advantages, such as fast growth (28-72 h is needed from hatching to the first results), cost-effectiveness, and long shelf-life of commercial eggs, that can be used all year round^3,10. On the other hand, since invertebrates have a primitive organ system and lack an adaptive immune system, they do not represent a perfect and reliable model for human cells¹.

However, it provides a preliminary evaluation method for the general toxicity of selected samples. Since it is widely used as a lethality assay, it can provide provisional indications about the toxic effects of potential anticancer agents. It is often also used to obtain feedback about the general toxicity of compounds endowed with any other biological activities for which it is essential to show the lowest mortality rate possible among the Artemia shrimps.

In an ongoing study from our group, different extracts from Plectranthus species showed antioxidant and antimicrobial activities (unpublished results). In parallel, isolated compounds were obtained by purification of the extracts and were then chemically modified. The extracts, pure compounds, and semisynthetic derivatives were then tested in terms of general toxicity. In this context, the present work aims to give an overview of the use of the Artemia lethality bioassay for the evaluation of general toxicity and potential cytotoxic activity of bioactive extracts and isolated compounds from different plants of the genus Plectranthus¹¹.

Figure 1: Artemia salina under the microscope. Newly hatched nauplii of A. salina as seen under the microscope (magnification 12x). Please click here to view a larger version of this figure.

Protocol

1. Equipment preparation

1. Acquire commercially available hatching equipment. Select a suitable place to set up the hatching equipment (Figure 2A). Place the funnel-shaped container in the black support (included in the set) and turn the funnel in a suitable direction to see the level mark and the tap.
2. To make hand-made migration equipment, cut the top of two 0.5 L (5.8 cm diameter) plastic bottles to obtain a final height of 12 cm. Create a hole of 1.5 cm diameter on one side at 7 cm from the bottom in each bottle and insert a 13 cm rubber tube (1.3 cm outer and 0.9 cm inner diameter) between the two openings. Seal the openings with hot glue (Figure 2B) and leave to dry for 15 min; put the bottles on a flat surface and fill them with water to verify that there is no leakage.

2. Preparation of artificial salt solution

1. In a glass beaker, prepare an artificial salt solution (brine shrimp salt) at a concentration of 35 g/L. To do this, add 28 g of the salt to 800mL of tap water, according to the manufacturer's instructions. Mix it with a stirring rod until all the salt is thoroughly dissolved.
   NOTE: Adjust the volume of the prepared saline solution according to the size of the available containers.

3. Sample preparation

1. Prepare all the samples in a microcentrifuge tube by dissolving a suitable amount of extracts (Plectranthus extracts, Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; and Pe- P. ecklonii) or compounds 1-5 (two natural compounds [1 and 2] obtained from Plectranthus spp. and three semi-synthetic derivatives [3, 4, 5]; Figure 3) in dimethyl sulfoxide (DMSO)¹², so as to obtain a final concentration of 10 mg/mL (if the sample is water-soluble, use of DMSO is not necessary).
2. Dilute 10 µL of each sample (and DMSO for the negative control) in a new microcentrifuge tube using 990 µL of artificial saline solution prepared in step 2.1, to obtain a final concentration of 0.1 mg/mL.
3. Under a fume hood, in an Erlenmeyer flask, prepare a solution of potassium dichromate (K₂Cr₂O₇) in distilled water at a concentration of 1 mg/mL^13,14,15.

4. Brine shrimp lethality bioassay

NOTE: This assay is developed from the works of several authors with modifications^{1,16,17,18,19}.

1. Fill the hatching vessel with the medium prepared in step 2.1 up to the level mark (500 mL) (Figure 2C).
2. Place one spoon (approximately 0.75 g) of brine shrimp cysts in the salt solution, and then close the container. Place a lamp (table lamp, 40 W, 230 V, 50 Hz, with a LED light bulb of 8 W, 4,000 K, 830 lm) pointing directly toward the equipment (Figure 2A) and turn it on.
3. Attach the air supplier system (3 W output, 50 Hz, 230 V) to the connector placed on the top of the equipment and turn the pump on.
4. Keep the room temperature at 25 ± 3 °C. Brine shrimp cysts hatch in the artificial salt solution, under vigorous aeration, continuous lighting, and stable temperature, after 24 h to 48 h.
   NOTE: Alternatively, a vertical incubator can be used.
5. Once the eggs have hatched, turn the air pump off and wait until the nauplii (moving toward the bottom of the funnel) are separated from the empty egg cases (floating at the top).
6. In order to separate the unhatched eggs from alive nauplii, open the outlet tap at the bottom and discharge the content of the funnel in one of the containers of the hand-made migration equipment container (described in step 1.2). Ensure that the solution containing the nauplii and the residual unhatched eggs is below the level of the tube. In the second container, add the residual salt solution from step 2.1 above the height of the tube.
7. Cover the container with the nauplii and the residual unhatched eggs using aluminum foil. Place the lamp on the second container with just the salt solution. The brine shrimp will be attracted by the light and migrate from one container to the other (harvesting container), leading to efficient separation between eggs (slowly sedimented to the bottom) and alive Artemia.
8. Then, place the equipment in the incubator under the same conditions used in step 4.4 for 4 h (Figure 2E). From the harvesting container, collect 900 µL of saline solution containing 10 to 15 nauplii. Place the saline solution with nauplii in each well of a 24-well plate (Figure 2F); all the samples are tested in quadruplicates.
9. Add 100 µL each of the negative control (DMSO), the positive control (K₂Cr₂O₇, potassium dichromate), the artificial salt solution, and each of the samples to respective well (Figure 2F)^13,14.
   NOTE: The samples in each well will be at a concentration of 0.01 mg/mL. The final concentration of the positive control in salt solution will be 0.1 mg/mL, to be sure that all the nauplii in the well are exposed to the toxic effect of potassium dichromate and die. The artificial salt solution will act as blank.
10. Incubate the plate at 25 ± 3 °C under illumination for 24 h (Figure 2G). After 24 h, register the number of dead larvae (non-mobile nauplii for 5 s) in each well under a binocular microscope (12x)²⁰ (Figure 2H). Alternatively, use a hand lens.
11. Add 100 µL of potassium dichromate solution, to induce the death of the remaining living larvae, and wait for 6 h. Count the total dead larvae in each well under a microscope. Determine the mortality rate according to the following equation.
    
12. Perform all the assay in triplicate. Calculate standard deviations (SD), and express the results as the mean of three independent experiments, each with internal quadruplicates (n = 12), ± SD. As mentioned by Meyer et al., consider crude extracts and pure compounds with LC₅₀< 1,000 µg/mL as toxic; also, take into account that the mortality rate of brine shrimp is proportional to the concentration of the tested samples²¹.

Figure 2: Artemia salina lethality bioassay method. (A) Commercially available equipment employed for the hatching of brine shrimp cysts; (B) Hand-made migration equipment; (C) Hatching vessel filled with saline solution; (D) Collection of unhatched eggs and nauplii; (E) Hand-made equipment in the incubator during the migration step. The container far from the lamp should be covered with aluminum foil; however, for a better view of the set installation here it was removed; (F) Harvesting of Artemia in wells prior to performing the assay. The compounds should be placed as shown: - refers to the negative control (DMSO), + to the positive control (K₂Cr₂O₇), salt to the artificial salt solution, and 1 to 3 to the samples to test (in this case compounds 1-3); (G) Incubation of the 24-well plate containing Artemia and the selected samples; (H) Artemia count under the binocular microscope. Please click here to view a larger version of this figure.

Figure 3: Structures of selected compounds. Structure of compounds 1-2, extracted from Plectranthus species, and compounds 3-5, obtained by semi-synthesis. Please click here to view a larger version of this figure.

Representative Results

The general toxicity of some natural products recently studied by our group was evaluated through the brine shrimp lethality bioassay. Four extracts (Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; and Pe- P. ecklonii) from Plectranthus genus, known for their antioxidant activity (unpublished results), were tested. Additionally, two natural compounds (1 and 2) obtained from Plectranthus spp., and three semi-synthetic derivatives (3, 4, 5; Figure 3), all described in another work³, were also investigated. Herein, their preliminary evaluation in terms of potential cytotoxic activity is reported.

All the tested extracts showed very encouraging results, with very low mortality rates, comparable to those registered for the blank (salt solution) and the negative control (DMSO; Figure 4). Conversely, among the pure compounds 1-5, only derivative 5 displayed a low mortality rate (2.30% at a concentration of 100 ppm) with no general toxicity (Figure 5).

Figure 4: Lethality of extracts on Artemia salina. Mortality rate of A. salina (%) after 24 h exposure to four methanolic extracts of Plectranthus spp., at 0.1 mg/mL (Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; Pe- P. ecklonii). All the extracts were acceptable in terms of general toxicity using this assay. Salt corresponds to the salt solution (blank); K₂Cr₂O₇ was used as the positive control and DMSO as the negative control. The results were expressed as the mean of three independent experiments, each with internal quadruplicates (n = 12) ± SD. Comparisons were performed within groups by the analysis of variance, using the one-way ANOVA with Dunnett's post-test. Significant differences between control and experimental groups were assessed using a commercial statistical analysis software. A probability level p < 0.01 was considered to indicate statistical significance. Please click here to view a larger version of this figure.

Figure 5: Lethality of compounds on Artemia salina. Mortality rate of A. salina (%) after 24 h exposure to five pure compounds at 0.1 mg/mL (1 and 2 are natural products and 3-5 are derivatives prepared from 1 and 2). It is evident that only 5 shows very limited toxicity using this assay. Salt corresponds to the salt solution (blank); K₂Cr₂O₇ was used as the positive control and DMSO as the negative control. The results were expressed as the mean of three independent experiments, each with internal quadruplicates (n = 12) ± SD. Comparisons were performed within groups by the analysis of variance, using the one-way ANOVA with Dunnett's post-test. Significant differences between control and experimental groups were assessed using a commercial statistical analysis software. A probability level p < 0.01 was considered to indicate statistical significance. Please click here to view a larger version of this figure.

Natural products 1 and 2 showed moderate lethality (36.68% and 30.95% at 100 ppm, respectively), whereas semisynthetic derivatives 3 and 4, obtained from 1 and 2, respectively, were more toxic than the original scaffold (64.02% and 36.64% at 100 ppm, respectively; Figure 5).

Overall data suggested that all the tested extracts, known for their antioxidant activity and showing no general toxicity, can be considered as candidates for further in vitro activity and toxicity studies on selected cell lines. If the absence of toxicity is also demonstrated in cutaneous models, the development of new bioactive products for cutaneous application can be carried out. On the other hand, the toxic effects observed for compounds 1-4 could make them suitable candidates for additional evaluations as anticancer agents.

Discussion

During the last years, the scientific community has increased its attention toward alternative models for toxicity screenings²¹. Beside A. salina lethality bioassay, other methodologies are usually performed for the evaluation of sample tolerability and include vertebrate bioassays (such as rodents), invertebrates (such as zebrafish), in vitro methods using yeast strains or cells, and in silico methods^22,23,24,25. All these methods have clear advantages and disadvantages, in consideration of spent money and time, reproducibility of the results, and the type of sample to be analyzed. For example, the in silico approaches represent a standardized low-cost methodology, requiring minimal equipment. Nonetheless, when such studies are focused on a single target, poor quality results are obtained, making this alternative out of the scope for preliminary screenings, as we present in this study^25,26. When it comes to microorganisms, Saccharomyces cerevisiae is considered one of the most extensively used models for the evaluation of toxic compounds²³. In fact, the use of in vitro yeast is generally fast and easy to carry out; however, it can be expensive, since it requires specific chemical products and equipment, and shows low sensitivity to minimal doses of cytotoxic compounds²⁷. In an alternative approach, human cells can be employed, in a fast, simple, and inexpensive way. Even so, these cannot represent an entire organism; thus, interactions between different cell types and systemic perturbations taking place in physiologic conditions are not appropriately taken into account^25,26. On the other hand, in vivo assays have the great advantage to take into consideration the whole organism, but animal models (such as rodents) require more time for the assay execution, are more expensive and imply complex ethical considerations²⁵. Conversely, in vivo assays employing aquatic organisms for toxicity screening are generally well accepted, considering also that the use of marine invertebrates undergoes less ethical concerns, and the methodology is easy to perform, fast, and cheap^21,24. In this context, the use of zebrafish for the study of general toxicity often represents the first choice in the field. In fact, compared to other animal tests, it can be considered a low-cost option, as zebrafish develop rapidly and reach the early larval stage around 72 h to 13 days post-fertilization^21,28. Nevertheless, maintenance of a zebrafish requires well-trained operators and specific conditions, such as a fish tank with a circulating system, able to aerate and filter the system water, to maintain the overall quality; moreover, several lids and drain covers are necessary, as zebrafish can jump, as well as specific lighting conditions (14 h light, 10 h dark), and pH levels need to be checked daily, among others^29,30. All together, these considerations make zebrafish models more time consuming and expensive than the Artemia model reported here, since these microcrustaceans are easier to handle and viable for cultivation in large populations using laboratory methods. This makes A. salina undoubtedly one of the most employed screening tools, mainly used to test the general toxicity of different samples, such as drugs and medicinal plant extracts¹.

In the protocol, A. salina nauplii have been reared, collected, and incubated with suitable samples for 24 h, to estimate the nauplii mortality rate induced by each sample. In the first step, the egg hatching is conducted in a commercially available funnel-shaped breeding vessel, in which vigorous bubbling was introduced (Figure 2A). This forced aeration is necessary since it allows the brine shrimp hatching to occur as successfully as possible. The hatching occurs at around 24 h at 25 ± 3 °C, whereas up to 48 h could be necessary at lower temperatures. Once the eggs hatch, the pump is turned off to allow the empty egg cases to float on the top, while nauplii and unhatched eggs are easily collected by opening the funnel tap at the bottom. During our experiments, complete egg hatching was never achieved, with the consequent presence of nauplii and non-mature eggs in the collected solution. For this reason, in order to efficiently separate the two forms of Artemia, a hand-made equipment for brine shrimps' migration is prepared (step 1.2). One container is filled up with the harvested organisms, and then covered with aluminum foil, while the other recipient is exposed to the direct light of a lamp. In these conditions, the unhatched eggs tend to settle down, whereas the alive nauplii are attracted by the light hitting the adjacent container, favoring the migration from one side to the other through the bridge connection. After 4 h, almost all live nauplii are inside the container exposed to the light and ready to be collected for the execution of the assay. At this stage, portions of the saltwater solution, containing ten to fifteen nauplii, are removed and incubated with each of the samples to be tested.

In this work, it has been shown how the method is efficient, economic, and easy to be carried out. However, some critical points should be acknowledged when performing this assay.

The assay is usually conducted with tap water. Depending on geographic and physico-chemical factors, tap water exhibits different hardness distribution, influencing the reproducibility of the assay and, in particular, the egg hatching step. For the same reason, the use of seawater could influence the reproducibility of the test. The varying salt concentration, the presence of contaminants, microplastics and other corpuscles would force the operator to undergo further steps, such as filtrations and other types of purification, that would make the experiment more complex and less easy to handle. All considered, optimal conditions should be settled based on the characteristics and availability of tap water, especially by a careful regulation of the amount of artificial salt added, that creates a suitable environment and acts as nourishment for the nauplii, too.

Wide variations in temperatures can result in lower hatch rates. As a consequence, low levels of living Artemia could be observed, mixed with a high number of unhatched eggs. Clearly, such conditions are not suitable for the development of reliable assays, so a controlled climatization of the room or the use of appropriate vertical incubators should be considered.

Vigorous aeration of the solution within the hatching vessel is required. The presence of continuous bubbling favors the contact between eggs and accelerates the hatching process.

Collecting both unhatched eggs and nauplii during the harvesting can strongly influence the reliability of the assay. This is due to the possibility of hatching during the incubation with the samples. In this case, not all the nauplii will have been exposed for the same amount of time, with consequent erroneous mortality rates. As eggs and nauplii are too small to be efficiently separated, higher incubation times in the hatching equipment or the use of the hand-made migration equipment are needed.

Each well should contain 10-15 nauplii for the incubation step, carefully counted by the use of a binocular microscope (12x magnification). The presence of too many nauplii in the same well can make the final counting difficult, or even erroneous, because of overlapping. On the other hand, small amounts of shrimps would lead to results with no statistical significance.

As is evident, most of the problems of this method are related to the hatching and growth stages, where more attention is required to avoid reliability issues. Even so, the method can tolerate modifications, especially in relation to the most critical points described above. Of course, when one of these parameters is changed, it could be necessary to conduct several attempts to optimize the original method. For example, changing the temperature can help control the overall time of the assay: increasing the temperature up to 28-29 °C will also increase the hatch rate and speed up the entire process.

Some limitations of A. salina bioassay are also related to the stability of the samples to the test conditions and time. The assay can only be carried out using samples that are stable at room temperature and under visible light. Moreover, it must be taken into account that the entire procedure lasts at least 4 days, and if the hatch rate is low, the assay could need an extra day for the hatching step.

Overall, in this study, we highlight two examples of the application of the general toxicity evaluation through the A. salina method. General toxicity has been established for all the samples tested. Extracts from Plectranthus plants and compound 5 displayed no general toxicity, while compounds 1-4 proved to be moderately or even highly toxic on brine shrimps. In particular, the negative effect of compounds 1 and 2 on Artemia was previously demonstrated by Sitarek and Matias by MTT and SRB/MTT assays, respectively^31,32. At the same time, the benzoylated analogs, compounds 3 and 4, show higher toxicity values than their precursors 1 and 2, highlighting that the ester functionalization could have an important role in terms of cytotoxicity, as confirmed for compound 4 in human NSCLC MDR cancer cell line by Garcia et al.³³. However, other assays are needed to confirm that such a cytotoxic activity could be exploited in cancer therapy. On the other hand, derivative 5, together with all of the assayed extracts, showed no toxicity and appears as the most promising pure compound of the series. However, further studies to evaluate the potential biological activity for cutaneous application are needed.

Here we demonstrated how the use of Artemia salina-based method as a screening tool could allow saving time and money, compared to other known methodologies. It represents an efficient and advantageous way to be exploited in preliminary toxicity contexts. It can be used in the presence of different and complex samples, synthetic, and semi-synthetic drugs, as well as for the bio-guided fractionation of natural product extracts and samples.

Materials

Name	Company	Catalog Number	Comments
24-well plates	Thermo Fisher Scientific, Denmark	174899	Thermo Scientific Nunc Up Cell 24 multidish
Aluminium foil	Albal	-	Can be purchased in supermarket
Artemio Set	JBL GmbH and Co. KG, D-67141, Neuhofen Germany	61066000	Can be purchased in pet shops
Binocular microscope	Ceti, Belgium	1700.0000	Flexum-24AED, 220-240 V, 50 Hz
Bottles	-	-	0.5 L Diameter: 5.8 cm; Height: 12 cm
Brine shrimp cysts	JBL GmbH and Co. KG, D-67141, Neuhofen Germany	3090700	Can be purchased in pet shops
Brine shrimp salt	JBL GmbH and Co. KG, D-67141, Neuhofen Germany	3090600	Can be purchased in pet shops
Dimethyl sulfoxide (DMSO)	VWR chemicals	CAS: 67-68-5	99% purity
Discartable tips	Diamond	F171500	Volume range: 100 - 1000 µL
Eppendorf microtubes	BRAND	7,80,546	Microtubes, PP, 2 mL, BIO-CERT PCR QUALITY
Erlenmeyer flask	VWR chemicals	4,47,109	volume: 100 mL
Glass beaker	Normax	3.2111654N	Volume: 1000 mL
Gloves	Guantes Luna	GLSP3	-
GraphPad Prism	GraphPad Software, San Diego, CA, USA	-	GraphPad Prism version 5.00 for Windows, www.graphpad.com, accessed on 5 February 2021; commercial statistical analysis software
Home-made A. salina Grower	-	-	Home made: two plastic bottles connected by a hose
Hot glue	Parkside	PHP500E3	230 V, 50 Hz, 25 W
Incubator	Heidolph Instruments, Denmark	-	One Heidolph Unimax 1010 equipment and one Heidolph Inkubator 1006
Light	Roblan	SKYC3008FE14	LED light bulb
Micropipettes	VWR chemicals	613-5265	Volume range: 100 - 1000 µL
Potassium dichromate (K2Cr2O7)	VWR chemicals	CAS: 7778-50-9	99% purity
Pump ProAir a50	JBL GmbH and Co. KG, D-67141, Neuhofen Germany	-	Included in the Artemio Set+1 kit
Rubber tube	-	-	1.3 cm outer and 0.9 cm inner diameter
Stirring rod	VWR chemicals	441-0147	6 mm, 250 mm
Termometer	VWR chemicals	620-0821	0 - 100 °C