Sometimes it can be difficult to quantify the environmental damage caused by uncontrolled and non-regulated waste discharging. That is why since some years before have begun to appear the ecotoxicological tests with Artemia Salina. The aim of this kind of tests is determine the toxicity of the samples to analyse, relating it with the mortality of these organisms.
Authors: Nuria Mengibar Guerrero and Mervi Pulkkinen
Artemia Salina is a kind of crustacean, which lives in salt water. This species it has probably not changed in 100 million years so it is considered one of the oldest species in the world. Their simple organism (they only have head, chest and abdomen) and its primitive nervous system make them suitable for toxicological tests since the damage caused to the animal is small or practically non-existent. Also some studies have demonstrated that Artemia Salina is sensitive to a wide range of heavy metals such as copper, zinc, chromium, cadmium or mercury making them perfect for quantifying the environmental damage caused by these harmful substances that are present in wastes of many industries or by products.
This study has analyzed the toxicity effects of copper and chromium on mortality and growth of Artemia Salina. In the case of copper it is known that it can bind directly to cellular structures and therefore interfere in the physiological functions of the organism tested. On the other hand, chromium is one of the most toxic heavy metals today and it could be present in two forms: Trivalent or Hexavalent chromium.
To prove and confirm this toxicity, three bottom ash samples provided by an energy plant in Lahti have been analyzed. Before doing the ecotoxicological tests, these three samples have been subjected to a batch test.
Material and methods
Artemia Salina eggs remain inactive until they find the necessary environmental conditions for their growth. Once these eggs find the proper conditions it takes at least 24 hours before the first nauplii appears, but they do not reach adulthood until after approximately 20-30 days.
Artemia Salina has to be cultivated before performing the test and for that it has been used JBL Artemio pur eggs and JBL Artemio salt. Three containers have been prepared with one liter of this salt solution and 5 spoonfuls of pure Artemia Salina eggs. After three days the first nauplii begin to appear. For that the growth of the brine shrimps will be possible, the containers should be kept at approximately 25 °C and the salt solution must be mixed gently from time to time. Also, if it is needed, more salt solution could be prepared and added the second or third day of life of Artemia Salina with the objective of keep the organism well nourished. Once they are grown, a cell plate is filled with 10 Artemia Salina in each cell, salt solution and the leachant solution obtained in the batch test (Table 1). To check how the age of Artemia Salina affects the test, the samples have been analysed three times in the same conditions but with Artemia Salina of different ages. Another variable that has been tested is the concentration of the leachant solution.
TABLE 1. Planning of the ecotoxicological test. Amount of leachant and salt solution
To compare the concentration of harmful substances with mortality, the concentrations of chromium and copper were estimated by a photometer in both solution of first leaching step and solution of second leaching step.
Results and discussion
After doing six tests (three for the first leachant solution and three for the second leachant solution) at different concentrations of the pollutant solutions, all the result that have been obtained are shown below in the form of different graphs (Figures 1 and 2).
Figure 1 (showing the amount of live Artemia Salina as a function of time testing sample 1) has been chosen as representative of the tests performed for the three samples. On the first day (color blue), Artemia Salina lasted 7 hours before dying, while on the third day of life (color red), following the same conditions of concentration, they lasted 4 hours this indicates that a higher age of the organism, there is less resistance to toxics.
FIGURE 1. Representation of the amount of live Artemia Salina as a function of time when sample 1 is being tested
On the other hand, in Figure 2 the percent of mortality in the minute 120 of the test is compared with the concentrations of copper and chromium present in each cell. As can be seen the mortality in cells 4, 5 and 6 (sample 2) is higher than in the others, indicating that because of a higher toxicity of the sample, the invertebrate organism dies in a shorter time. If the three graphs are compared, it can be seen that the three trend lines follow practically the same form. This is because the mortality of the Artemias Salina is directly proportional to the concentrations of the harmful substances present in the sample.
FIGURE 2. Representation of the percent of mortality in the minute 120 of the test compared with the concentrations of copper and chromium present in each cell
The chromium and the copper are accumulated by diffusion in the Artemia Salina. These substances penetrate through the cell membrane (a very fine skin, which makes them especially sensitive to toxics) of Artemia Salina following the Fick’s laws of diffusion (diffusion is the movement of a substance from a region of high concentration to a region of low concentration) and causing damage and different alterations in these organisms, ranging from difficulties in mobility to death.
Finally the results obtained from the toxicological analysis of the leachant solution of the second step of the batch test do not differ significantly from those obtained in the first part. The only difference is that in this second part, the concentrations of chromium and copper present in the samples are smaller and therefore the life of the tested animals is longer than in the first ecotoxicological test. They can live between 24 and 12 hours depending on their age.
It has been proven that the ecotoxicological test with Artemia Salina is a good method to determine the toxicity of a sample. Furthermore the test confirms that sample number 2 is the most toxic. Also the test provides evidences that variables such as the sample concentration or the age of the organism tested are directly proportional to the mortality of Artemia Salina and therefore must be taken into account when this type of ecotoxicological analysis are carried out.
Mengibar Guerrero, N. 2017. Utilization of concrete and ash waste in geotechnical construction – Legislation, methods and analysis requirements in Finland and Spain. [Online document]. Bachelor’s thesis in Environmental Technology. Lahti University of Applied Sciences, Faculty of Technology. Lahti. [Cited 2 June 2017]. Available at: http://urn.fi/URN:NBN:fi:amk-2017053011100
Nuria Mengibar Guerrero is an exchange student from Polytechnic University of Catalunya, Barcelona (UPC) in Lahti University of Applied Sciences (Lahti UAS).
Mervi Pulkkinen is a senior lecturer in the Faculty of Technology of Lahti UAS.
Article picture of Artemia Salina by Xavier 2010: https://www.flickr.com/photos/xavipat/4451152486.