Lethal Effects of Toxic Plants ( Neem , Datura , Acanthus ) on Artemia Species

Effects of the water-soluble extracts (dry leaf powder of Azadirachta indica, Datura stramonium and Acanthus ilicifolius) were studied on the behavioral pattern and mortality of Artemia species. The extracts of all the plants brought significant change in the behavioral pattern of Artemia. The Artemia become agitated just offer exposure to the extract and started to move upwards and downwards vigorously. The Artemia exposed to the various concentration of water-soluble plant extracts were unable to maintain dorsoventral position and dropped to the bottom finally. The mortality rates recorded after 24 h exposure to the extract Azadirachta indica were 30% at 1500 mg/L and 70% at 3000 mg/L. Similarly, after 24 h exposure to the extracts of Datura stremonium, the mortality rates were 40% at 2000 mg/L and 75% at 3500 mg/L. Again, after 24 h has exposure to the extracts of Acanthus ilicifolius, the mortality rates were 35% at 2500 mg/L and rose to 80% at 4000 mg/L. The 24 h LC50 values of the water-soluble extracts of Neem, Datura and Acanthus were 2331.930, 2357.948 and 2922.957. Artemia was more sensitive to the watersoluble extract of Neem than Datura and Acanthus.


Introduction
Artemia is one kind of zooplankton popularly known as brine shrimp [1].It is widely distributed on the five continents in hyper saline biotopes including salt lakes coastal or inland waters rich in chloride, sulphate or carbonate and especially in coastal salinas [2].It is about 12 mm in length.Artemia is a high saline organism (20-300 ppt) [3,4].They can survive short periods of time in the fresh water but cannot reproduce.The optimum temperature is 20 to 55 C for Artemia to survive.They have two modes of reproduction [5].Artemia life cycle begins by the hatching of dormant cysts, which are encased embryos that are metabolically inactive.The cysts can remain dormant for many years as long as they are kept in dry and oxygen-free condition [6].Surgeloss [7] reported that Artemia has a very efficient ecological defense mechanism.It has great practical value because of its availability as day cysts.However, since the demand perennially treats, it is always in short supply and thus very expensive.
Artemia used as feed in the hatchery and they can be used as feed mixed with artificial diets as a source of protein as an attractant [8].Artemia is an excellent food for newly hatched fish [9] and shrimp larvae [10].The nauplii of Artemia are readily taken by aquatic animals in nursery stage.
There are many kinds of toxic plants in the natural environment [11].These toxic plants have a great impact on the brine shrimp Artemia [12].Toxicity of plants to planktonic fauna (Artemia salina) is different from plants to plants [13].In the present experiment Azadirachta indica (Neem), Datura stremonium (Datura) and Acanthus ilicifolius (Acanthus) are used as toxic plants to assess the extent of toxicity of these plants on Artemia.

Materials and Methods
The experiment was conducted in the laboratory of the Institute of Marine Sciences and Fisheries, the University of Chittagong from May to August 2016.The fresh leaves of Neem, Datura, and Acanthus were collected from the south campus of the area of Chittagong University and Coastal area of Cox's Bazar.The collected leaves of the plants were dried at room temperature and then heated in an oven at (50-60)C until a constant weight of the samples were reached.The dried samples were powdered with the help of a powder driven grinder.
Three water-soluble extracts were prepared from the dried powdered leaves of Neem, Datura, and Acanthus.For the preparation of a stock solution, the dried powders were mixed thoroughly with distilled water at a ratio 1:10, that means one gram powder was mixed with 10 mL of distilled water.The mixture was left at room temperature for 24 h.The mixture was then filtered with fine mesh clothing.Thus a crude extract was obtained.It was collected in a conical flask and stored in a refrigerator at 4 C as a stock solution.

Saline water preparation
Concentrated brine was collected from the salt bed of Chakaria coast, Cox's Bazar.The brine was mixed with tap water. 1 kg of mud was mixed with ten liters of the tap water in an aquarium for the better adaptation.Gradually, 5 kg mud was mixed with 50 L of the tap water.

Experimental setting
Three aquaria of equal size (30″18″16″) washed with tap water were set up in a ventilated and damp proof room.Necessary equipment and chemicals were collected and set in a laboratory for measuring the water parameters of the aquaria.Each saline water provided with a mechanical aerator.Experimental setup for the toxicity test with different plants is shown in Fig. 1.

Artemia collection and acclimatization
Artemia was collected from local market of Cox's Bazar [14].After collection, Artemia was put in a plastic bowl.Then a jar was taken and three liters of saline water (20 ppt) was put in the jar.Then Artemia cysts were poured in the jar.Then an aerator was linked in the jar for aeration and waited 2 days for hatching.After hatching, the cysts of Artemia was poured in the aquarium containing saline water (20 ppt).Gradually after hatching, Artemia started to grow large [15].Then egg sperm was given as a feed for newly hatched Artemia.Salinity, temperature, pH were measured with the help of refractometer, thermometer and pH meter.Then waited for three weeks until the Artemia gained matured size.After gaining matured size, the toxicity test was executed.

Observation of physico-chemical variables
Salinity, temperature and pH of the aquaria water were measured with the help of refractometer (Model: DR103, The United Kingdom), thermometers, and Hanna pH meter (HI 9124 HI 9125, China) respectively.Dissolved oxygen is determined by the titrimetric method developed by Winkler [16].

Calculation
Dissolved oxygen ppm = volume of sodium thiosulphate consumed in mL  its normality  0.008  10 6 /volume of sample in mL.

Toxicity test
For the toxicity test, four jars were taken with four liters saline water.Then 20 species of Artemia was given into each jar.After that, different concentration (e.g.1500, 2000, 2500, 3000, 3500 mg/L) of leaf powder was given to the Artemia species in each jar with micro pipette.Then reading was taken after 6, 12, 18 and 24 h.After that, the mortality rate was calculated.The measured water parameter of the aquarium was (salinity 20-70) ppt, temperature (28-30)C, pH (6.8±0.13) and dissolved oxygen (8.5±0.20)mg/L.The experiment was repeated twice for the three plants extracts.

Statistical analysis
All the data were analyzed statistically.Statistical analyses were done by Probit analysis [17].Probit analysis suggested by Fisher and Yates [18] was followed graphically and for determining the LC 50 values mathematically.The values for empirical Probit, working Probit were taken from the tables given by Finney [19].

Results and Discussion
From the present study it was found that the water-soluble extracts of the leaf powder of the indigenous plant's species (Neem, Datura and Acanthus) were effective in killing the Artemia at low concentration 1500 mg/L in Neem, 2000 mg/L in Datura and 2500 mg/L in Acanthus.Behavioral changes become gradually apparent with the increase in the exposures of time and concentration.At medium concentrations 2000 mg/L to 2500 mg/L in Neem and 2500 mg/L to 3000 mg/L Datura and 3000 mg/L to 3500 mg/L in Acanthus, Artemia subjected to change in their behavioral pattern.The Artemia began to move up and down vigorously soon after they were released into the higher concentration and came up to the surface repeatedly.After a considerable period of time, the Artemia lost their stability to swim.Their movement becomes slow down and closed their mouths, consequently, the whole body becomes paralyzed.Finally, mortality occurred at a different interval of time.
Different concentrations of the water extracts caused varying rates of Artemia mortality during the 24 h exposure period.In all cases, the mortality rate was low at low concentration and it increased with the increased concentration and exposure time.The mortality recorded after 24 h exposures to the extracts of Azadirachta indica were 30% at 1500 mg/L and increased to 70% at 3000 mg/L (Table 1).After 24 h exposure to the extracts of Datura stremonium mortality was 40% at 2000 mg/L and raised to 75% at 3500 mg/L (Table 2).

Conclusion
Toxic plants have a great impact to brine shrimp Artemia species.After giving the concentration of leaf powder, Artemia species gradually lose their surviving attitude.Then mortality rates recorded after 24 h exposure.Lethal effects of toxic plants on Artemia is an exceptional task and it will bring new dimensions for the researcher in the future.

Table 1 .
Mortality of Artemia exposed to the water-soluble extracts of Azadirachta indica.

Table 2 .
Mortality of Artemia exposed to the water-soluble extracts of Datura stremonium.

Table 3 .
Mortality of Artemia exposed to the water-soluble extracts of Acanthus ilicifolius.