We are Laura Manfra and Federica Selli researchers from the Higher Institute for Environmental Protection and Research, ISRA, department of Environmental Quality Monitoring Laboratory of Phytoplankton, echo Toxicology and Ecology. Our research activities target the use of biological methods for the evaluation of environmental quality with particular reference to salt water, brackish water, and sediment. Our latest study concerns the development and standardization of a valuable methodological protocol to determine long-term 14 days lethal toxicity exerted by chemical substances, industrial waste, wastewater, or sewage and liquid environmental samples on the salt water crustacean artia Franciscan.
This video describes the performance of procedural toxicity testing. Our testing involves exposing artia larvae to an interval of substance concentrations or a test sample for the purpose of determining the concentration or dilution levels that would cause the death of 50%of the organisms exposed over 14 days and meeting the conditions defined by this method. If necessary and possible the following may also be determined.
The concentration level that causes the death of 20%of organisms exposed the highest analyzed concentration level that does not determine a higher mortality rate than that of the negative control, the lowest tested concentration level that determines after 14 days a higher mortality rate than that of the negative control. To perform testing, we required our team Franciscan larvae of less than 48 hours old at stage two or three obtained from newly hatched eggs. You may use synthetic salt water as dilution water, preparing it by dissolving recognized analytical quality reagents or a commercially available formulation in distilled or deionized water.
After variation for 48 hours and filtration in filters with a porosity of at least 0.45, you may preserve the dilution for a maximum of 30 days in the dark. At a temperature of zero four degrees, you must feed test organisms Ella Ter microalgae, which grows exponentially with densities of between 1.3 multiplied by 10 raised to the power of six and 2.0 multiplied by 10 raised to the power of six cells milliliters. The reference substance in this test is sodium dosel sulfate.
In addition to the usual laboratory equipment, you will also need the following testing containers, including 100 millimeter bur silicate glass beakers, 500 millimeter flask to prepare the standard solution, six to hundred millimeter flasks to prepare the testing solutions, glass or poly five centimeter diameter Petri dishes with removable covers to use for activating the cysts and transferring the artia from the hatching dish to the testing containers. Glass poster pipettes, which tips rounded by flame for transferring the noli glass cannelly or past three milliliter disposable plastic pipettes to be cut for transferring the artia micropipets and graduated pipettes to prepare the testing solutions. Six 50 milliliter graduated cylinders for transferring testing solutions from the flasks to the testing containers To generate test organisms.
Put 20 milligrams of cysts into a Petri dish containing 12 milliliters of dilution water 48 hours before testing. Maintain the dish at 25 degrees and at a 4, 000 lumens per square meter light intensity for one hour. After 24 hours, transfer the hatching larvae into a new Petri dish filled with artificial water.
Perform the transfer with the microscope using a light source so that the hatching phototropic larvae will migrate towards the light bean. Use a glass past pipet to make the transfer ensuring that you only transfer the nascent larvae and not the cysts themselves or larvae still in membranes. Put the dish containing the larvae in a dark thermostatic chamber for another 24 hours at 25 degrees.
The standard solution for the testing substance must be prepared by dissolving a half gram of the substance in a 500 milliliter flask. Fill it up with deionized or distilled water and stir until completely dissolved. Solutions must be prepared at the time of use unless you do not know whether the substance is stable in solution.
In that case, you may prepare the standard solution up to two days before testing. You must prepare the negative control in a 200 milliliter flask by adding an adequate of an algal suspension to the dilution water Such that you obtain a density of 10 raised to the power of five cells. Milliliters testing solutions must be prepared in five to hundred milliliter flasks by adding the standard solution to the dilution water in the quantity specified so that you will obtain the desired concentrations for testing.
To feed the organisms, you must use quis of duna teta microalgae suspension, adding them when you prepare testing solutions until they reach a density of 10 raised to the power of five cells milliliters. But before you start, we recommend you follow this order of addition dilution, water algal suspension, then standard solution. Introduce equal quantities, 50 milliliters of the testing solutions into the testing containers utilizing the graduated cylinder.
Make three replicas for each concentration for each series of tests. Prepare a control container with an amount of dilution water equal to the volume of the test solutions. Introduce equal volumes about 12 milliliters of the test solutions into the Petri dishes.
For each series of tests, make a control Petri dish. 48 hours after cyst activation, the nly will reach the second, third larval stage and are now usable for testing. Take the Petri dish you used for cyst activation out of the thermostatic chamber.
Transfer a small quantity of larvae into the Petri dishes containing the control and the testing solutions. In this phase of the test, you may perform the transfer with a glass past pipee, which tip rounded by flame and a laterally positioned light source that encourages the artia to aggregate. Transfer 10 larvae from the testing solutions.
Petri dishes to the testing containers. Also perform this operation utilizing the past pipet and microscope cover the testing beakers with paraphilia, leaving a gap for air passage, maintaining them at a temperature of 25 degrees for the whole duration of the test. And at an illumination of 900 lumens per square meter with that photocopied ratio of 14 hours of light to 10 hours of darkness after two days from the start of testing.
And then after 5, 7, 9, and 12 days, observe the artia under the microscope and verify their survival rate. Replacing the medium and food supplement organisms that do not show some movement for about 10 seconds should be considered dead while testing, you must periodically replace the test solutions, preparing them the same day as they are to be used while testing. You must periodically replace the test solutions, preparing them the same day as they are to be used.
Make up the test solutions from the standard solution you previously prepared. Perform the ARTIA transfer using EPI PET with a sufficiently wide diameter so as not to damage the organisms during this phase. You may use a three millimeter plastic poster pipe pett to be cut after 14 days of exposure.
At the end of testing, count the number of surviving larvae and report the result on your worksheet. Calculate lc 50 and or lc 20 NOEC and LOEC at 14 days and report their values. Confidence limits at 95%were appropriate and calculation methods.
If at the end of testing the following conditions have been met, the controls average mortality rate is less or equal to 20%Were using sodium doda cell sulfate. The lc 50 at 14 days is included in the 8.0 milligram liter interval. If the proceeding conditions have not been met, all data obtained with the same batch of organisms should be considered invalid and testing repeated.
