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Biol Res , Chronic toxicity bioassay with populations of the crustacean Artemia salina exposed to the organophosphate diazinon. A chronic toxicity bioassay was conducted with the microcrustacean Artemia salina as the testing organism for the toxic organophosphate diazinon in order to determine if the species is an appropriate indicator of pollution in aquatic environments.
Tests of animal exposure to different concentrations of the toxicant were performed for 24, 48 and 72 hours after larvae hatching. Registered mortality data was used to obtain the lethal dose 50 LD 50 of the organophosphate for each exposure time, considering the immobilization of A.
The lethal concentration LD 50 in the same exposure times was calculated by evaluating morphological changes on the three initial stages of larval development. Both doses were determined by using probit statistical analysis. Results indicate greater dose-response exactitude after 24 hours of exposure to the toxicant. High sensitivity of the organism to the toxicant was determined, thus indicating that A. Key terms: Diazinon, Artemia salina, ecotoxicology, bioassay.
Toxicological bioassays are currently the most commonly used tests to determine the effectiveness of certain species as bioindicators and to evaluate pollution in the environment.
These studies are intended to obtain results rapidly and at a low cost. A bioassay is a test that involves living organisms to analyze substances in terms of the biological response they produce Silva, They evaluate the degree of damage of a chemical substance to living organisms after chronic or acute exposure. Acute tests quantify the lethal concentrations of a xenobiotic on any particular species.
Currently, if herbicides, fungicides and insecticides are considered as a whole, all cases of acute intoxication are due to insecticides that inhibit enzyme acetylcholinesterase, with the consequent accumulation of elevated levels of acetylcholine in nervous synapses. Within this group, organophosphorous pesticides are particularly important because of the rise in sales of products with this kind of substance since the ban of organochlorinated pesticides Ortega et al.
Although the purpose of pesticide usage is to eliminate undesired organisms that could damage crops or transmit diseases to animals and humans, there are other living organisms -including humans- whose physiological or biochemical functions are similar to those of the species of interest to be eliminated and that are sensitive to different degrees to the toxic effects of chemical pesticides, thus their use as chemical weapons Barnes, ; Ortega et al.
The preferred route of propagation of organophosphates is the aqueous pathway. The contamination of water bodies is generally due to crop treatments, farm disinfections and industrial wastes. The brachiopod crustacean Artemia sp. The common habitats for this species are coasts and shallow saline water, which are usually close to agricultural areas. Organophosphorous pesticides, commonly used in agriculture, are a danger for Artemia populations due to its toxicity, especially considering that this is a target species for its capacity of bioaccumulation.
Therefore, a chronic toxicity bioassay with the microcrustacean Artemia salina as testing organism for the toxic organophosphate diazinon was performed in the present work in order to determine if that species is an appropriate indicator of pollution in aquatic environments.
As a test organism, populations of the brachiopod microcrustacean Artemia salina were obtained from dehydrated eggs purchased in a specialized store. Eggs were developed under proper conditions for hatching and manually separated into subpopulations of 20 individuals at 24, 48 and 72 hours of life.
They were not fed during those periods since their digestive system is still non-functional. Culture flasks liter polypropylene flasks were used.
Culture density - the number of cultured eggs was 1 gram per liter of water. Air supply - a saturated oxygen condition was provided by an aquarium air pump. Once the eggs were hydrated under this condition, Artemia salina cyst hatching was produced. After 24 hours of egg hydration, the culture exhibited individuals in the stage of nauplium I, the first larval stage of early normal development.
Nauplii were left in the solution for 10 minutes, the time necessary to produce immobility in the specimens in order to analyze their normal morphology at this stage of development, using a microscope with 10X magnification.
The same methodology was applied at 48 and 72 hours post-hatching to compare our own morphological observations to prior bibliographical reports. Results in each interval showed that all individuals exhibited the normal morphological characteristics reported for their respective stages of normal early development, as illustrated in photographs of the individuals at the three aforementioned stages:.
First larval stage: Photograph taken 24 hours post-hydration Fig. The presence of the nauplium eye, the first pair of appendages or antennae, the second pair of appendages or antennae and the third pair of appendages or mandibles were observed. The characteristic brownish color was noted as an index of good health.
Appendages were symmetrical in length and width. There was no evidence of developmental anomalies. Second larval stage:. Photograph taken 48 hours post-hydration Fig.
The presence of the same appendages of the previous stage was noted, with a brief growth that maintained symmetry in length and width.
The anal opening, a characteristic phenomenon in the normal development of stage II nauplium, was noted. The specimens showed the normal brownish color, without any evidence of developmental anomalies.
Photograph taken 72 hours post-hydration Fig. The three pairs of appendages were noted, with greater development and growth. The presence of a lineal digestive system was observed, as characteristic of this stage. Specimens were normally brownish in color. It was concluded that under ideal culture conditions, eggs hatched normally and Artemia salina nauplii exhibited a morphologically normal development during their first three developmental stages.
In the preliminary toxicity assay, a population of Artemia salina was cultured under the following conditions: a 5-liter polypropylene container was filled with 2 liters of tap water with 65 grams of iodide-free salt diluted to obtain the proper salinity; 2 grams of dehydrated Artemia salina eggs were introduced.
With the data obtained from this assay, the LD 50 was calculated for the organophosphate diazinon in the nauplii of Artemia salina at 24, 48 and 72 hours of exposure, using probit analysis as the statistical method.
The assay was performed by hydrating the eggs under ideal conditions for two hours to stimulate nauplii hatching. Then 20 nauplii of Artemia salina were manually removed from flasks and subdivided into 4 groups of 5 individuals per each dilution and placed in sterile glass flasks filled with water obtained from the main culture flask containing serial dilutions of diazinon for dosage testing in a total volume of 20 mL per sample controlling nauplii survival and morbidity rates every 8 hours until completing a total of 72 hours of exposure.
In the assay, 8 dilutions were tested with decreasing concentrations of diazinon, considering a control group without exposure to the pesticide for every 2 dilutions of diazinon. Artemia salina populations of 20 individuals each subdivided into 5 subgroups were used for every diazinon concentration tested.
For each dilution, 3 groups of 20 individuals were assayed: one with 24 hours of exposure, another with 48 hours and the last with 72 hours of exposure. A total of 10 dilutions were evaluated, with 3 different concentrations of diazinon and were compared to the control group of 20 individuals unexposed to the pesticide.
Replicas of 5 individuals each were incubated in 25 mL glass flasks filled with 20 mL of a dilution of diazinon in saline water from the main culture. Preliminary bioaasay LD 50 : The first group of A. After the first trial, a new larval population was exposed to lower concentrations. The control group showed no mortality until 72 hours of assay.
The respective control groups did not show any variations. Thus, this preliminary trial gave the parameters for the definitive assay to determine the EC 50 ; this assay must be performed by intoxicating Artemia populations to analyze early developmental alterations after 24, 48 and 72 hours of exposure to diazinon. The determination of the LD 50 or ED 50 requires of quantal statistics, so it is necessary to transform the response values into anglit, logit or probit units the latter were used in this work.
As well, the doses used need to be transformed into logarithmic units known as metametric doses. Specialized computer software resolves this type of calculation in our case, we employed the program Microsoft Excel , but the reliability depends on the person or institution. The comparison of the procedures employed is necessary, as well as developing media for their simplification. The general objective of our research is to provide parameters for the development of a bioactivity analysis using the probit method.
The main objective of this kind of analysis is to evaluate the necessary level of stimulus to obtain a response in a group of individuals of a population. The duration of exposure to the stimulus must be specified e. With the data obtained in the preliminary assay, LD 50 was obtained through probit analysis at 24, 48 and 72 hours of exposure to diazinon for Artemia salina larvae.
Figure 2A represents the data obtained at 24 hours of exposure. The Y-axis probit indicates the percentage of dead individuals after 24 hours of exposure to the toxicant, while the X-axis represents the logarithm of every dose used in the bioassay. The greater the R 2 value, the more significant the results are, accepting as significant an R 2 value starting from 0.
To obtain LD 50 , the equation was calculated for a y value of 0. Subsequently, data obtained from the exposure of larvae to the toxicant for 48 hours were graphed, calculating the LD 50 by probit analysis in 4. Figure 2C shows that after 72 hours of exposure, the probit analysis gave an LD 50 of 4.
Results of the final bioassay EC Results obtained and registered in the control card are presented. A dose of 2. Group D received a dose of 2. Group F received a dose of 1. Group G received 1. Statistical analysis. With all registered data, the EC 50 was calculated for the 3 exposure periods with the probit analysis.
Figure 3A shows the results obtained after 24 hours of exposure of the individuals to diazinon. The Y-axis represents the percentage of individuals with morphological alterations and the X-axis shows the doses used, expressed as logarithms.
The result of the analysis gave an EC 50 of Morphological alterations in the population exposed to diazinon Nauplii 1 24 hours. Augmentation of appendage width Figure 4A - Longer filtrating chaetae produced by tissue retraction Figure 4B. Nauplii 2 48 hours - Failure in development of the third pair of appendages, with mandible function Figure 5A.
Metanauplii 72 hours -Agenesia of the first pair of appendages antennae with sensory function and the third pair of appendages mandibles with food capturing function Figure 6A. There is no doubt that currently ecotoxicological bioassays have become an increasingly common research topic for scientists worldwide, mainly because of growing pollution of global environments, thus augmenting the number of toxicological studies and assays in this matter.
Therefore, our group from the Laboratory of Reproduction Biology of the School of Medicine, University of Chile, proposed this study as a first step to determine and control polluted aquatic environments.
Artemia salina was chosen as the testing organism, considering the feasibility of culturing large populations using laboratory methods, facility to manage, short term results given its short life cycle, its availability, low cost and the fact that it is found worldwide.
The test substance, the organophosphorous pesticide diazinon, was chosen because of its massive and indiscriminate use, without proper legislation. Among the methods developed to evaluate the potential effect of toxic substances are acute and chronic toxicity assays, in which the damaging effects of such substances are estimated through the response of the organisms exposed to a range of concentrations of the evaluated substance during brief periods of exposure related to the length of the life cycle of the organism under study.
Standardized toxicity assays allow for calibrating the toxicity of substances and the sensitivity of species to the substance or comparing the results to observations in the real environmental.
Detección de metabolitos fúngicos con actividad tóxica mediante bioensayo sobre Artemia salina
Skip to search form Skip to main content You are currently offline. Some features of the site may not work correctly. DOI: Resumen El objetivo fue aplicar un ensayo de toxicidad sobre Artemia salina para la deteccion de metabolitos fungicos toxicos, obtenidos a partir de hongos contaminantes de hierbas medicinales y alimentos. Los extractos fungicos fueron clasificados, segun el porcentaje de mortalidad hallado, como: no toxico NT , levemente toxico LT , toxico T y muy toxico MT. En los tipos T y MT se investigo la presencia de micotoxinas. View via Publisher.
Effect of enriched Brachionus plicatilis and Artemia salina nauplii by The growth, developmental stages and survival rates of Sparus aurata larvae fed with Brachionus plicatilis and Artemia salina nauplii enriched by microalga Tetraselmis chuii were studied. Two experiments were carried out; the first concerning with culturing the microalga T. Eggs of Artemia salina L. Ecdysis takes place in two stages: a extrusion of the inner membrane, and b ecdysis of the nauplius from that membrane. The conditions which allow for the former are much more varied than those for the latter. Nauplii form in only solutions of a few sodium salts; and, in Mg, Ca, and Sr salts, potassium is very toxic.