The Toxicological Studies Of Acephate On Paramecium Caudatum And Paramecium Bursaria

Human's living environments contain large amounts of pesticide residues,which cause various destructions to environment and creatures.Many current monitoring reports about animal toxicity experimentation limited on higher animal group,while less is focused on the toxicity mechanism of non-target animals like aquatic protozoan.Acephate is a relative safe organophosphorous pesticide,however,its affecting mechanism on the physiology,behavior,propagation,etc of aquatic protozoan are still not clear.Using Paramecium to study the effect of acephate on its different administrative levels,hoping to provide evidences for the analyses and evaluations of pesticide toxicity,and the research also has essential scientific meanings on thorough investigation of the inherent mechanism of toxicity effect on cellular level.1 The development of automatic counting software of protozoan and its realizationThe traditional method of counting the protozoan cannot accurately calculate the figure of living protozoan.To advance the counting efficiency and minish the counting error,this paper will adopt the technology of processing the digital images and continuously take two or more pictures.After the technology of figure processing such as image subtracting,enhancement,filtering and binarizing,they were transformed into a black and white picture.The resulting image was further processed to identify and label connected regions,followed by automatically counting the regions and statistically revising the result.An automatic counting system was developed to count the dynamic change of ciliates,such as poremecium's population,and obtained more accurate and efficient results as compared to the manual counting method. (1)Acute toxicity effect The living time of Paramecium caudatum and Paramecium bursaria which are exposed in acephate shortened with the rise of solutions,Under the toxicity,the behavior of Paramecium caudatum and Paramecium bursaria are badly affected.Within the concentration between 5mg/L to 25mg/L,Parameciums swiftly moved at first.Besides the normal straight and axial moving,the speed of rolling round itself rapidly advanced.Then the speed gradually decreased.Within the concentration of 35mg/L.to 65mg/L,as the body of Paramecium caudatum complete immersion in the concentration,its moving speed rapidly decreased.With the breach of contractile vacuole and food vacuole,the content mixed with cytoplasm,and volume of the whole cell enlarged.Then the cell membrane rived,the content of cell gradually overflowed to the offside of the cell, rived and dissolved.The swift change of the behavior and shape of Parameciums in the high concentration marked the acute toxicity effect of acephate to the aquatic organism.(2)Confirmation of median lethal concentration The median lethal concentration LC₅₀of Paramecium caudatum and Paramecium bursaria for 1h and 12h are measured.Regressive analysis indicated that there is a linear relationship between death probability unit and concentration logarithm.Compare the 1h and 12h Paramecium caudatum,the former is higher.Therefore,Paramecium bursaria's drug resistance is lower than Paramecium caudatum while,its sensitivity to the acephate is higher.The result of this experiment confirmed that Paramecium can fleetly and directly reflected the toxicity of pesticides and can be a kind of hopeful biological indicator for estimating the pollution of water environment.3 The change of population growth under chronic toxicity effect of acephateACPS system was used to study the population changes.Under chronic toxicity effect of acephate,Paramecium in every solution showed an "S" curve.Comparing the time spent to reach the stationary phase of population growth,the former one is about 11days,and the latter is about 13 days.With rising concentration of acephate, this time became shorter and shorter.Under five different concentrations of acephate,the largest population density of Paromecium caudatum and Paramecium bursaria during its stationary phase were tested.There was a significant negative correlation between the concentration of acephate and the Paramecium caudatum's largest population density.Thus,the increased concentration of acephate inhibited the reproduction of the Paramecium thereby constrained the increase of their population density.Paramecium bursaria's population growth was different. Although the overall trend showed similar characteristics,when the concentration was 0.005 mg/L,its population density was 1,532/mL,a little larger than the density of its control group(1,490/mL).4 The change of individual characteristics under chronic toxicity effect of acephate(1)Morphological variation The study cultured Paramecium coudatum on acephate under five different concentration gradients.The results showed that both the length and width(which is wider than the controls)of Paramecium caudatum shortened with the growth of concentration,whereas the length and width of Paramecium bursaria shortened significantly.Either the variation of length or width of Paramecium caudatum and Paramecium bursaria had a linear relation with the concentration of acephate.The chronic toxicity of acephate to Paramecium could significantly induce the variation of its individual form and size.(2)The change of contractile vacuole's constriction The frequency of the contractile vacuole's constriction verified obviously and normally to the Paramecium exposed in the acephate solutions.The figure indicated that with the rising of concentration the Paramecium caudatum's frequency of the contractile vacuole's constriction decreases in turn;Under every concentration the frequency of constriction exposed for 20 minutes was obviously higher than that of 10 minutes(except the comparison group).Presumption:the activity of Paramecium's contractile vacuole was obviously affected.With the protraction of the time,its frequency of constriction slightly increased.According to the result of exposed time comparison,Paramecium gradually adjusted and adapted under the menace of pesticide and developed on the way to the formal frequency of constriction.On the other side,the change of the contractile vacuole's constriction frequency of Paramecium in the acephate hinted that the change of contractile vacuole's activity can be an efficient method of estimating pesticide rapidly and effectively. (3)The change of glycometabolism This research used capillary electrophoresis technology to analyze the concentration of sugar after Paramecium caudatum mixed with Paramecium bursaria under the 5 concentration grads of the acephate.The result indicated that the content of glucose of Paramecium caudatum's comparison group was the highest.With the gradual rise of the acephate concentration and the reduce of the glucose concentration,the content of maltose was not detected during the experiment.Except that when Paramecium bursaria was in those two sugars of 0.005mg/L the content was higher than the comparison group,the content of glucose and maltose had the trend of decreasing with the rise of drug concentration.Under the concentration of 0.25mg/L,the content of glucose reached rock bottom and that of maltose could hardly be estimated.Comparing these two,we might find out that under the same stress of drug concentration or comparison group,the content of glucose in Paramecium bursaria was higher than that in Paramecium caudatum,which was due to the difference between two species.Furthermore,there might be relationship between the decrease of maltose content and that of glucose content.The content of maltose in Paramecium bursaria decreased with the rise of drug concentration(except 0.005mg/L).This guideline reflected the effect of maltose metabolism method under the stress of drugs.(4)Changes of the number of symbiotic algae in Paramecium bursaria Ordinary light microscope and Fluorescence microscope were used to measure the acephate's toxicity effect to the Symbiotic Algae in Paramecium bursaria under low concentration.The numbers of Chlorella were different in Paramecium cultured under different concentration.When the concentration was 0.005 mg/L,the number of Chlorella was much larger than the number of its control group.Under three concentration(0.01mg/L,0.05 mg/L,0.25 mg/L),there was a significant negative correlation between the concentration of acephate and the number of Chlorella.So, there was almost no Chlorella under the concentration of 0.25 mg/L.Accordingly inferred,the chronic toxicity effect of acephate lead to the decrease of Chlorella's number.(5)The change of ultrastructure Observation on the test group was studied by using Transmission Electron Microscope,under the concentration of 0.005mg/L and 0.25mg/L,the ultra structure's pellicle structure of Paramecium bursaria was injured and the trichocyst vesicle was obviously larger.Mitochondria of the test group appeared to be shallow electron density and the matrix lacuna became bigger or fuzzy,the structure of cristae became hard to recognize.It showed that the functions of Mitochondria were affected by toxicity effect.A large number of Gorky and endoplasmic reticulum in the experimental group appeared.Accordingly inferred,the effects of toxicity were also shown in these important organelles,thus affected its growth and the increase of its population number.There was no abnormal changes on the pellicle and the pellicle alveolus,the trichocyst and the Mitochondria's Ultra structure of Paramecium bursaria.The number of Chlorella of the test group (0.005mg/L)was significantly increased compared with its control group,but this of the test group(0.25mg/L)was greatly reduced.Moreover,the dividing Chlorella's number of the test group(0.005mg/L)was also larger than its control group. Digesting Chlorella could be seen in the test group,the vesicle around the Chlorella taking up most of the Chlorella's space,this was identical with the number of Chlarella shown by Fluorescence microscope.Inferring from the result of sugar content studied by Capillary Electrophoresis Interaction:The chronic toxicity effect of acephate lead to the decrease of Chlorella's number,thus resulting in the intensification of Paramecium bursaria's toxic effect.5 Summary and conclusionThis research developed Automatic Counting Software of Protozoan(ACPS),which improved the accuracy and efficiency of the experiments and solved how to research the protozoan quickly and quantitatively.Using this software quantitatively researched the concentration of acute toxicity effect and median lethal effect along with the active change of the population growth.With the rise of drug's concentration Paramecium's living time shortened and its behavior was badly affected.When measuring the median lethal concentration LC₅₀of Paramecium caudatum and Paramecium bursaria for 1h and 12h,we found that Paramecium caudatum's drug resistance was higher than that of Paramecium bursaria.Under the chronic toxicity,the maximal species density had the trend of decreasing and in the concentration of 0.005mg/L the density of Paramecium bursaria was slightly higher than comparison group.The effect of the acephate chronic toxicity to the individual Paramecium appeared in morphology,behavior of contracrtile vacuole, glycometabolism,quantity of phycobiont and ultrastructure.The mechanism of the acephate chronic toxicity effect to the individual Paramecium can be further concluded.The pellicle structure of Paramecium caudatum been destroyed,the trichocyst vesicle being obviously large,Golgi apparatus and endoplasmic reticulum largely appearing,the quantity of chlorella in the Paramecium bursaria experimental group(0.25mg/L)greatly decreasing,the quantity of chlorella in the split condition being more than that of the comparison group,the interspaces of perialgal vacuole enlarging all fundamentally changed the physiological functions,behavior and multiplication of Paramecium.The quantity of chlorella in the Paramecium bursaria experimental group(0.005mg/L)was obviously larger than that in the controlled group,which tallied the result under the fluorescence microscope.Combining with sugar content measured by the capillary electrophoresis,we may conclude that the slight acephate in the environment won't restrain the chlorella's breed but instead,it has the promoted effect to them.Protozoan is one-celled individuality.They directly contact to the substances in the environment.Even slight change of the condition will cause the rapid change of its shape,structure and behavior.The result of the experiment proved that Paramecium could speedily and directly reflect the drug toxicity and could be a kind of hopeful biological indicator for estimating the pollution of water environment.This paper provides detailed information of toxicology for researching the toxicity of acephate to the non-target organisms.The result has the scientific meaning of clarifying the toxicity of the acephate to the infusorium such as Paramecium and deeply discussing the infusorium's function of estimating the environment in the pesticides.