This survey explores the usage of juveniles of the apple snail Pomacea canaliculata, an invasive animate being from South America, as a theoretical account being for toxicological research. This species was selected because it is ubiquitously distributed in the wetlands of Asia including Hong Kong, it is easy to keep in the lab, juveniles can last for up to two hebdomads without nutrient supply, and the juveniles are sensitive to pollutants. I compared the toxicity of three heavy metal compounds ( Cu sulphate, Cd chloride and zinc chloride ) to the juveniles of P. canaliculata. The information will be utile for an appraisal of the suitableness of this being as a pollutant bio-indicator. Furthermore, this survey will besides pave the manner for understanding the toxic mechanism or biomarkers happening. My survey is divided into three parts.
The first portion tests the acute toxicity of the three heavy metals on the endurance of the juvenile snails. The consequences show that the juveniles are most sensitive to copper ( 96-h LC50: 50.10 I?g/ L ) and least sensitive to zinc ( 96-h LC50: 3.14 mg/L ) . The 2nd portion consists of chronic trials concentrating on the effects of the heavy metals on the growing of apple snails. The consequences show that Cu ‘s consequence is similar to H2O control ; Cd is more resist the growing of juveniles than Zn. I measured the metal concentration in situ of the cultural medium ( Cu 2.5, 40, 100 I?g/L, Cd 10.6, 100, 500 I?g/L and Zn 141, 1000, 4000 I?g/L ) in a presentation experiment at hr 2 and 72. The consequences show that there were no important alterations of metal concentrations.
P. canaliculata, the household of Ampullariidae, is herbivorous feeding freashwater snail. They are originated in Argentina ( Martin et al. , 2001 ) and chiefly distributed in humid tropical and semitropical countries in Africa, South and Central America.
They are omnivorous when include macrophytes ; carnal affair and debris could in their provender ( Carlsson and Bonmark 2006 ) . P. canaliculata is covetous and Renaissance man in feeding wont comparing with other Ampullariidae ( Cowie 2002 ) .
P. canaliculata shows immense ecological malleability. It could aestivation during dry season up to 12 months and increases the opportunity of endurance. The medium size of grownup P. canaliculata could up to 50 millimeter shell tallness, and some of them may be larger than this size ( Cowie et al. 2006 ) .
The function of Bioassay in Ecological Risk Assessment
Since polluted H2O class will harm wildlife and homo, it may do toxic or carcinogenic consequence ; H2O quality monitoring is important concern. Nowadays, wastewater licensing plans are chiefly depending on chemical-based monitoring. However, bio-assay and the usage of toxicity proving for outflowing quality appraisal is deriving credence ( Hickey 1995 ) . Recently, bioassay becomes a new tendency and bit by bit substitutes the chemical appraisal for bioavailability and assesses chemical mixture interactive in the H2O ( Cairns 1995 ) . The H2O quality appraisal could be conducted by the handiness of proving populating beings. This type of monitoring is more inexpensive and environmental friendly comparing with chemical based trial. On the other custodies, there are some demands of utilizing bio-assay. The effectual toxicity trials of wastewaters require sensitive sufficient trial species, to give out effectual response at low degrees of contaminations. Besides, the trial coinage must be available to keep either in research lab or field aggregation throughout the twelvemonth. In add-ons, when the biological trials are relevant to the ecology, the trials should be performed utilizing native or widespread species ( Richardson and Martin 1994 ) .
P. canaliculata in Hong Kong
P. canaliculata invaded to Asiatic states in the early 1980s ( Carlsson and Bonmark 2006 ) . It is ubiquitously distributed in the wetlands of Asia including Hong Kong ( Kwong et al, 2008 ) . They colonized in the cardinal, northern, south-eastern and western portion of New Territories. Most of them could be found in Long Valley and cause serious agribusiness lost by high ingestion ability.
Key: Solid symbol: Presence ; Open symbol: Absence of P. canaliculata.
Figure 1. Distribution of the apple snail in Hong Kong ( Kwong et al 2008 )
Reasons of taking P. canaliculata juvenile as the theoretical account being for toxicological research
For the demand being the bio-assay which described in 1.2, P. canaliculata is a good theoretical account being for toxicology research. One of the grounds is that it is widely distributed in tropical and semitropical country, particularly in Hong Kong. They were easy to catch it throughout whole twelvemonth. Besides, they were easy maintained in research lab and juvenile can last for up to two hebdomads without nutrient supply harmonizing to my preliminary experiment. They have rapid growing rate to make sexual adulthood by 82 yearss and length of service of 1 to 4 old ages. Furthermore, big sum of eggs could be found in field and the laboratory civilization, the hatching success of 95.8 % which provide sufficient supply of eggs for immense informations set experiment. Since juvenile are sensitive to pollutants ( Humphrey et al. 1995 ) and the features of P. canaliculata ; P. canaliculata juvenile could be proposed as the theoretical account being for bio-assay research to supervise the H2O taint degree beside the usage of high cost instrument in the research lab.
Reasons of taking Cu, Cd and Zn as the theoretical account tested metals
Both Cu and Zn are indispensable for most cells with metamorphosis, and could bring on toxic consequence in little saddle horse comparing with other component, and involved in metabolic activities. ( Francis and Harrison 1988 ) . Copper is the indispensable component of most works and animate beings, it becomes toxic at high concentrations ( White & A ; Rainbow 1985 ) . However, improper dosage or over expose to it may take to abnormally metabolise the fats, triglycerides and neglect to synthesis melanin and Dopastat. The major beginning of Cu in H2O pollution is paint. The major beginnings of Cd pollution in H2O are waste batteries, incinerators ash, plating, surfacing and plastics escapes. Human intake extra value of it may do liver harm, nephritic failure ( Taiyi and et Al. 2002 ) and carcinogenic ( United States Public Health Service 1993 ) . On the other custodies, Zn is besides indispensable component for normal organic structure demand. Excessive consumption of Zn could do zinc toxicity ; which could take Fe and Cu lack. Since, fresh water is the major beginning of human and wild life imbibing, metal pollutant may go through to the consumer really easy. Besides, it ‘s clearly shown that improper consumption of these metals may do serious or fatal consequence. Water quality monitoring of these three metals is required to supply safety information to consumer.
Importance of the undertaking
P. canaliculata is widely distributed in New Territories Hong Kong. This provides sufficient sum of eggs for the toxicology research which need a batch of sample for big the information set and farther supplying the profile of heavy metal toxicity effects for this coinage. Besides, it fits the demand to move as bio-assay ; utile for an appraisal of suitableness of this being as a pollutant bioindicator. For the farther survey, this set of informations pave the manner for understanding the toxic mechanism or biomarkers happening of P. canaliculata.
Undertaking ‘s aims
In this survey, three theoretical account heavy metals, including Cu sulphate, Cd chloride and Zn chloride were used for the toxic appraisal. The survey was separated into 2 parts: ague and chronic trial.
The purpose of acute trial was to happen out the LC50 and NOAEL of different metals to the juveniles.
Chronic trial aimed to carry on the growing experiment of the juveniles with different intervention and found out the suppression ability of the metals to the juvenile. In add-ons, auxiliary metal concentration measuring was conducted, because Metal ion may attach to the container ‘s inner wall.
Materials and Methodology
Egg sampling and aggregation
During the entire procedure in this undertaking, the 4-day-old energetic juvenile snails were used ( Melo et al. , 2000 ) . The juvenile hatched from the eggs which collected indiscriminately from Long Vally ( 22A° 30 N, 114A° 6 ‘ W ) and cultured in the consistent environment with 28a„? in filtered field H2O via 0.30 I?m filter. Snails were cultured without any nutrient supply before the trial began. Healthy juveniles were transferred into cultural medium with different containers for different trial. On the other custodies, de-chlorine pat H2O with air exposure for one hebdomad were used for medium dilution and cultural mixture, which were filtered by 0.30 I?m filter. As the projective stated above, the survey was separated into 2 parts: acute trial and chronic trial. In add-ons, auxiliary metal concentration measurings were conducted.
Acute trial focused on the relationship between the mortality of juvenile snails and the concentration of heavy metals. In between, the information of Range Finding and Dose-Response were used to pull out the Dose-Response Curve of each metal. Furthermore, harmonizing to the curve, we found out the LC50 and the No Observed Adverse Effect Level ( NOAEL ) of each metal intervention. BD Falcona„? 351006 Petri dish was used for Range determination and Dose-Response toxic effects appraisal.
Figure 2. Pomacea canaliculata eggs in Long Valley
Range Finding experiment
For the Range determination experiment, it conducted crossing the concentration of 1, 0.1, 0.01 and 0.001 mg/L for Copper, 0.85, 0.085, 0.0085 and 0.00085 mg/L for Cadmium, and 28.2, 5.64, 0.564, 0.0564, 0.00564 and 0.00056 mg/L for Zinc. For each concentration, three replicates which contain 15 snails per replicate wholly had been exposed for 96 hours. Death had been assumed if the snails were observed which remain immobile, operculum closing, no motion of tentacles and the bosom round under electronic microscope. The survivorship of each metal had been calculated for the finding of the Dose-Response concentration scope.
Figure 3a. BD Falcona„? 351006 Petri dish
Figure 3b. BD Falcona„? 351006 Petri dish
After the Range happening trial, Dose-response experiments of three heavy metals had been conducted later. Three replicates were used. For each replicate, 15 snails were subjected to at least four different metal concentrations for 96 hours each replicate. Dose-response experiment conducted crossing the concentration of 20, 40, 60 and 80 I?g/L in Copper ; 170, 340, 510 and 680 I?g/L in Cadmium, and 1.128, 2.256, 3.384 and 4.512 mg/L in Zinc. The survivorship had been calculated every 24 hours as the clip dependent toxic consequence profiles. At last, Dose-Response curve of three metals could be found. Furthermore, harmonizing to the curve, the LC50 and NOAEL could be found.
Chronic trial focused on the growing rate of juvenile snail with different heavy metal exist ; based on the NOAEL we had found to plan a growing experiment with different metal intervention. This survey purpose is to look into the toxic consequence of different heavy metals which could impact or advance the juvenile growing. BD Falcona„? Conical Centrifuge Tubes 50 milliliter was used for growing experiment.
Figure 4. BD Falcona„? Conical Centrifuge Tubes 50 milliliter
This experiment focused on the consequence of 3 heavy metals on growing of P. canaliculata juvenile. The juvenile had cultured for four hebdomads with metal concentration 2.5 I?g/L for Cu, 10.6 I?g/L for Cd, and 141 I?g/L for zinc.15 replicates of each metals plus control will be tested. The medium had exchanged and every 3 yearss during cultivation period and the juveniles were cultured with 50 milliliters consistency medium. Besides, they were feed by same sum and quality of Head boodle ( Lactuca sativa volt-ampere. capitata L. ) with 1.5 centimeters diameter each ( see Figure 6 ) . The diameter of the juveniles ‘ shell length, breadth and aperture were measured at twenty-four hours 0, hebdomad 2 and hebdomad 4.
Figure 5. Head boodle ( Lactuca sativa volt-ampere. capitata L. )
Figure 6. Same sum and quality film editing of Head boodle ( Lactuca sativa volt-ampere. capitata L. )
GTA/AA metals ‘ concentration measuring
Harmonizing to the mention, chemicals may attached to the container ‘s interior wall ; “ after 2 and 48 hours intervention, the exposure solutions in two sets of containers were extracted for examine chemical concentrations severally. The container wall ‘s TBT sorption ability could be estimated ” ( Lau and et Al. 2007 ) . Since BD Falcona„? Conical Centrifuge Tubes and BD Falcona„? 351006 Petri dish are made of CHO compound ; metal ion may attach to the Falcon ‘s inner wall. Therefore, I had measured the metal concentration in situ with the cultural medium, 10, 40 and 100 I?g/L for Cu, 5, 100 and 500 I?g/L for Cd, and 141, 1000 and 4000 I?g/L for Zn in a presentation experiment at hr 2 and hr 72 by utilizing Varian Spectr AA 220Z Graphite Tube Atomizer ( GTA ) and Varian Spectr AA 220FS Atomic Absorption Spectrometer ( AA ) , the method was modified based on ( Lau and et Al. 2007 ) . Since there is the restriction of mensurating instrument in GTA, Cu demand to increase the concentration which could fall into the measurement scope ( utilizing 10 alternatively of 2.5 I?g/L of the growing experiment ) . All standard solutions were diluted by Milli-Q H2O.
For Varian Spectr AA 220Z Graphite Tube Atomizer ( GTA ) , Cu and Cd were measured. Standard solution had been prepared to graduate the criterion curve for each measuring with clean and mix standard solution of 20 I?g/L of Cu and Cd.
For Varian Spectr AA 220FS Atomic Absorption Spectrometer ( AA ) , standard solution had been prepared to with concentration 0, 0.2, 0.5, 1, 1.5 and 2 mg/L of zinc standard solution.
Figure 7. Varian Spectr AA 220Z Graphite Tube Atomizer ( GTA )
Figure 8. Varian Spectr AA 220FS Atomic Absorption Spectrometer ( AA )
All statistical analysis was conducted by package SPSS 16 for Windows. In acute experiment, LC50 values for the juvenile with 3 metals were calculated by Log transmutation and correlativity analysis. The Dose-Response curves of three metals were plotted by Sigma Plot 2001. In chronic experiment, the shell length, breadth and aperture alterations, and GTA/AA metals ‘ concentration measuring were analysis by Microsoft Excel 2007. The growing alterations of informations from different interventions were transformed into ranks and compared with one-way analysis of discrepancy, and so multiple comparings utilizing Tukey trial.
Based on the informations collected from the Range determination and Dose-Response experiment. The Dose-response curve of 3 metals had been drawn.
Figure 9. Dose-Response Curve of Copper
Figure 10. Dose-Response Curve of Cadmium
Figure 11. Dose-Response Curve of Zinc
LC50 and NOAEL of each metal
Harmonizing to the Dose-Response curve of each metal above, the LC50 of each metal was found. The 24 hr LC50 values for the juvenile were 92.30 I?g/L of Cu, 780.30 I?g/L of Cd and 3.85 mg/L of Zn. The 48 hr LC50 values for the juvenile were 81.20 I?g/L of Cu, 467.20 I?g/L of Cd and 3.76 mg/L of Zn. The 72 hr LC50 values for the juvenile were 64.50 I?g/L of Cu, 398.50 I?g/L of Cd and 3.21 mg/L of Zn. The 96 hr LC50 values for the juvenile were 50.10 I?g/L of Cu, 342.10 I?g/L of Cd and 3.14 mg/L of Zn.
By construing the Dose-Response curves, the NOAEL had been found as 10 I?g/L of Cu, 50 I?g/L of Cd and 1 mg/L of Zn.
For the chronic trial, the diameter of juveniles ‘ shell length, breadth and aperture were measured at twenty-four hours 0, hebdomad 2 and hebdomad 4. Harmonizing the figure 8, 9 and 10, the growing rate of 4 hebdomads intervention showed that showed the same tendency of growing for the corresponding metal. The growing rate by falling order were control & gt ; Cu & gt ; Cd & gt ; Zn.
Figure 12. Shell Length alterations of 4 hebdomads Growth Culture with 3 metals
Figure 13. Shell Width alterations of 4 hebdomads Growth Culture with 3 metals
Figure 14. Shell Aperture alterations of 4 hebdomads Growth Culture with 3 metals
GTA/AA metals ‘ concentration measuring
For metals ‘ concentration measuring, the highest standard divergence of Hour 2 was 4000 I?g/L of Zn with A±46.7 and the lowest was 5 I?g/L of Cd with A±0.1. Besides, the highest standard divergence of Hour 72 was 4000 I?g/L of Zn with A±50.0 and the lowest was 5 I?g/L of Cd with A±0.1.
Table 1. GTA/AA metals ‘ concentration measuring
Nominal ( I?g/L )
40.9 A± 0.8
40.0 A± 0.2
102.6 A± 0.6
100.2 A± 1.2
105.6 A± 2.2
104.2 A± 1.6
504.4 A± 1.9
501.7 A± 2.8
1011.3 A± 6.2
1007.3 A± 4.7
4239.9 A± 46.7
4233.1 A± 50.0
10.5 A± 0.6
9.9 A± 0.6
5.1 A± 0.1
5.1 A± 0.1
142.3 A± 2.0
142 A± 2.1
Harmonizing to the consequence of the LC50 of the P. canaliculata juvenile, it showed that they could excite by the trial metal in really low concentration. The LC50 of 24, 48 and 96 hr were 92.30, 81.20 and 50.10 I?g/L of Cu, and 780.30, 467.20 and 342.10 I?g/L of Cd. By comparing the LC50 of the Chinese Toad ( Bufo gargarizans ) Tadpole, the LC50 of 24, 48 and 96 hr were 201, 138 and 138 I?g/L of Cu, and 32.1, 23.3 and 18.9 mg/L ( Yang et al. 2003 ) . P. canaliculata juvenile is much sensitive to the Cu and Cd in the low concentration and much suited to being bio-assay. Besides, juveniles are most sensitive to copper ( 96 hours LC50 50.10 I?g/L ) and least sensitive to zinc ( 96 hours LC50 3.14 mg/L ) .
After the 4 hebdomad civilization, juveniles showed different growing response with different metals ( Cu for 2.5 I?g/L, Cd for 10.6 I?g/L and Zn for 141 I?g/L ) . It showed that Cd ‘s growing suppression to the juvenile was important ( One Way ANOVA statistic analysis by SPSS ) , and could suppress the growing more than Zn ; and Zn could suppress the growing more than Cu. Besides, Cu showed similar consequence with the control.
by SPSS 16
– : no important difference P. & gt ; 0.05
i?S : important difference P. & lt ; 0.05
Table 2. Mean One Way ANOVA statistic analysis of the alteration of shell length
Beside the acute trial, snails absent in the contaminated site were proposed as other mechanism ( Laskowski & A ; Hopkin 1996 ) . The factors were turning away and decrease of nutrient consumption and consequence in suppression of growing and/or fruitfulness ( Gomot-de Vaufleury and Kerhoa 2000 ) . This explain that why snails were inactive after two hebdomad by observations, and besides provided the ground of suppression growing. In mention, Copper is related to the binding of metallothionein in the soft tissue or organ ( Yap et al. 2009 ) , it may bring on the alteration of the protein sequence and farther inert the normal enzymatic activities. Therefore, it ‘s sensitive to copper in low concentration. Cadmium could used up glutathione and protein edge sulfhydrl groups and ensuing farther production of reactive O species, for illustration, hydroxyl groups, H peroxide and superoxide ion ( Stohs, et al 2001 ) . Cadmium could replace zinc in batch of critical enzymatic activities ; break and halt of activities were farther exist ( Moore and Ramamoorthy 1984 ) ; this provides a ground to explicate juvenile is sensitive to cadmium.
GTA/AA metals ‘ concentration measuring
Harmonizing to the Table 1 consequences, there was no important alteration in medium concentrations at hr 2 and hr 72 which were about indistinguishable. This interpreted the Falcon container ‘s inner wall was inert to the fond regard of the metal ions. The values were stable and consistency during class of experiment. In add-ons, these two containers could be used for Cu, Cd and Zn related civilization with the consistency cultural medium environment.
In this survey, toxic profiles of this species were found. The LC50 of 24, 48 and 96 hr were 92.30, 81.20 and 50.10 I?g/L of Cu, 780.30, 467.20 and 342.10 I?g/L of Cd and 3.85, 3.76, 3.21 and 3.14 mg/L of Zn. It found that the sensitive degree by falling order are Cu & gt ; Cd & gt ; Zn. This species is really sensitive to three heavy metals particularly copper and Cd. Besides, the NOAEL were found as 10 I?g/L of Cu, 50 I?g/L of Cd and 1 mg/L of Zn.
For the chronic growing experiment, it found that juveniles were shown different limitation response for growing due to different metal interventions. The sensitive degree by falling order are Cd & gt ; zinc & gt ; Cu & gt ; control. Cadmium showed important different with other interventions.
In this survey besides provided sufficient grounds to P. canaliculata juvenile can be used as a pollutant bioindicator every bit good as bio-assay in future, particularly in tropical and semitropical country with high copiousness of this coinage. On the other custodies, these toxic profiles may be used for future protein biomarkers happening.