This study explores the use of juveniles of the apple snail Pomacea canaliculata, an invasive animal from South America, as a model organism for toxicological research. This species was selected because it is ubiquitously distributed in the wetlands of Asia including Hong Kong, it is easy to maintain in the lab, juveniles can survive for up to two weeks without food supply, and the juveniles are sensitive to pollutants. I compared the toxicity of three heavy metal compounds (copper sulfate, cadmium chloride and zinc chloride) to the juveniles of P. canaliculata. The information will be useful for an assessment of the suitability of this organism as a pollutant bio-indicator. Furthermore, this study will also pave the way for understanding the toxic mechanism or biomarkers finding. My study is divided into three parts.
The first part tests the acute toxicity of the three heavy metals on the survival of the juvenile snails. The results show that the juveniles are most sensitive to copper (96-h LC50: 50.10 Î¼g/ L) and least sensitive to zinc (96-h LC50: 3.14 mg/L). The second part consists of chronic tests focusing on the effects of the heavy metals on the growth of apple snails. The results show that copper's effect is similar to water control; cadmium is more resist the growth of juveniles than zinc. I measured the metal concentration in situ of the cultural medium (copper 2.5, 40, 100 Î¼g/L, cadmium 10.6, 100, 500 Î¼g/L and zinc 141, 1000, 4000 Î¼g/L) in a demonstration experiment at hour 2 and 72. The results show that there were no significant changes of metal concentrations.
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P. canaliculata, the family of Ampullariidae, is herbivorous feeding freashwater snail. They are originated in Argentina (Martin et al., 2001) and mainly distributed in humid tropical and subtropical areas in Africa, South and Central America.
They are omnivorous when include macrophytes; animal matter and detritus could in their feed (Carlsson and Bonmark 2006). P. canaliculata is avaricious and generalist in feeding habit comparing with other Ampullariidae (Cowie 2002).
P. canaliculata shows huge ecological plasticity. It could aestivation during dry season up to 12 months and increases the chance of survival. The medium size of adult P. canaliculata could up to 50 mm shell height, and some of them may be larger than this size (Cowie et al. 2006).
The role of Bioassay in Ecological Risk Assessment
Since polluted water course will harm wildlife and human, it may cause toxic or carcinogenic effect; water quality monitoring is significant concern. Nowadays, effluent licensing programs are mainly depending on chemical-based monitoring. However, bioassay and the use of toxicity testing for effluent quality assessment is gaining acceptance (Hickey 1995). Recently, bioassay becomes a new trend and gradually substitutes the chemical assessment for bioavailability and assesses chemical mixture interactive in the water (Cairns 1995). The water quality assessment could be conducted by the availability of testing living organisms. This type of monitoring is more cheap and environmental friendly comparing with chemical based test. On the other hands, there are some requirements of using bioassay. The effective toxicity tests of effluents require sensitive sufficient test species, to give out effective response at low levels of contaminants. Also, the test specie must be available to maintain either in laboratory or field collection throughout the year. In additions, when the biological tests are relevant to the ecology, the tests should be performed using native or widespread species (Richardson and Martin 1994).
P. canaliculata in Hong Kong
P. canaliculata invaded to Asian countries 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 central, northern, south-eastern and western part of New Territories. Most of them could be found in Long Valley and cause serious agriculture lost by high consumption 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 choosing P. canaliculata juvenile as the model organism for toxicological research
For the requirement being the bioassay which described in 1.2, P. canaliculata is a good model organism for toxicology research. One of the reasons is that it is widely distributed in tropical and subtropical area, especially in Hong Kong. They were easy to catch it throughout whole year. Also, they were easy maintained in laboratory and juvenile can survive for up to two weeks without food supply according to my preliminary experiment. They have rapid growth rate to reach sexual maturity by 82 days and longevity of 1 to 4 years. Moreover, large amount of eggs could be found in field and the laboratory culture, the hatching success of 95.8% which provide sufficient supply of eggs for huge data set experiment. Since juvenile are sensitive to pollutants (Humphrey et al. 1995) and the characteristics of P. canaliculata; P. canaliculata juvenile could be proposed as the model organism for bioassay research to monitor the water contamination level beside the use of high cost instrument in the laboratory.
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Reasons of choosing copper, cadmium and zinc as the model tested metals
Both copper and zinc are essential for most cells with metabolism, and could induce toxic effect in small mount comparing with other element, and involved in metabolic activities. (Francis and Harrison 1988). Copper is the essential element of most plant and animals, it becomes toxic at high concentrations (White & Rainbow 1985). However, improper dose or over expose to it may lead to abnormally metabolize the fats, triglycerides and fail to synthesis melanin and dopamine. The major source of copper in water pollution is paint. The major sources of cadmium pollution in water are waste batteries, incinerators ash, plating, coating and plastics leakages. Human intake excess value of it may cause liver damage, renal failure (Taiyi and et al. 2002) and carcinogenic (United States Public Health Service 1993). On the other hands, zinc is also essential element for normal body requirement. Excessive intake of zinc could cause zinc toxicity; which could lead iron and copper deficiency. Since, freshwater is the major source of human and wild life drinking, metal pollutant may pass to the consumer very easy. Also, it's clearly shown that improper intake of these metals may cause serious or fatal effect. Water quality monitoring of these three metals is required to provide safety information to consumer.
Importance of the project
P. canaliculata is widely distributed in New Territories Hong Kong. This provides sufficient amount of eggs for the toxicology research which need a lot of sample for large the data set and further providing the profile of heavy metal toxicity effects for this specie. Also, it fits the requirement to act as bioassay; useful for an assessment of suitability of this organism as a pollutant bioindicator. For the further study, this set of data pave the way for understanding the toxic mechanism or biomarkers finding of P. canaliculata.
In this study, three model heavy metals, including copper sulfate, cadmium chloride and zinc chloride were used for the toxic assessment. The study was separated into 2 parts: acute and chronic test.
The aim of acute test was to find out the LC50 and NOAEL of different metals to the juveniles.
Chronic test aimed to conduct the growth experiment of the juveniles with different treatment and found out the inhibition ability of the metals to the juvenile. In additions, supplementary metal concentration measurement was conducted, because Metal ion may attach to the container's inner wall.
Materials and Methodology
Egg sampling and collection
During the total process in this project, the 4-day-old energetic juvenile snails were used (Melo et al., 2000). The juvenile hatched from the eggs which collected randomly from Long Vally (22Â° 30 N, 114Â° 6' W) and cultured in the consistent environment with 28â„ƒ in filtered field water via 0.30 Î¼m filter. Snails were cultured without any food supply before the test began. Healthy juveniles were transferred into cultural medium with different containers for different test. On the other hands, de-chlorine tap water with air exposure for one week were used for medium dilution and cultural mixture, which were filtered by 0.30 Î¼m filter. As the projective stated above, the study was separated into 2 parts: acute test and chronic test. In additions, supplementary metal concentration measurements were conducted.
Acute test focused on the relationship between the mortality of juvenile snails and the concentration of heavy metals. In between, the data of Range Finding and Dose-Response were used to draw out the Dose-Response Curve of each metal. Furthermore, according to the curve, we found out the LC50 and the No Observed Adverse Effect Level (NOAEL) of each metal treatment. BD Falconâ„¢ 351006 Petri dish was used for Range finding and Dose-Response toxic effects assessment.
Figure 2. Pomacea canaliculata eggs in Long Valley
Range Finding experiment
For the Range finding experiment, it conducted spanning 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 totally had been exposed for 96 hours. Death had been assumed if the snails were observed which remain immobile, operculum closure, no movement of tentacles and the heart beat under electronic microscope. The survivorship of each metal had been calculated for the determination of the Dose-Response concentration range.
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Figure 3a. BD Falconâ„¢ 351006 Petri dish
Figure 3b. BD Falconâ„¢ 351006 Petri dish
After the Range finding test, Dose-response experiments of three heavy metals had been conducted subsequently. 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 spanning the concentration of 20, 40, 60 and 80 Î¼g/L in Copper; 170, 340, 510 and 680 Î¼g/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 time dependent toxic effect profiles. At last, Dose-Response curve of three metals could be found. Moreover, according to the curve, the LC50 and NOAEL could be found.
Chronic test focused on the growth rate of juvenile snail with different heavy metal exist; based on the NOAEL we had found to design a growth experiment with different metal treatment. This study aim is to investigate the toxic effect of different heavy metals which could affect or promote the juvenile growth. BD Falconâ„¢ Conical Centrifuge Tubes 50 mL was used for growth experiment.
Figure 4. BD Falconâ„¢ Conical Centrifuge Tubes 50 mL
This experiment focused on the effect of 3 heavy metals on growth of P. canaliculata juvenile. The juvenile had cultured for four weeks with metal concentration 2.5 Î¼g/L for copper, 10.6 Î¼g/L for cadmium, and 141 Î¼g/L for zinc.15 replicates of each metals plus control will be tested. The medium had exchanged and every 3 days during cultivation period and the juveniles were cultured with 50 mL consistence medium. Also, they were feed by same amount and quality of Head lettuce (Lactuca sativa var. capitata L.) with 1.5 cm diameter each (see Figure 6). The diameter of the juveniles' shell length, width and aperture were measured at day 0, week 2 and week 4.
Figure 5. Head lettuce (Lactuca sativa var. capitata L.)
Figure 6. Same amount and quality cutting of Head lettuce (Lactuca sativa var. capitata L.)
GTA/AA metals' concentration measurement
According to the reference, chemicals may attached to the container's inner wall; "after 2 and 48 hours treatment, the exposure solutions in two sets of containers were extracted for examine chemical concentrations respectively. The container wall's TBT sorption ability could be estimated" (Lau and et al. 2007). Since BD Falconâ„¢ Conical Centrifuge Tubes and BD Falconâ„¢ 351006 Petri dish are made of CHO compound; metal ion may attach to the Falcon's inner wall. Thus, I had measured the metal concentration in situ with the cultural medium, 10, 40 and 100 Î¼g/L for copper, 5, 100 and 500 Î¼g/L for cadmium, and 141, 1000 and 4000 Î¼g/L for zinc in a demonstration experiment at hour 2 and hour 72 by using 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 limitation of measuring instrument in GTA, copper need to increase the concentration which could fall into the measuring range (using 10 instead of 2.5 Î¼g/L of the growth experiment). All standard solutions were diluted by Milli-Q water.
For Varian Spectr AA 220Z Graphite Tube Atomizer (GTA), copper and cadmium were measured. Standard solution had been prepared to calibrate the standard curve for each measurement with blank and mix standard solution of 20 Î¼g/L of copper and cadmium.
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 software SPSS 16 for Windows. In acute experiment, LC50 values for the juvenile with 3 metals were calculated by Log transformation and correlation analysis. The Dose-Response curves of three metals were plotted by Sigma Plot 2001. In chronic experiment, the shell length, width and aperture changes, and GTA/AA metals' concentration measurement were analysis by Microsoft Excel 2007. The growth changes of data from different treatments were transformed into ranks and compared with one-way analysis of variance, and then multiple comparisons using Tukey test.
Based on the data collected from the Range finding 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
According to the Dose-Response curve of each metal above, the LC50 of each metal was found. The 24 hour LC50 values for the juvenile were 92.30 Î¼g/L of copper, 780.30 Î¼g/L of cadmium and 3.85 mg/L of zinc. The 48 hour LC50 values for the juvenile were 81.20 Î¼g/L of copper, 467.20 Î¼g/L of cadmium and 3.76 mg/L of zinc. The 72 hour LC50 values for the juvenile were 64.50 Î¼g/L of copper, 398.50 Î¼g/L of cadmium and 3.21 mg/L of zinc. The 96 hour LC50 values for the juvenile were 50.10 Î¼g/L of copper, 342.10 Î¼g/L of cadmium and 3.14 mg/L of zinc.
By interpreting the Dose-Response curves, the NOAEL had been found as 10 Î¼g/L of copper, 50 Î¼g/L of cadmium and 1 mg/L of zinc.
For the chronic test, the diameter of juveniles' shell length, width and aperture were measured at day 0, week 2 and week 4. According the figure 8, 9 and 10, the growth rate of 4 weeks treatment showed that showed the same trend of growth for the corresponding metal. The growth rate by descending order were control > copper > cadmium > zinc.
Figure 12. Shell Length changes of 4 weeks Growth Culture with 3 metals
Figure 13. Shell Width changes of 4 weeks Growth Culture with 3 metals
Figure 14. Shell Aperture changes of 4 weeks Growth Culture with 3 metals
GTA/AA metals' concentration measurement
For metals' concentration measurement, the highest standard deviation of Hour 2 was 4000 Î¼g/L of zinc with Â±46.7 and the lowest was 5 Î¼g/L of cadmium with Â±0.1. Also, the highest standard deviation of Hour 72 was 4000 Î¼g/L of zinc with Â±50.0 and the lowest was 5 Î¼g/L of cadmium with Â±0.1.
Table 1. GTA/AA metals' concentration measurement
40.9 Â± 0.8
40.0 Â± 0.2
102.6 Â± 0.6
100.2 Â± 1.2
105.6 Â± 2.2
104.2 Â± 1.6
504.4 Â± 1.9
501.7 Â± 2.8
1011.3 Â± 6.2
1007.3 Â± 4.7
4239.9 Â± 46.7
4233.1 Â± 50.0
10.5 Â± 0.6
9.9 Â± 0.6
5.1 Â± 0.1
5.1 Â± 0.1
142.3 Â± 2.0
142 Â± 2.1
According to the result of the LC50 of the P. canaliculata juvenile, it showed that they could stimulate by the test metal in very low concentration. The LC50 of 24, 48 and 96 hour were 92.30, 81.20 and 50.10 Î¼g/L of copper, and 780.30, 467.20 and 342.10 Î¼g/L of cadmium. By comparing the LC50 of the Chinese Toad (Bufo gargarizans) Tadpole, the LC50 of 24, 48 and 96 hour were 201, 138 and 138 Î¼g/L of copper, and 32.1, 23.3 and 18.9 mg/L (Yang et al. 2003). P. canaliculata juvenile is much sensitive to the copper and cadmium in the low concentration and much suitable to being bioassay. Also, juveniles are most sensitive to copper (96 hours LC50 50.10 Î¼g/L) and least sensitive to zinc (96 hours LC50 3.14 mg/L) .
After the 4 week culture, juveniles showed different growth response with different metals (copper for 2.5 Î¼g/L, cadmium for 10.6 Î¼g/L and zinc for 141 Î¼g/L). It showed that cadmium's growth inhibition to the juvenile was significant (One Way ANOVA statistic analysis by SPSS), and could inhibit the growth more than zinc; and zinc could inhibit the growth more than copper. Also, copper showed similar effect with the control.
by SPSS 16
-: no significant difference P. > 0.05
ï¼Š: significant difference P. < 0.05
Table 2. Mean One Way ANOVA statistic analysis of the change of shell length
Beside the acute test, snails absent in the contaminated site were proposed as other mechanism (Laskowski & Hopkin 1996). The factors were avoidance and reduction of food intake and result in inhibition of growth and/or fecundity (Gomot-de Vaufleury and Kerhoa 2000). This explain that why snails were inactive after two week by observations, and also provided the reason of inhibition growth. In reference, Copper is related to the binding of metallothionein in the soft tissue or organ (Yap et al. 2009), it may induced the change of the protein sequence and further inert the normal enzymatic activities. Thus, it's sensitive to copper in low concentration. Cadmium could used up glutathione and protein bound sulfhydrl groups and resulting further production of reactive oxygen species, for example, hydroxyl radicals, hydrogen peroxide and superoxide ion (Stohs, et al 2001) . Cadmium could replace zinc in lot of vital enzymatic activities; disruption and stop of activities were further exist (Moore and Ramamoorthy 1984); this provides a reason to explain juvenile is sensitive to cadmium.
GTA/AA metals' concentration measurement
According to the Table 1 results, there was no significant change in medium concentrations at hour 2 and hour 72 which were almost identical. This interpreted the Falcon container's inner wall was inert to the attachment of the metal ions. The values were stable and consistence during course of experiment. In additions, these two containers could be used for copper, cadmium and zinc related culture with the consistence cultural medium environment.
In this study, toxic profiles of this species were found. The LC50 of 24, 48 and 96 hour were 92.30, 81.20 and 50.10 Î¼g/L of copper, 780.30, 467.20 and 342.10 Î¼g/L of cadmium and 3.85, 3.76, 3.21 and 3.14 mg/L of zinc. It found that the sensitive level by descending order are copper > cadmium > zinc. This species is very sensitive to three heavy metals especially copper and cadmium. Also, the NOAEL were found as 10 Î¼g/L of copper, 50 Î¼g/L of cadmium and 1 mg/L of zinc.
For the chronic growth experiment, it found that juveniles were shown different restriction response for growth due to different metal treatments. The sensitive level by descending order are cadmium > zinc > copper> control. Cadmium showed significant different with other treatments.
In this study also provided sufficient evidence to P. canaliculata juvenile can be used as a pollutant bioindicator as well as bioassay in future, especially in tropical and subtropical area with high abundance of this specie. On the other hands, these toxic profiles may be used for future protein biomarkers finding.