Ecological Effect Of Copper In Litpenaeus Vannamei Recirculating Aquaculture System

The purpose of present study was to investigate the performance of Litopenaeus. vannamei recirculating aquaculture system (RAS) under copper pollution , the effect of copper on bacteria community in biological aerated filter (BAF) and shrimp, and the copper budget, distribution and emission facters in the shrimp RAS. Furthermore, under the same copper concentration in both RAS and static aquaculture system, comparing the difference of shrimps'immunity, digestive tract, histological changes, physiological metabolism and heavy metal accumulation was also covered in the study. The main results include:1. The Medium Lethal Concentration (LC₅₀) of Cu to Litopenaeus. vannamei (body length was 4.0±0.4cm, weight was 0.94±0.02g) was evaluated and the study showed that 24h LC₅₀, 48h LC₅₀ , 72h LC₅₀ and 96h LC₅₀ of Cu was 54.81mg/L, 42.92mg/L, 23.91 mg/L and 13.11mg/L respectively. Acute toxicity of Cu²⁺ can cause death , MT production in hepatopancreatic, ammonium excretion increase, inhibition in oxygen consumption rate and activity of Na+-K+-ATPase within gills of shrimp, however, there was no evidence to illustrate that molting behavior was also result from it.2. After Litopenaeus. vannamei (body length was 8.5±0.76cm, weight was 7.6±0.5g) were exposed in 0.5mg/L and 5mg/L Cu²⁺ solution for 7 days, oxygen consumption rate decreased by 24.62% and 44.031% and ammonium excretion was elevated by 78.26% and 175.69%, respectively. The study showed that copper can be accumulated within hepatopancreas, gills, carapaces and muscles increasingly, and the amount of accumulated Cu in hepatopancreas was 100 times higher than it in muscles. So it was sufficient to conclude that Cu accumulation was most significant in hepatopancreas out of the four organs in the study for Litopenaeus. vannamei in the specific life conditions as we had.3. Three months'experiment was conducted for intensive culture of shrimp (L. vannamei) in RAS to study the effect of Cu polluted water (0.3mgL^-1) on the biofilm. The results indicated that there was no significant difference in COD, NH₄-N and NO₂-N removal rate between regular RAS system and the one worked with biofilm. Also the number of Heterotrophic Bacteria(HB), Ammonium-oxidation bacteria(AOB) and Nitrite-oxidizing bacteria(NOB) in the biofilm was not effected dramatically. The number of Heterotrophic Bacteria, Ammonium-oxidation bacteria and Nitrite-oxidizing bacteria were only 103, 102-103 and 100-101 per gram packing at the beginning, and it took 30 days for the number of Heterotrophic Bacteria and Ammonium-oxidation bacteria to reach the stable level, which were 106 and 104-105 per gram packing respectively. But for Nitrite-oxidizing bacteria, it took nearly 45 days to reach the stable number of 104-105 per gram packing.4. Harvesting was obtained after 98 days'culture, and it showed that there was no significant difference in mean body weight (11.08±0.76 mg vs 10.52±0.51 mg), yield (6.42±0.33 kg/m³ vs 5.99±0.39 kg/m3), survival rate (67.78±5.29 % vs 66.39±1.93 %) and FCR (1.2±0.060 vs 1.29±0.082) between the Cu polluted systems and the control systems (P>0.05). Shrimp growth was not limited by any of the water quality parameters in the study. TAN and NO2-N concentrations were 0.28±0.13mg/L and 4.02±5.60mg/L in the control systems and the values were 0.26±+0.1 mg/L and 4.42±6.09mg/L respectively. Juveniles of the Pacific white shrimp, L. vannamei, can be raised in limited water exchange regime with healthy conditions, low feed conversion rate, fast growth, and high biomass loading in RAS.5. Cu concentration decreased from 0.3 mg/L to 0.089±0.012mg/L at the harvesting time and most of the Cu had been removed out from the water treatment processes. The major output of Cu was in the mechanical filter and the draining effluent in the capture, which accounted for 41.72% and 15.05% of the total inputs respectively. As a result, the cultured shrimp had not been effected on immunity, digestive tract with Cu accumulation in tissues. The molality of Cu in the shrmps was at last 42.91±6.10 mg/Kg, and it meet the requirment of the Green Food and Pollution-Free Agriculture Products Standard, which gave the molality limitation as≤50 mg/Kg.6. Cu budget in the shrimp RAS revealed that the most source of Cu to the systems was the aqua feeds, which accounted for 86.55% of the total inputs. The major output of Cu was in the mechanical filter, renewal waters and the draining effluent during the capture activity, which accounted for 22.33%, 12.43% and 11.83% of the total inputs respectively. Another, Cu loading releasing from the three ways was 14.7 mg /cycle, 7.94 mg/ cycle and 7.56 mg /cycle respectively.7. Harvesting was also obtained after 112 days culture, and there was significant difference in mean survival rate (40.00±2.22 % vs 15.44±3.07 %), body weight (13.36±0.74 g vs 10.34±1.15 g) and FCR (2.03±0.12 vs 6.52±0.32) between the RAS and the static aquaculture system under 0.8 mg/L of copper pollution (P>0.05). The mean concentrations of TAN (1.63±1.15 mg/L vs 6.48±8.41 mg/L), NO₂-N (1.03±0.39 mg/L vs 6.09±1.32 mg/L) and COD (10.93±4.80 mg/L vs 24.36±13.87 mg/L) were also significant to each other in the two systems. Juveniles of the Pacific white shrimp, L. vannamei, had good aqueouse environment achieved faster growth rate with lower feed conversion rate in RAS comparing to those in static aquaculture system.8. PO activity, SOD activity, quantity of large granular cells and total haemocyte count of Litopenaeus vannamei had significantly decreased after shrimps been cultured for 112 days with 0.8mg/L copper molality in both RAS and static aquaculture system. The result showed that PO activity (0.008±0.003U/min/mg pr) of shrimp in static aquaculture system was significantly lower compared to in RAS (0.011±0.007 U/min/mg pr). In RAS, SOD gene expression levels of shrimp was 1.41±0.57, and it had been elevated very much; mRNA levels of Lys and ProPO increased to 1.49±1.32 and.40±1.19 respectively but no significant differences were detected. In static aquaculture system, SOD and Lys gene expression levels of shrimp were dramatically decreased to 0.71±0.42 and 0.64±0.53, however, the mRNA level of ProPO significantly increased to 1.64±1.12. The upregulation of proPO mRNA by the shrimp may be resulted in decreased PO activity. The present study documented that the concentration of Cu²⁺ at 0.8mg/L decreased the humoral immunity of Litopenaeus vannamei both in RAS and static aquaculture system, the Litopenaeus vannamei can regulate the expression levels of gens to banlance the decreased enzyme activity as immune modulation to enhance the immune ability. In addition, the mRNA transcription of SOD and Lys gen in shrimp is sensitivity to the copper pollution compared to proPO gen.9. The toxicity of Cu²⁺ at 0.8mg/L inhibited oxygen consumption, Na⁺-K⁺- ATPase activity and elevated in ammonium excretion in Litopenaeus vannamei both in RAS and static aquaculture system. The gills of shrimps collected in static aquaculture system after been cultured in 0.8 mg/L copper pollution water for 112 days showed a high disorder and a high degree of melanization. Absence of pillar cells, loss of regular structure of epithelium and multifocal necrosis on gills of shrimp were observed in the RAS. To the histological changes of hepatopancreas, atrophy and irregular tubular structure were observed in shrimps collected in static aquaculture system at the harvest. In the RAS, there was an absence of storage and secretory cells, as well as multifocal necrosis and loss of tissue in some zones of the tubules, separation between myoepithelium and epithelium.The amount of MT induced in the hepatopancreas was 2.27±0.62nmol/g and 2.75±0.55nmol/g respectively in RAS and static aquaculture system, and the Cu concentration accumulated in hepatopancreas was 834.52±267.41mg/kg dry weight and 1398.31±263.90mg/kg dry weight respectively, which showed a significant difference with each other. There was significant linear correlation between the Cu concentration and MT, Which may be used as a valid biomarkers to compared the RAS and static aquaculture system under heavy metal pollution.