Relationship Between Scallop Quality And Marine Environment In Zhangzidao Island Maricultural Area And Characteristics Of Uptake And Depuration Of Bacteria In Scallop

Bivalves tend to accumulate harmful substances and microorgnisms via food-chain transmission due to their filter-feeding habitual behavior. This biological characteristic not only affects negatively the growth and immunity of the shellfish, but may also threaten the consumers'safety. Seafood safety has become a key issue of sustainable development of aquaculture. In order to evaluate the relationship between food safety of scallop (Patinopecten yessoensis) and aquatic environmental factors in the cultural area of Zhangzidao Island, and to clarify the mechanism of uptake and depuration of bacteria in scallop, we have conducted experiments on environmental monitoring and evaluation of the relationship between the marine pollution level and food safety of the scallop, studying the accumulation of opportunistic bacteria and their distribution in different tissues of scallop, the effect of diets on the bacterial depuration in the scallop, and the effect of bacteria on activities of digestive and immune enzymes of the scallop. The findings of this study are useful for developing aquacultural probiotics and will provide scientific basis for sustainable aquaculture of bivalves. The results are as follows:1. In April, June, August, October and November, 2008, six samplings were carried out respectively, to detect the seasonal variation of shellfish toxins (paralytic shellfish poisoning toxin, PSP; diarrhoeic shellfish poisoning toxin, DSP), heavy metals (copper, Cu; lead, Pb; cadmium, Cd; zinc, Zn) and bacteria (total bacteria and coliforms) both in the seawater and the scallop Patinopecten yessoensis. The monitoring results showed that the active phosphate was at concentrations between 1.20-28.20μg/L, met the grade II of Seawater Quality Standard (GB3097-1997) throughout the year. Dissolved inorganic nitrogen (DIN) was at concentrations between 33.65-663.44μg/L, and all samples taken in June and two inshore samples taken in November greatly exceeded the grade II of Seawater Quality Standard. The heavy metal levels in seawater and sediment both met the grade I of Seawater Quality Standard and Marine Sediment Quality, respectively. Compared with coliforms, the number of total bacteria in seawater was more related to environmental factors, showing a strong negative correlation with dissolved oxygen in seawater (R= -0.788), and a positive medium correlation with pH (R = -0.584) and water temperature (R = -0.572). Dissolved oxygen was the main limiting factor of marine bacteria under the survey conditions; pH and water temperature also had a certain influence on it. Scallop monitoring results showed that the levels of four heavy metals and two shellfish poisoning toxins were in agreement with grade I of Marine Biological Quality, the average contents of Pb (0.11 mg/kg) and Cd (0.20 mg/kg) in August and that of Zn (19.81 mg/kg) in November were the highest during the survey. Accumulation of heavy metals was related to mariculture patterns, environmental conditions and the physiological state of the shellfish. Raft-cultured one-year scallop can accumulate Pb and Cu most easily, while the bottom-cultured was easier to accumulate Cd. Contents of Cu and Zn in scallop had a strong positive correlation with that of the sediment, and the content of Pb in scallop has a moderately positive correlation with that in sea water (R = 0.436), Cd showed a lack of correlation. PSP content in inshore bottom-cultured scallops (73.20μg/100g) showed a maximum in June, but was undetectable in November. The average level of PSP in scallop was negatively correlated with active phosphate in seawater (R =- 0.655), indicating that active phosphate may be a major limiting factor for toxic alga producing PSP. The highest level of PSP was detected in the digestive gland, about 4.92 to 8.17 times higher than the average content of total soft tissue, gills and mantle, ranked in a descending sequence, and the lowest was in the adductor, about 60 percent of the average content. For seasonal variations, two top levels of total bacteria in scallop were detected in August (3490 cfu/g) and in October (3150 cfu/g ); The highest number of coliforms in scallop (80 cfu/g) were found in station 1 in June, and coliforms were detectable until October. Both culture pattern and growth stage can affect the bacterial accumulation in scallop. Raft-cultured and young scallops were more easily contaminated by bacteria than the bottom-cultured ones. The coliforms in scallop were significantly correlated to those in seawater (R = 0.947, P < 0.01), and were also positively correlated with total bacteria in scallop (R = 0.693 ), but negatively correlated with the seawater pH, showing a lack of correlation with the total bacteria in seawater. Accordingly, the relationships between coliforms in scallop (y, cfu/g) and those in seawater (x1, cfu/mL), and the total bacteria in scallop (x2,×103 cfu/g) were expressed as follows: y = 1.4689x1 + 2.6488 (R2 = 0.9422), y = 8.3402x2 + 2.5719 (R2 = 0.7786); The relationship between total bacteria in scallop (y,×103 cfu/g) and coliforms in seawater (x, cfu/mL) was as follows: y = 4.3612x2 - 75.457x + 326.62 (R2 = 0.9017).In April and November the ecological environment quality was excellent in Zhangzidao Island cultural area, and maintained a―good‖level in other months. The potential ecological risk of heavy metals was at a bottom level in surface sediments, while the N/P value was most suitable for growth of harmful alga from August to October, the risk of harmful alga bloom was at a top level. The annual quality of scallop was at a―good‖level on the whole, the limiting factors of which were the contents of PSP toxin and coliforms in scallop tissues from June to October.2. After being challenged by exposures, the accumulation and depuration of Escherichia coli and Enterococcus faecalis in P. yessoensis were studied, and the results showed that the number of bacteria reached a top level at 1 to 3 d. The bioconcentration factor (BCF) and the rate of accumulation in E. faecalis treatments were both higher than those of E. coli treatments, suggesting that the scallop accumulated E. faecalis more easily from the environment. The largest numbers of both bacteria were detected in the digestive tract, and the smallest numbers were detected in hemolymph. The contents of E. coli and E. faecalis in the digestive tract were 5.7 and 12.9 times higher than those in the total soft tissue; while those in the hemolymph were 6.0 and 13.9 percent of the total soft tissue, respectively. Furthermore, the level of E. faecalis was about 85 times higher than that of E. coli in hemolymph. After 24h of depuration, the highest depuration rates were observed in all treatments. The depuration rate of both the 0.03% and 0.05% Spirulina-feeding treatments exceeded 90 percent, higher than other groups, while the latter group obtained a top rate of 93%. Therefore, in the early days of depuration, Spirulina can significantly increase the depuration rate, and under the experimental conditions feeding Dunaliella salina powder went against the depuration of the bacteria from the scallop.3. The effect of long-term exposure to bacteria on digestion of scallops was studied. The analysis of enzyme activities showed that after the long-term exposure to higher than 3 log10CFU/mL E. coli and E. faecalis, the digestive enzyme activity in scallop was obviously inhibited. Compared with E. faecalis, the inhibition of digestive enzyme activities was more significant after exposure to E. coli. The trypsin activity in the E. coli treatment was markedly lower than to the E. faecalis treatment after exposure challenge, while no significant difference was observed between the two treatments after injection challenge.4. By using exposure challenge, secondary exposure challenge and injection of antigens of disrupted whole cell and bacterial genomic DNA bacterial antigens, the relationship between bacterial retention and scallop immunity was studied. (1) The secondary exposure results showed that at 1 d of secondary exposure to E. coli, SOD and ACP activities in cell-free hemolymph significantly increased (P < 0.05), but this up-regulation of immune enzyme activity may maintain shorter than two weeks during the secondary challenge of the same bacteria.(2) At 4 h of injection, SOD activities in cell-free hemolymph of all treatment groups were higher than the control (Ct), but the difference was not significant (P > 0.05). After injected with different cell contents of the two bacteria, the rank of SOD activities in cell-free hemolymph was as follows: live bacterial cell > antigens of disrupted whole cell > bacterial genomic DNA. The SOD activity in scallop injected with live E. coli cells were lower than with E. faecalis cells, but it was vice versa among the treatments with the antigen of disrupted whole cell and bacterial genomic DNA. At 72 h, the SOD activity in scallop injected with antigens of disrupted whole cell was significantly higher than all the other groups (P < 0.05), no significant difference was observed between the other groups.(3) At 21 d of exposure and 18 h of injection, scallops were tested for the immune enzyme activities in haemolymph. After a 21d exposure to bacteria, the immune enzyme activities in cell-free hemolymph were significantly higher than those in hematocyte. Compared with E. coli treatment, E. faecalis-exposed scallops showed a lower ACP activity.