risk management of transgenic fish

转基因鱼风险评估及风险管理Risk assessment and

management of transgenic fish

梁利群LIANG LI-QUN

Introduction•• Aquaculture is a major industry in China. With the

improvement of people’s life, there need more and more marine products, especially fish, which will need more aquaculture species with quality product characteristics. Gene engineering breeding, an effective tool, will play an important role in the preparation of new aquaculture species.

• The study on transgenic fish began in 1980s in our country with the chief purpose of improving the growth rating. Growth hormone genes of Oncorhynchus keta and Ctenopharyngodon idellus are used to construct transgenic carp. Due to the special characteristics of gene engineering products, transgenic fish will be significant in foods and medicines.

• However, the fish, living in an open water area with good reproductive capacity, are not handily managed. Therefore, it is necessary to make the scientific assessment and management of non-target effect, gene flow and ecological adaptation to transgenic fish, which will protect human health, ecological environment of water and the genetic resource of fish.

一、assessment of the non-target effect on transgenic fish

principle and purpose

• As one kind of foods, transgenic fish will have a direct or indirect effect on human body. According to the principle of substantial equivalence in regulating transgenic fish, it is necessary to make a non-target effect assessment and normal toxicology experiment. Considering the specialty of transgenic fish, the examination is proceeded with a little difference from “Procedures and methods for toxicological assessment on food safety”, which shows higher proportion of the object under testing in the feed. Feeding transgenic fish to the rat and mouse makes the toxicology experiment. It contains general toxicity (rat acute toxicity, rat subacutetoxicity) and reproductive toxicity (F2 reproductive experiment of mouse). After the test of Physiology and Biochemistry values of experimental animals, we can assess the effect of transgenic fish to human and other animals as foods, medicines and materials.

1 Experimental animals: rank 2, clear rat and mouse. Rat: 200, mouse 200.

Testing animals: good nourishment and health/same age/male: female(♀:♂): 1:1/body weight gap less

than 20% between group and 10% within group.Before the experiment, all experimental animals were detected for a week and the unhealthy animals were replaced.

一、assessment method of non-target effect on transgenic fish

• 2 Experimental group and Control group• Rat subchronic toxicity testing: 3 experimental groups with 20

animals per group and 2 control groups with the same amounts of animals.

• Rat acute toxicity testing: 3 experimental groups with 20 animals per group and 2 control groups with the same amounts of animals.

• Mouse F2 reproductive testing: 3 experimental groups with 30 animals per group and 2 control groups with the same amounts of animals.

一、assessment method of non-target effect on transgenic fish

3 Experimental time• Rat subchronic toxicity testing: 90 days• Acute toxicity testing: stomach perfusing one time• Mouse F2 reproductive testing: 90days4 Confirmation of the experimental dose• In the Rat subchronic toxicity testing and Mouse F2 reproductive

testing, feeding dose was confirmed according to the actual circumstance. At a week before experiment, we feed the general feeding per day and record the ingestion amounts. Then the average ingestion amounts were used as the control experimental dose.

• Rat acute toxicity testing dose: perfuse the stomach 20g/kg bodyweight per time

一、 assessment method of non-target effect on transgenic fish

5 Testing index• Rat subchronic toxicity testing and Acute toxicity tesing by once

perfusing stomach

General detection: detecting animals’general appearance/behavior/activity/hair colour/poisoning symptom/death condition/food-intake/body weight, calculating food-utilizing rate.

Hematology Test: rat red blood cell counting/white blood cell counting/blood plate counting/ hemoglobin/category of white blood cell.

Blood biochemical test: rat serum asparate amino transferase (AST)/ alanine aminotransferase (ALT)/ alkine phosphatase (ALP)/blood urea nitrogen (BUN)/total protein (TP)/albumin (ALB)/blood glucose (GLU)/ total bilirubin (TBI)/ Creatinine (CRE)/CHO

Urine test: occult blood/nitrate/pH value/urobilinogen/bilirubin/protein/glucose/ketone.

Pathology test: weight of the heart, liver, kidne, spleen, orchis and the organ coefficient.

• Pathology and histology test of liver, kidney, spleen, orchis, ovaria, hystera, stomach, dodecadactylon, pancreas.


5 Test indexMouse F2 reproductive test• General detection was the same with 90 days feeding experiment.

In addition, accouchment, body weight, food consumption, male parent sperm activity, shape, amounts were also detected.

• Filial mouse: detection of the amounts, sex, miscarriage amounts, stillbirth amounts, megascopic abnormality and the cause of death. General test and pathology testof genital organ.


二、assessment of gene flow


• The water area used to culture the transgenic fish will be investigated seriously. The reproductive time will be confirmed according to the climate of cultured water area. After comparing the reproductive time of transgenic fish and wild fish, we can find whether they overlap in the time. If overlapping doesn’t occur, it is impossible to find gene flow; by contraries, If overlapping occurs, then it is necessary to investigate their reproductive characteristics. The relation between transgenic fish and wild relatives belongs to intra, inter-specific relationship. And the hybridization affinity and fertility of the filial will directlyaffect the gene flow of the exogenous object genes to wild relatives. To protect the ecology and fish resource of cultured water area from destruction, it is important to study the affinity relation between transgenic fish and their wild relatives, which will maintain the genetic stability of fish and ensure the sustainable development of fish foods, medicines and materials.

• Background information investigation of transgenic fish and their wild relatives

• Comparable study of reproductive characteristics on transgenic fish and their wild relatives

• Study of affinity relation on transgenic fish and their wild relatives• Study of gene flow on transgenic fish and their wild relatives


procedure


method

1 Background information investigation of transgenic fish and their wild relatives

The wild relatives of transgenic fish in the culturedwater area are investigated in detail, including their kinds and amounts, so that we can confirm if there exists overlapping area of the transgenic fish parents and the wild relatives. If there does not exist the wild relatives of transgenic fish in the water area, then the following assessment is unnecessary, otherwise, it is necessary to make the assessment as following.

2 Comparable study of reproductive characteristics on transgenic fish and their wild relativesThere are many factors to affect the fertility of fish gamete, such as time, temperature, sun exposure and so on. Therefore, it is necessary to carry through tissue slice analysis of ovary and spermarium between transgenic fish and the wild relatives with the same age in a year.The preparation method of tissue slice: add Bouin to fix 1cM tissue; slice the fixed tissue to 7µm width; HE staining; detecting under microscope.Finally, the gamete fertility time to different stage is confirmed.


method

2 Comparable study of reproductive characteristics on transgenic fish and their wild relatives During the reproductive season of the transgenic fish, when the water temperature can satisfy the demand of egg deposition, a circumstance will be constructed to lay eggs according to the reproductive characteristics of fish. The mature transgenic fish, wild relatives, close relatives are cultured in the same pond with enough oxygen following the ratio of ♀:♂3:2. The egg deposition is detected periodically. If there comes through ovulation and sperm expulsion in three kinds of fish, then it shows that the overlapping in the reproductive time occurs. If the transgenic fish escape, it is possible to inflict gene flow through the hybridization. Combining tissue slice and reproductive time, we can confirm the reproductive characteristics of transgenic fish and the wild relatives.


method

3 Study of affinity relation on transgenic fish and their wild relatives In artificial reproduction condition (conclude temperature, stream and so on), mature transgeneic fish and closed species were artificial ripening by injecting oxytocin to thoracic cavity and abdominal cavity. Then the fish appeared ovulation and spermatorrhea. The sperm and egg were collected by expressing their abdominal and artificial inseminated to fertilized eggs (direct and back-cross). According to the fish cultivated order, the fertilized eggs were hatched and cultivated in man-made condition. Hatching rate = the number of hatch / the number of fertilized eggs×100%. The phenotype of F1 generation was decided by observing the phenotype and morphology measurement, just as full-length, body length and so on. The fertility was decided by ensure the phenotype of F1generation, detecting the ploidy (flow cytometry, FCM), detecting the gonad tissues dissection and whether or not reproduced. If the F1 generation hatching rate was 100% and procreated, the affinity between the transgeneic fish and the wild close species were 100%. In these procedures, the transgeneic fish might lose the exogenous aim gene. And the aim gene was complete likely shifted to their wild close species.


method

• 4 Study of gene flow on transgenic fish and their wild relatives• The aim gene shift could be detected by the following methods. in

the transgenetic fish breeding season, according to the transgeneticfish hatch style in the breeding release region, the male maturetransgenetic fish were natural hybridization with non-transgeneticfish in the rate of 1%, 10% and 25%. In our experiment, the ratio of female and male was 3:2. We obtained 3 group fish young plantin all. When the fish had 1.5cm, we gathered 200 from 3 group randomly. They were detected exogenous gene integration with molecular biology method, which involve Dot Blot, Southern Blot and PCR. The detection concluded aim gene, promoter gene and terminator gene. The positive fish, which contain exogenous aim gene, would present specific band with the specific primer in PCR reaction.

• shift ratio = the number of positive fish / 200 ×100%• The discrepancy from transgenetic fish to non-transgenetic fish in

different experimental group was decided by the variance analyses method.

二、assessment of gene flowmethod

• According to the actual principle, the experiments are proceeded to find the major risk factors by comparing the competing capacity of transgenic fish and normal fish, including plunderage of food, growth condition, survival rate, prolificacy under the several different environments. Through the assessment of ecological adaptation to the transgenic fish, we can understand the effect on the ecological system of the area scientifically.

三、assessment of ecological adaptation


• Comparable test of survival competing capacity between female parents of transgenic fish and normal fish

• Comparable test of environment adaptation capacity between female parents of transgenic fish and normal fish

• Comparable test of prolificacy between female parents of transgenic fish and normal fish


procedure


method

• 1 Comparable test of survival competing capacity between female parents of transgenic fish and normal fish

• enclosed watersChoosing a 0.5 to 1 mu yield enclosed waters to do the survival

competitive capacity comparative test between transgenetic fish and non-transgenetic fish. According to the criteria of breeding density in this area, we released adequate one year old transgenetic fish and non-transgenetic fish with the same body weight and numerical measure. The fish were fed in semistarvation (the compactedness of intestine was two degree) and enough food (the compactedness of intestine was four degree) in a period. The experiment included the competitive ability of food, growing attitude, and survival rate and so on. The competitive ability was decided by analyzing the food occupation, i.e. the measurement of their compactedness of intestine. The discrepance of growing attitude between two groups was decided by measuring their body length and body weight. The viability was confirmed by calculating the survival rate, survival rate = the number of survival fish / the number of release fish × 100%. By analyzing the experimental data dispersion, we could identify the survival competitive capacity of transgenetic and non-transgenetic fish.


method

• 1 Comparable test of survival competing capacity between female parents of transgenic fish and normal fish

• the opening water with the necessary risks control approach• Choosing a above 0.5 mu yield opening waters, with the necessary flood control,

prevention of burglary and escape-proof approach, to do the survival competitive capacity comparative test between transgenetic fish and non-transgenetic fish. The same body weight and numerical measure adequate one year old transgenetic fish and non-transgenetic fish were released. The water should contain some food (haloplankton, fish, crevette and shellfish). The fish were fed in semistarvation in a period. The experiment included the food habit, the competitive ability of food, growing attitude, and survival rate and so on. The food habit experiment was major on the range of transgenetic and non-transgenetic fish (by detecting the food classes of fish intestinal canal with microscope). The competitive ability was decided by analyzing the food occupation, i.e. the measurement of their compactedness of intestine. The discrepance of growing attitude between two groups was decided by measuring their body length and body weight. The viability was confirmed by calculating the survival rate, survival rate = the number of survival fish / the number of release fish × 100%.By analyzing the experimental data dispersion, we could identify the survival competitive capacity of transgenetic and non-transgenetic fish.

• 2) the method of detecting the food classes of fish intestinal canal with microscope:• Diluting the food from 1/3 intestinal canal, and identify the classes of food

• 2 Comparable test of environment adaptation capacity between female parents of transgenic fish and normal fish

• According to the physiological character and water area condition (which contains temperature, salinity, basicity, pH, and dissolve degree of oxyzen), comparable test of environment adaptation capacity between female parents of transgenic fish and normal fish was done to ensure the risk factor and environment adaptability of transgenetic fish in the enclosed water. The experimental was done in the condition of temperature control, and with oxygen system in the less than one cubic meter container. The experimental fish, which contained transgenetic and normal fish, were all one year old and with the same body length and body weight. We assayed temperature, pH, and dissolve degree of oxyzen by equipment. The pH gage could measure the pH value lifting and fall when add some NaOH or HCl solution in to the water slowly. We chose different environment factor for the different fish. For example; transgenetic fish are cold water fish, so we chose the temperature range above their survival temperature. The transgenetic fish live in high base water region, so the basicity should be chosen and the salinity was abandon. The assay method consulted <the monitor and analyze method of water and sewage> which composed by the national office of environmental protection. The experiments were done in the controllable temperature and oxygen container, so the alteration of environment factor was slowly.


method

• 3. Comparable test of prolificacy between female parents of transgenic fish and normal fish

• Researching the prolificacy of transgenetic and normal fish in enclosed condition

• Choosing a 0.5 to 1 mu yield enclosed waters an according to the criteria of breeding density in this area, we released adequate one year old transgenetic fish and normal fish with the same body weight and numerical measure, similar habitus, and labeled by injecting electronic label below their skin or hanging a label in vitro. In their breeding season, we detected the transgenetic and allogenic normal fish: 1.Ensured the age of sex maturation: Before the breeding season, the transgenetic and allogenic normal fish ovary and spermarium were detected or analyzed by cut film to determine their development phase. Meanwhile, whether or not sex maturation also coule be determined by egg laying and release sperm condition. 2. Anatomize the female parent to find the amount of eggs.

• The ascertain of amount of eggs: weigh the female fish ovary (A), fish weight excluded ovary (B) and definite ovary weight (C) respectively, and count the eggs (M).

• K=M B /C A (pellet per gram)

三、 assessment of ecological adaptation

method


method

3 Comparable test of prolificacy between female parents of transgenic fish and normal fish

• the opening water with the necessary risks control approach• Adequate one year old transgenetic fish and normal fish with the same

body weight and numerical measure, similar habitus, and labeled by injecting electronic label below their skin or hanging a label in vitro were released into the opening water area with the necessary flood control, prevention of burglary and escape-proof approach. The fish were fed in semistarvation. The comparable test of prolificacy was done when they were sex maturation. In breeding season, the transgengtic and normal fish were detected 1.ensure the age of sex maturation: Before the breeding season, the transgenetic and allogenic normal fish ovary and spermarium were detected or analyzed by cut film to determine their development phase. Meanwhile, whether or not sex maturation also coule be determined by egg laying and release sperm condition. 2. Anatomize the female parent to find the amount of eggs.

四、 risk management of transgenic fish

• The concrete measures of risk management are confirmed through following aspects: biological control measures, Physical control measures, chemical control measures and urgent measures. It mainly contains type, purpose, facilities, and certification method of the measures.

• 1 Physical control measures• The breeding facilities must be very firm, and there

should not be less than 3 layers protecting nets in the water exits. Facilities used to control natural disasters such as flood water should be set up in the water area culturing transgenic fish. In addition, it is necessary to set up some correlative preventive measures to prevent transgenic fish from escaping.

四、Risk management of transgenic fish

• 2 biological control measures• In the water exits of the transgenic fish culturing

aquatorium, a culture pond with some feral animals feeding by fish is set up. When the three layers protecting net in the water exits are out of work, the culture pond can play a role in preventing transgenic fish from escaping.


• 3 chemical controlling measures• A enclosed cistern is set up in the downstream of the

biological controlling water. When the facilities of physical and biological control are out of work, we can add in high concentration burnt lime, which can lead to the death of transgenic fish, to accomplish the purpose of preventing fish from escaping.

四、Risk managementof transgenic fish

• 4 urgent measures• To prevent transgenic fish from escaping, some urgent

alarming measures should be established. The appearance of escaping should be reported to higher-up or national administrative department immediately.

• Ensure the salability of the network communication of the transgenic safety.

• Set up safety burning facilities of transgenic fish


Documents

risk management of transgenic fish