Problem Statement
The steady growth in per capita consumption of fish has caused increased attention to the safety of these products. There is always a potential for exposure to pesticides and environmental pollutants originating either from direct or indirect sources. Researchers have shown that the concentration of residues present in the raw fish fillet is not necessarily the same concentration present in the fillet following preparation. Food preparation has been shown to reduce non-polar organic pesticides, such as DDT and dieldrin, in fish by as much as 70%. While regulatory agencies commonly use raw whole fish or fillets to issue health advisories, it is important to assess the influence of food preparation on final contaminant concentration. While significant research has determined the losses of non-polar contaminants during preparation, more research is required to measure the influence of processing on intermediate polarity contaminants so that exposure can be estimated through human risk assessment efforts.

Detection of contaminants in fish generally requires costly and time-consuming methods which rarely provide results prior to consumption. Thus, there is a need to develop low cost, rapid methods for measuring pesticide residues in fish so that contaminated foods and be identified prior to consumption. Development of low-cost assays will increase the number of samples which can be assayed and improve attempts to determine the fate of pesticides during processing and preparation and improve the estimates of human exposure.

Enzyme Linked Immuno-Sorbent Assays (ELISA) have been used for several years to measure contaminants in foods, including fish. ELISA’s are currently available to measure PCB in fish. As with most analytical methods, ELISA’s are sensitive to interferences which result from the sample matrix and development of an extraction/preparation procedure is necessary to enable the ELISA to be useful for various foods. When analyzing fish for residues, the lipid can interfere with extraction and analysis of pesticides. As part of this proposed study, attempts will be made to use a commercially available ELISA kit to measure a moderately polar organophosphate pesticide (i.e., chlorpyrifos) in fish samples. ELISA’s can also be useful to provide sufficient data for an estimate of human exposure to pesticides as a part of risk assessment efforts. Part of the risk assessment process is to determine the concentration of residues, which are present at the point of consumption.

The objectives of this study are to: adapt a commercially available ELISA kit for measurement of chlorpyrifos in fish; using the ELISA to verify residue concentrations in catfish samples which contain chlorpyrifos; and use the ELISA to determine the fate of chlorpyrifos during the preparation (baking, frying and smoking) of dosed catfish.

Approach

In a recent study, rainbow trout and channel catfish samples were collected and analyzed for organochlorines, organophosphates and pyrethroids. Analysis costs (approximately $150/sample) limited the number of samples that could be tested. In addition to the high cost for testing, analytical methods took from weeks to months to be completed, which limited their usefulness when attempting to evaluate food samples prior to consumption. During the study, residues of chlorpyrifos (DursbanÒ ) were detected in 10.5% of the channel catfish sampled. Since chlorpyrifos is not registered for use in fish, it is considered a violative residue as regulated by the FDA.

Channel catfish will be raised at Purdue University. Twenty-one fish will be fed a 32% protein, floating catfish feed at the rate of 0.5-1% body weight. Chlorpyrifos will be incorporated into feed at 5 mg kg^-1. Thoroughly ground feed will be mixed with chlorpyrifos and made into pellets using 3% carboxymethyl cellulose as a feed binder. The total amount of treated feed consumed by the fish during the study will be measured. A set of control fish will be fed commercial feed with no added residues.

At harvest, a total of 21 fish for each organochlorine will be collected and divided into 3 groups of 7 fish to be processed using one of three preparation methods. Fish will be weighed and measured for length then skinned and hand filleted into two similar fillets keeping the belly flap intact. Matched fillets will be subsequently analyzed for chlorpyrifos with one fillet being maintained raw while the other fillet will be cooked using one of the three preparation methods. Corresponding fillets from each fish will be wrapped in aluminum foil and placed in zip-lock plastic bags.

Frying of samples will be done using a regular deep fryer in canola oil. Breaded fillets will be fried at 190^oC for about 7-10 min until golden brown. An internal temperature of 71^oC will be obtained and measured by a iron-constantin thermocouple. Samples will be baked at 190^oC in an oven for about 45 min until golden brown. Prior to smoking, fillets will be soaked in a 25% salt solution at 10^oC for about one hour and air dried before smoking. All cooked fillets will be packaged and stored at -23^oC until further analysis.

Individual fillets will be ground using a meat grinder. All utensils will be cleaned thoroughly with detergent, rinsed with water, dried and then rinsed with 2-propanol between each sample. The samples will be placed in glass bottles and stored at -23^oC. For moisture determination, 1g of sample was weighed and placed in a vacuum oven at 98^oC and 1.5"Hg for 6 h. Change in weight during drying will be used to determine moisture content.

Extraction of fat and chlorpyrifos residues from fish will be performed by a soxhlet extraction. After thawing overnight and mixing, a 5g of sample will be weighed into a 100 ml glass beaker and mixed with 55 g of anhydrous sodium sulfate. Samples will then placed in a desiccator at room temperature for at least 12 h. The mixture will be transferred into a pre-rinsed 33 x 80 mm cellulose thimble and plugged with pre-rinsed glass wool. Samples will be extracted with a soxhlet apparatus for at least 7 h with 250 ml hexane at a turnover rate of 4 to 5 times per hour. The extract will be concentrated to less than 10 ml at 45^oC and transferred to a pre-weighed, 50 ml screw cap centrifuge tube. The remaining solvent will be evaporated under a constant flow of nitrogen for 2 h at room temperature and weighed to determine fat content.

Chlorpyrifos will be extracted from fat by liquid-liquid partitioning. The sample volume will be adjusted to 15 ml with petroleum ether. The extract will be partitioned 3 times with 30 ml of acetonitrile saturated with petroleum ether, by shaking vigorously for 2 min. Each time, the acetonitrile layer will be transferred into a 1 L separatory funnel containing 650 ml of deionized water, 40 ml of saturated NaCl solution and 100 ml of petroleum ether. The residues will then be partitioned back into petroleum ether, the extract filtered through anhydrous sodium sulfate and concentrated to about 5 ml.

For cleanup of residue extract, a 22 mm i.d. chromatographic column will be prepared with 20 g, activated (at 130^oC for 24 h, and cooled to room temperature) florisil^® (F-100, 30-60 mesh), topped with 1 cm layer of anhydrous sodium sulfate. The prepared column will be rinsed with 50 ml of petroleum ether and the extract transferred onto the column. Residue elution will be performed using two 200 ml solutions (6% diethyl ether in petroleum ether and 15% diethyl ether in petroleum ether). Two fractions of 200 ml each are collected. The two fractions are combined and final volume is reduced to 5 ml by evaporation at 42^oC. The volume is adjusted and an aliquot of the extract is transferred to 2 ml injection vials for gas chromatographic analysis.

Samples will be analyzed for chlorpyrifos using an HP-5, 30 m x 530 µm column in a gas chromatograph with an electron capture detector. An isothermal temperature program will be used with inlet, oven, and detector temperatures at 230^oC, 220^oC and 300^oC, respectively. Helium will be used as carrier gas at a flow rate of 1.5 ml min^-1 and nitrogen (Grade 5.0) will be used as the makeup gas with flow rate of 60.0 ml min^-1. The standards for chlorpyrifos will be prepared from an EPA certified, 1000 ppm standard solution in methanol. Quantitation will be done based on peak area.

ELISA kits from a commercially available source will be adapted to measure chlorpyrifos in catfish. Homogenized tissue (1.0 g) will be weighed into a 40 ml centrifuge tube and 10 ml of 75% methanol added. Samples will then be sonicated with a 650 watt sonic disruptor for 30 sec and centrifuged for 4-5 min. Supernatant will be removed and filtered, diluted and stored at 4^oC prior to analysis. Anaylsis will be conducted as described in the commercial method manual.

Statistical analysis will be performed using Statview 4.02 software. One way analysis of variance with Fisher protected-LSD (p-LSD) test at 95% significance level will be used to determine significance differences among treatment means. Multiple regression analysis will be performed to find correlation coefficients (p<0.05) between dependent and independent variables.

Impact

Organophosphate pesticides, including chlorpyrifos, are currently being reviewed by the EPA for safety as required by the Food Quality Protection Act. Preliminary recommendations by a scientific review panel indicates that chlorpyrifos in food poses a significant risk to infants, children and pregnant women. The EPA will soon decide if the tolerance for chlorpyrifos in selected foods should be reduced to allow for a 10-fold safety factor. The risk assessment being conducted by the EPA will include human exposure from all food sources for chlorpyrifos.

Efforts to measure chlorpyrifos and determine its fate during fish preparation are necessary to assess the human health impact from exposure to this pesticide in fish from the Great Lakes region.

Relevance to Indiana-Illinois Sea Grant Efforts

The number of State health advisories for fish consumption have increased many-fold in recent years with comprehensive measurement of PCB’s and mercury in river and Great Lake fish. These advisories continue to be issued from data collected by traditional, slow and expensive laboratory methods. The resources applied to this effort are extensive due to the cost associated with collection of fish and analysis of these contaminants. Further studies are necessary to monitor currently registered agricultural pesticides in wild fish. Since additional resources for this expanded testing are unlikely to be appropriated, rapid and low-cost screening methods are necessary to preliminarily screen fish samples which would then be followed by traditional methods to validate preliminary data.

ELISA’s provide a low-cost and rapid screening tool for analysis of single contaminants in fish. ELISA’s are available for a number of contaminants, however, few have been adapted for measurement of contaminants in fish. Screening of fish, through ELISA’s, can be coordinated with traditional multiresidue methods to assess residues in a greater number of fish and to measure a greater number of contaminants.

Chlorpyrifos is used on cotton, soybeans and alfalfa to kill insects (e.g., corn borer and earworm, lesser cornstalk borer, cutworms, seed corn maggot and bettle and corn rootworm). Since these are heavily planted crops in Indiana and Illinois, there is a likelihood that residues from agricultural sources will be detected in river and lake fish from this region. Contamination of fish by this organophosphate has been detected in other regions of the country. Chlorpyrifos has been banned in the Mississippi Delta region due to non-point source agricultural residues which were detected in catfish.