Index
- The Use of HPLC in Biotoxin Analysis
- Mussel Survey Efforts
- Dealer Inspection Changes
- Shellfish Closures: Why? How? When?
- Upcoming Events
The Use of HPLC in Biotoxin Analysis - By DMR Laboratory Technician Jill MacLeod
As indicated previously, Maine DMR has changed the routine toxin assessment in shellfish from the Mouse Bioassay method to an analysis utilizing High-Performance Liquid Chromatography (HPLC). Formally known as High Pressure Liquid Chromatography, this method has evolved from an early 20th century plant-pigment separation into the high-tech process that it is today. Thanks to the expertise of Bigelow Analytical Services in East Boothbay, we were able to implement this chemical testing tool as outlined by the National Shellfish Sanitation Program (NSSP).
Currently, liquid chromatographic separation of compounds within an extract of shellfish is used before detection and quantitation of all of the toxins that are monitored in shellfish in Maine. The preparation, conditions, and detection for each analysis vary, however. The most common toxins in Maine over the past decades have been those associated with Paralytic Shellfish Poisoning (PSP). Saxitoxin is the parent compound to the class of toxins that cause PSP. There are more than 50 related compounds with varying levels of toxicity, meaning that high levels of certain toxins are more dangerous than others. All of these toxins seem to work to paralyze movement in the same manner as saxitoxin by blocking certain voltage-gated sodium channel units at the neuromuscular junction, thus preventing cellular signal propagation. The efficacy in blockade of this channel varies across compounds, resulting in the differences in toxicity and therefore, risk to consumers. The NSSP calls for quantification of 12 of these PSP toxins, requiring two separate HPLC systems, two individual portions of the cleaned sample extract, and six different solutions to exploit the chemical properties of the various compounds for optimal detection.
To start, a 5 gram portion of blended homogenate of several whole shellfish is boiled in low-concentration hydrochloric acid. The sample is then centrifuged and the liquid is separated from the tissue. This liquid, or extract, contains the target toxins as well as other compounds that are soluble in the acid solution. To clean the sample of some of these extra components, a small amount of extract is treated and filtered so it can be injected into the HPLC systems.
Clean extracts are pipetted into small vials that are compatible with the automated sampling hardware. A robotic arm selects a vial from a tray that is mapped by computer software and moves it temporarily to the injector. A needle moves into the vial and draws up a small amount of the extract. Continuing with the PSP analysis example, 10 microliters are injected into the aqueous "mobile phase," the solution of chemicals that is constantly flowing through the system. The mobile phase passes through a "stationary phase" which is tiny particles of silica packed inside a column. These solutions and columns are formulated to aid in binding the target compounds to the stationary phase based on the polarity or hydrophobicity of each compound. The toxins that do not bind well with the stationary phase pass through the column quickly. Each toxin passes through the column at a specific rate, resulting in reliable division of the toxins within the flowing solution as it leaves the column.
Once toxins are separated within the stream of mobile phase, they need to be measured in some way. For PSP analysis, our method utilizes a chemical reaction that makes the toxins fluoresce. An oxidizing agent is mixed into the flow and the solution passes through a heated block that helps the conversion. Once oxidized, the toxin solution flows into a unit that emits an excitation wavelength and then reads the amount of light emitted by the now fluorescing compounds.
The end result of this entire process is a chromatogram: a graphical output of the separation and detection of the target toxins in a sample (Figure 1). Each toxin is identified by its position in the chromatogram, which is based on the differing amount of time it takes for toxins to pass through the column. Those that pass quickly are seen first, with each sample requiring about 30 minutes to separate on the column and to pass through the detection unit.
Toxins are then quantified by a calculation utilizing the area of the "peak" seen on the chromatogram and the area of a reference standard peak. Each compound quantity is adjusted for its relative toxicity to saxitoxin and the total levels of PSP toxin is reported as micrograms of saxitoxin "equivalents" per 100 grams of shellfish meat. If the total in these units is greater than or equal to 80, the shellfish from the area where the sample was collected are considered unsafe for consumption.
Years of work by many scientists worldwide contributed to the development and implementation of this testing method for PSP analysis. As technology advances, methods will continue to evolve and improve upon sensitivity and precision of measurement. Every new adaptation of methods to the monitoring of biotoxin analysis allows more efficient monitoring and management of our shellfish resources along the coast of Maine.
Chromatogram of a PSP toxin-containing mussel from the Maine Coast. The x-axis is time in minutes and the y-axis is the measure of luminosity at the target wavelength. The annotated peaks along the chromatogram represent different compounds that cause PSP.
2016 Mussel Survey Efforts - By DMR Program Supervisor Denis-Marc Nault
The Shellfish Management program oversees mussels and DMR has made it a priority to better evaluate this resource. The first steps are to determine the size and quantity of the inter-tidal mussel resource. We have looked at past survey methods and determined that aerial photography would not be cost effective. The advent of drones and digital video and photographs is being evaluated as a possible tool for DMR to quickly and cost effectively evaluate Maine's mussel resource.
DMR worked with a contractor to design a drone survey in the mouth of the Jordan River in the spring of 2016 but was not able to get it "off the ground" due to Federal Aviation Administration regulations and the very close proximity of the Bar Harbor Airport. The Shellfish Management staff spent considerable time and effort designing and placing 40 sample stakes over 4000 ft transects for this survey. In July, it was decided to collect mussel samples by hand for 50% of the established sites because the drone survey was not going to happen. The last week of July, twenty 1'x2' plots were collected from both the Trenton and Lamoine sides of the Jordan River, counted and measured.
The initial data shows a tremendous amount of small mussels with very few market size animals. This could be due to the survey sites being high on the flats, as larger mussels were observed at the lower tide range and into the sub-tidal areas.
Efforts are again underway to implement the drone survey in 2017.
Get the Scoop on Dealer Inspection Changes- By DMR Seafood Inspector Melinda Cook
As some of you have noticed, since DMR's last US Food and Drug Administration program evaluation in September, we are completing our inspections a little differently. Although we have run the program recently, the same as the past several years, we had new FDA evaluators in 2016 who requested changes.
We have therefore altered the way we perform our inspections in order to comply with the National Shellfish Sanitation Program (NSSP). From here on out, each inspection will be more thorough, more like your annual recertification inspection. The inspections will follow the model ordinance more closely, and will ensure that you and our inspection program staff are following all of the codes and keeping all of us in compliance with the NSSP.
The biggest part of this new approach will be adding correction dates to your inspection forms instead of noting "next routine inspection." This means that if you have a debit (or debits) listed on your inspection form, we will schedule a follow up visit to verify all of the corrections. You will have to have debits corrected at that time or risk losing your certificate. We will be working with you to get things corrected. We are here to help you stay in compliance, not shut you down. The follow up visits will be scheduled with consideration for your schedule and when you think you can reasonably have the debit(s) corrected. We will also implement other means of verifying your corrections such as emails or text messages with attachments like corrected forms or photos.
And lastly, we have been informed that you need to add another column to your receiving logs. You will now have to log the "time of harvest" from the harvester tag in addition to the "time received." This allows you, as well as us and the FDA to quickly see that the time harvested and received are in compliance; which is especially important to ensure product safety during the summer months with warmer waters and rising vibrio numbers. To make this transition easier, we have posted some example receiving logs on our website that you can print off and use. If these do not work with your process, contact us and we will be happy to work with you to create something that does.
As clichÇ as it sounds, we are all in this together. The NSSP is a collaborative program that requires the cooperation of industry and regulators to be successful. This industry and this fishery are important to us and this state and we want to see them continue well into our future and for future generations to come. Help us remain in compliance by diligently working with us to keep yourself debit free and/or to correct any debits you may have. As always, thank you for reading and please don't hesitate to call us with any questions, comments or concerns.
John Fendl, Dealer Inspection Program Supervisor and Downeast Maine Region
Cell: 207-592-8934
Melinda Cook- Shellfish Sanitation Officer and Central/Midcoast Maine Region
Cell: 207-557-3558
Arthur Rowe- Plant Inspector and Southern Maine Region
Cell: 207-557-3557
Angel Wilson - Shellfish Program Coordinator and Plant Inspector (assigned region varies with need)
Cell: 207-350-7902
Shellfish Closures: Why? How? When? - By DMR Scientist Erick Schaefer
Why: As shellfish feed by filtering food from the water surrounding them they accumulate whatever is in the water into their digestive systems. People then consume these shellfish effectively transferring whatever may be in the shellfish into our system. Shellfish closures exist to protect the public from bacterial, viral, naturally occurring harmful algal blooms, and other deleterious substances (lead, mercury, PCB's etc.) that may impact human health.
Maine operates under the National Shellfish Sanitation Program Model Ordinance or NSSP. This program is overseen by the FDA and Maine must comply with the standards set forth by this program to not only protect public health but to also sell our product across state lines and internationally.
How: Maine is divided into 47 shellfish growing areas and every 12 years each one of these growing areas goes through an evaluation process that consists of a sanitary survey. This sanitary survey looks at and evaluates all actual and potential sources of pollution that may impact the waters and ultimately shellfish of the growing area. Waste water treatment plants, private septic systems, animal farms, streams, and licensed discharges are just a few of the potential sources evaluated along the entire coast. Next, bacterial water quality is monitored through the testing of over 1500 samples sites scattered from Kittery to Calais. These sample sites are located to monitor known or potential pollution sites as well as resource areas. Samples are taken throughout the year under all tide and environmental conditions. The data from these sites is evaluated yearly and includes analysis of how weather conditions, tides, currents, and other factors may affect the distribution of pollutants in the area. Each station also has a P90 score (weighted average) based on the most recent 30 samples from that station. This score is used to determine the classification of the area and how or if shellfish may be harvested or grown in that growing area. Currently there are five possible classifications for shellfish growing areas.
- Approved: P90 score of 31 cfu/100 ml (colony forming units of bacteria per 100 milileter of water sampled) or less and no point sources of pollution present. Allows direct harvest and consumption of product.
- Conditionally Approved: P90 score of 31 cfu/100 ml or less but only open when certain conditions are met. Examples would be one inch rainfall conditional areas, seasonal conditional areas, and WWTP conditional areas. These are all based on predictable and manageable events such as rainfall, WWTP performance, tide height, or season.
- Restricted: P90 score of greater than 31 cfu/100 ml and less than 164 cfu/100 ml with no point sources of pollution. This classification allows what is called "depuration" harvest. Only licensed depuration companies may harvest shellfish from these areas with a DMR issued permit. Shellfish are than taken to a depuration plant where they are allowed to naturally cleanse themselves of any pathogens that may be present before being consumed or sold. Testing is done both before and after this process to ensure the product is safe for consumption.
- Conditionally Restricted: P90 score greater than 31 cfu/100 ml and less than 164 cfu/100 ml. Same as restricted except that harvest can only occur during certain conditions and a point source may be present. An example would be that when a WWTP plant is operating within its permit parameters shellfish could be harvested from certain areas and when the plant was operating outside its permit conditions shellfish could not be harvested.
- Prohibited: P90 score greater than 163 cfu/100 ml or a known point source of pollution is present. Shellfish from these areas cannot be harvested. Note that if there is a point source of pollution present, shellfish cannot be harvested regardless of the P90 score.
When: There are five types of closures that we deal with for Public Health.
- End of year closures based on updated water quality: Each growing area is reviewed on an annual basis. During this review, which takes place in December and January, all stations are evaluated by their P90 score which is calculated with a minimum of the most recent 30 samples. Based on the results of this evaluation, new closures will be promulgated as needed. This process involves a data analysis of the area to be closed to determine if certain hydrographic, environmental conditions or seasons are contributing to the poor water quality. Factors that are considered include: suspected pollution source, distance to receiving waters, whether impact is constant or intermittent, pollution concentration, shoreline survey information, pollution transport dynamics, and water quality of nearby stations. Once all the data has been reviewed a closure will be promulgated based on the unique circumstances of that area. Examples would be closures based on stream dilutions, non- point runoff issues or impacts from wildlife or waterfowl. These end of the year closures will generally be finalized and go into place in the March- May time period. Affected towns will generally be given a two week notice before closures go into place. This same process is also used to upgrade shellfish harvest areas if water quality has improved.
- Emergency Closures: These closures go into effect because of emergency situations like WWTP bypasses, failing septic systems that are identified during shoreline survey work, oil spills, or flood events. These closures go into effect immediately and will be lifted when the event is no longer deemed to negatively impact the receiving waters.
- Conditional Area closures: These types of closures are known closures based on predictable and manageable conditions. Examples would be one inch rainfall areas, marina conditional areas, or seasonal conditional areas. They exist as closed areas only when certain conditions are violated or exceeded. These conditional areas are reviewed annually to ensure they still meet the criteria for their unique condition.
- Permanent or Static Closures: These closures exist on a permanent basis and are related to known point sources of pollution that were identified during sanitary survey work. Examples would be the prohibited zones around WWTP outfalls and licensed overboard discharges.
- Biotoxin Closures: These closures are like emergency closures and will be put in place when toxin levels in shellfish exceed the quarantine standards set by the FDA. Closures will stay in place until such time that the Department deems it no longer a threat to public health.
This sums up the why, how and when of shellfish closures.
Upcoming Events
Vibrio Training:
DMR Regulations Chapter 115: "Vibrio parahaemolyticus Control Plan" took effect January 2016. You can find a copy of the rule on the DMR website.
This Regulation is intended to prevent Vibrio illness outbreaks related to the consumption of oysters and hard clams from the Damariscotta River. Section 115.04(D) requires mandatory, annual training of harvesters and certified shellfish dealers who take or are the initial purchasers (primary dealers) of oysters or hard clams from the Damariscotta River.
DMR has scheduled 2 in-person training sessions in 2017: January 25, 2017 from 10am-12pm; and February 23rd, 2017 from 10am-12pm. The training will be held at the DMR Boothbay Harbor facility at 194 McKown Point Rd., West Boothbay Harbor, ME 04575.
Please RSVP to Angel Wilson by email or by phone at 207-633-9515. Walk-ins are also welcome. Attendance will be recorded and submitted to Marine Patrol. Failure to participate in one of these sessions will make you ineligible to harvest or be the primary buyer of Damariscotta River oysters or hard clams from May-October 2017.
Fisherman's Forum Shellfish Focus Day:
March 2nd, 2017 at the Samoset in Rockland, ME. Agenda will be sent out in February.
NESSA (Northeast Shellfish Sanitation Association) Conference:
April 11-13th, 2017 at Harasseeket Inn in Freeport. Agenda will be sent out in February.
NESSA Conference Registration Form
NESSA Conference Save the Date Information
Shellfish Advisory Council Meeting:
February 9th, 2017 at ME DMR in Augusta, 10am-1pm.
2017 Shellfish Warden Certification Training
The Department of Marine Resources will be holding the annual municipal shellfish warden certification class on March 14 and 15 at the Hutchinson Center in Belfast from 8am-5pm each day. There are currently 27 wardens eligible for recertification, and 9 new wardens signed up to become certified.
Again this year, DMR has hired Mike Pinkham to lead the training. Mr. Pinkham is a former DMR Marine Patrol officer and the current municipal shellfish warden for Gouldsboro and Steuben. He has over 35 years of experience enforcing marine resource laws and regulations. He served as a Marine Patrol officer for over 33 years, and the past three years as a Shellfish Warden. The in-person training is coupled with the online training component developed in 2013.
If there are any towns that currently are in need of a new shellfish warden, or have one that is planning to leave soon, please feel free to contact Angel Wilson who will be happy to walk you through the nomination process to add a prospective warden to the upcoming training. For current and former shellfish wardens, please keep in mind that you may retain your certification even if you are not currently employed by a town.