“Inconsistent and contradictory results from nutrition studies conducted by different investigators continue to emerge, in part because of the inherent variability of natural products, as well as the unknown and therefore uncontrolled variables in study populations and experimental designs.” — The Challenge of Reproducibility and Accuracy in Nutrition Research: Resources and Pitfalls
- Develop a set of protocols and best practices to avoid PDC contamination in sourcing and preparation,
- Determine how the food chain gets contaminated with PFCs, and
- Ascertain how food processing adds PDC contamination.
Non-food exposures
Numerous non-food sources of BPA are present in the environment and likely pose significant “background” contamination which will vary depending upon the test subjects’ lifestyles and environments.
Sanitation/Hygiene
As with all appropriate bench science work, preparation surfaces should be non-reactive, non-porous, resistant to chemicals, heat, and easy to clean. Stone or ceramic tiles can accumulate contaminants in the grout. Sealants will contain polymers. Polymer-based surfaces cannot be used.
Dietary interventions also require that preparation surfaces must be efficient to maintain hygiene in the event that food makes incidental contact.
Stainless steel offers all of those characteristics which is why professional and health-department approved kitchens are almost totally stainless steel.
Environment – Floor, walls, ventilation PM2.5
Constant ventilation of HEPA-filtered air is vital to prevent contamination of food with polymer-based particles which can confound results. Air should be constantly monitored throughout the trial and records kept.
The type and age of the HVAC system should be noted as well as the type of filter used and the ducting materials (metal versus plastic).
Immediately after dusting, the HVAC filter should be changed and the system fan run continuously for four hours to collect as much dust as possible which has been stirred up from cleaning. A fresh filter should then be installed.
HEPA-class air purifier use should report the make, model, capacity, and whether it is portable or installed with the HVAC system. If portable, the location should be noted along with the distance from the HVAC air return.
PurpleAir is an accurate and cost-effective solution for monitoring. Other than the cost of the sensor ($179), monitoring is free and historical data can be downloaded and saved.
Cleaning Protocol – Floors
Polymer-based materials are universally found in professional and institutional kitchens given to their durability and ease of cleaning. However, friction against the floor caused by shoes and rolling carts inevitably creates ultra-fine particles many of which qualify as PM2.5 which — regardless of chemical composition — cause inflammation when inhaled. Significantly, many of the polymers from which flooring is made (such as polyvinyl chloride) have known health effects and can contaminate food destined for test subjects.
For this and standard hygiene reasons, floors should be wet-mopped at the end of every food preparation day. The cleaner for this task and food preparation surfaces should be hypochlorite-based and free of surfactants such as ethoxylates that have been shown to cause health effects.
Spills and other unanticipated incidents should be cleaned with water — minus cleaning agents — when possible. Exceptions should be noted in the list of materials.
Cleaning Protocol – Preparation Surfaces
The same cleaning agents and protocols should be employed. No paper towels.
Towels – Sourcing and Washing
Only 100% cotton towels should be used. The source should be noted in the list of materials. Before first use, study towels should be washed and dried twice to remove any potential contaminants.
Towel, apron, and other cloth washing
A new washer and dryer should be purchased for a dietary intervention study because difficult-to-remove fabric softener and detergent residues can build up on the internal surfaces up from phthalates and other chemicals contained in detergents and fabric softeners. These can be basic, no-frills consumer-level, budget units. Note the brand/model of both.
Dishwashing
Replacing an institutional dishwasher is impractical and economically not necessary.
Before first use, the dishwasher (note brand/model/age noted in equipment) should be run twice with an empty load using the detergent selected for the study. This will minimize deposits of undesirable chemicals.
The dishwasher should use the most vigorous setting (pots/pans), hottest final rinse. Do not use a heated dry cycle which may “bake on” organic contaminants. Instead, remove items immediately upon cycle finish when they are still wet. Then rinse all items under a running stream of carbon-filtered water and place in a stainless-steel dish rack to dry.
Age of clothes washers, dryers, and dishwashers is important
The age of clothes washers, dryers, and dishwashers with plastic components should be noted because polymer components — including seals — will leach more chemicals and additives as they age because many of the additives compounds are not covalently bound.
Equipment and Materials – Acceptable Materials
Plastics of any sort must be prohibited except in inevitable situations such as the seals in washers, dryers, and dishwashers. These utensils and items.
This includes so-called “BPA Free” items which are often manufactured with one or more of the 24 analogs of Bisphenol A and which have been found to exhibit similar harmful effects. This also includes the polymer Tritan which is a popular “BPA free” material that has also been shown to exhibit similar estrogenic, endocrine disrupting activity[vii].
Avoid any materials that are not glass or stainless steel. Exceptions may be made for the use of wooden cutting boards (with a minimum of glue joints) and utensils. Bamboo is not acceptable because the raw material is processed in an unknown number of methods and the resins used are not disclosed.
Pots, pans, cooking preparation utensils, mixing bowls, measuring cups and spoons, eating utensils, serving plates, bowls, coffee/tea cups, and beverage glasses should be either stainless steel or glass.
Cutting boards should be tight-grained maple composed of wide slats of wood to minimize glue joints. Hand washing is required to avoid damage to the cutting board. Olive oil which is selected as a menu ingredient should be used to coat boards when dry. Do not use mineral oil or any other substance.
Note specific item name, model, and source in the equipment section.
Many basic glass items familiar to bench science (beakers, watch glasses, funnels, graduated cylinders) can facilitate meal preparations.
Soap and Detergents
Must avoid polymer ingredients as well as phthalates (including odor enhancers), surfactants with nonylphenol connections, nanoparticles, color enhancers, enzyme cleaners, and fragrances.
Prohibited equipment and materials – Dishes, pots, pans, utensils, and items with any sort of plastic or non-stick coatings including ceramics
The following are forbidden:
- Sous vide
- non-stick pans
- most cooking oils
- plastic utensils
- plastic prep bowls
- synthetic gloves
- plastic bags
- plastic wrap
- Beverages in cans, plastic bottles, or glass.
Avoid food contact with plastics or recycled paper or cardboard. Recycled paper and cardboard contain Bisphenols because thermal cash register receipts are frequently included with other papers.
Avoid contact with any sort of box or wrapping with brightly colored graphics or type. Phthalates are contained in most packaging inks.
Do not wear any cloth garment or apron or use any towel or fabric with a design printed on them. Phthalates are contained in most of those design inks.
Food or other items that arrive in paper packaging should not be opened in the food preparation area.
Just as with plastic polymers, ceramic compositions are suspect.
No standards exist, nor any regulations for disclosure of constituents. Many contain chemical compounds that can leach out and contaminate foods, especially if they are predominantly alkaline or acidic. Some ceramic glazes can leach heavy metals such as lead, cadmium, and other colorant compounds.
So-called “stone” non-stick coatings have not been subjected to peer-reviewed investigations. However, these coatings are created by mixing ceramic or stone dust with a polymer. A slurry of those components is used to coat the interior of a cooking pot or pan. The moist coating is heated to a high temperature resulting in a non-stick surface.
Manufacturers are stubbornly non-transparent about materials because they consider the manufacturing process a trade secret.
Consumers and consumer groups have offered anecdotal opinions that cadmium or other heavy metals as well as organic polymer chemicals can leach into foods. Remember that the plural of anecdote is not data.
Very limited use should be made (and noted) for appliances such as a glass blender with a plastic seal at the bottom for steel blades. When used, aluminum foil should cover the mouth before inserting the plastic top.
Most aluminum foil used in cooking has a very light coating of release agent used in the rolling process. The identity and composition of that release agent is unknown <<cite + contact Reynolds>>
Food processors such as Cuisinart cannot be used unless all components that contact food (including the bowl) are stainless steel or glass. We have been unable to locate a food processor with stainless steel bowl components. It is important to note that food processors marketed as “BPA” free — such as those like the Cuisinart with a Tritan bowl — leach estrogenic compounds that can alter blood profile measurements for inflammation and endocrine measurements.[vii]
Metal shredders, graters, or a mandoline are acceptable as long as all food contact surfaces are metal.
In addition, many food processor tasks (such as meat grinding) can be performed with a KitchenAid mixer which has a stainless steel bowl and attachments. Some KitchenAid attachments, such as the pasta maker, have plastic parts and should not be used. A manual, all-stainless pasta maker is preferred.
All cups, saucers, plates, bowls, serving, measuring, and storage containers should be glass. Measuring cups and spoons may be stainless steel.
Also avoid:
- Electric drip coffee makers. Even those marketed as “BPA” free contain either BPA analogs that are just as harmful, or Tritan which has been found to be estrogenic like BPA. Electric and stove percolators, glass and steel French Press. Hand-poured drip using a glass carafe and either glass or metal grounds holder. A paper filter is not recommended because of unknown chemicals which can leach out.
- Sodastream,
- Keurig and other “pod” beverage makers.
Plastic components of all those can leach chemicals of emerging concern.
Precise Measurements
Covered in this section: Best practices – Precise measurements.
Recipes are protocols
Cooking is a complicated organic chemical manufacturing process whose final product is determined by myriad processes that need to be controlled as tightly as possible and detailed notes taken at every step. New organic compounds are formed at every step.
Processing steps out of order, at an inappropriate temperature, prepared in the wrong manner or cooked the wrong amount of time will affect the ultimate organic composition. In a non-study environment, that just affects the taste. But in a dietary intervention where very low levels of chemicals can affect physiological processes, the failure to follow the protocol may affect outcomes and replicability.
As one example, take spices: The process of “blooming” — heating spices in hot oil before adding other ingredients — will favor the extraction of lipid-soluble compounds that many cooks flavor for a particular intensity. Smaller particles offer different faces of the particles and a greater surface area from which to extract the desired flavor compounds.
A precision recipe will approximate particle size (in millimeters or fractions of an inch). “Finely diced” is not precise. The amount of the spice, the temperature of the oil (in degrees, not “low heat”), and the elapsed time will alter — and determine — the chemical composition of the final product.
The next step in the protocol (recipe) will determine whether the oil and bloomed spices are to be reserved for later use (weight of final product noted) or whether other ingredients are to be added immediately after the elapsed blooming time. If meat is added at this point, one set of organic compounds will be created. If tomatoes, another variation; if potatoes yet another.
The variations are legion. If wine is added, the alcohol will extract a certain set of flavor compounds before evaporating. The remaining polyphenols and other compounds will react with others present to form another set of unknown compounds. Those compounds are a set of “unknowable unknowns.” Precision cooking protocols will help make those unknowable unknowns the same from trial to trial and increase the likelihood of replication if the protocols are followed exactly.
The important lesson is that each step and the timed order, temperature, and method in which ingredients are added (“diced” is not precision. “half-inch cubes” (or “approximately a centimeter”) will all exert their own effects on the final organic manufacturing product called a meal.
Traditional American and English kitchen staff will be more familiar with non-metric quantities. Using English units for everything but weight may help avoid confusion among those also learning to cope with the demands of precision and documenting every step.
These steps were developed and tested in a small “proof-of-concept” dietary intervention to determine (among other things) that experienced cooks could adapt to the more precise demands with little trouble. That experience also showed that cooks with successful backgrounds in baking adapted most easily to the needed precision.
Ingredients as reagents
Each food element must be treated as a reagent. That means they must be exactly the same (or as close as possible) in the:
- identity.
- purity,
- conformance with common food consumption practices
- consistency, availability, and capability of being reliably, economically, and easily obtained from sources accessible to other investigators.
Identity
Identity includes the vendor from which food is purchased. Preference should be granted to fruits, vegetables, grains, and other items that are branded and nationally available. Determining a varietal or cultivar identity and provenance transparency traceable to a specific and established source is desirable.
Obtaining the food directly from a nationally available grower/source who is willing to ship is the best option but may be seasonal. Farmers’ markets, while a valuable and healthy resource, are generally not acceptable unless their goods can be shipped nationally.
Purity
While the Organic Regulations set by the United States Department of Agriculture have been substantially degraded by the addition of scores of chemicals and pesticides, it is the only nationwide standard in the U.S.
Another consideration is the fact that most supermarket fruits and vegetables are irrigated with recycled municipal wastewater and are grown with commercial fertilizers. Furthermore, an increasing percentage are fertilized with biosolids — a euphemism for the human feces sludge from municipal sewage treatment plants once the recycled water has been removed.
Both biosolids and recycled wastewater are sources of undesirable unhealthy chemical and biological contaminants as well as micro- and nanoplastics, and their resulting plastic chemical contamination. (Micro- and Nanoplastics: Health Implications)
Currently, USDA organic regulations do not list biosolids and recycled wastewater in either allowed or prohibited substances.
There have been efforts by large agricultural corporations to have those included as allowed. However, biosolids and recycled wastewater contain scores of prohibited substances including pharmaceuticals and illegal narcotics.
Conformance with common food consumption practices
For the broadest applicability, foods chosen for a dietary intervention study should reflect the most common consumption habits of the studied population. As one example, our proof-of-concept study attempted to emulate a “typical” American diet. To create that diet, investigators relied, upon the U.S. Department of Agriculture’s 2016 study: Americans’ Eating Patterns and Time Spent on Food.
We did not include most “niche” foods such as plant-based meat and milk substitutes because they are not mainstream dietary items. In addition, those are both highly processed items that experience substantial contact with plastic.
Consistency & Availability
Foods for a precision dietary study are capable of being reliably, economically, and easily obtained from sources accessible to other investigators.
While every effort should be made to reduce confounding factors — especially for chemicals targeted for examination in a study — extreme sourcing to avoid supply chain contamination can make a study more time-consuming, expensive, and non-replicable.
One example of an extreme sourcing (and ultimately impractical) protocol can be found in: Revised Stealth Syndromes Human Study Protocol – APPENDIX 2 – Detailed parameters of intervention diet selections. The effort to develop that selection protocol took more than six months — including personal visits to baseline food sources.
However, our proof-of-concept investigation demonstrated that the protocol’s extreme measures and emphasis on obtaining food directly from the source were impractical to implement fully. The time and expense, plus the seasonal variation in foods, would render such a study incapable of being replicated by other investigators.
Examples of Adjustments to Facilitate Replicability
The need for widespread availability of study foods demands national sourcing. That meant that — for our study — the food for the intervention leg of a study needed to come from direct sources willing to ship food nationwide, or from large grocery chains.
To date, Whole Foods Markets are the only U.S. supermarket chain that bans its fruit and vegetable vendors from using biosolids.
Biosolids and recycled wastewater contain hundreds of chemical contaminants. Whole Foods has not specifically addressed the issue of recycled wastewater.
Despite that, we chose Whole Foods for most of the intervention study ingredients because of its biosolids ban and a wide variety of USDA-certified organic products.
We did test milk from Whole Foods against the Safeway brand and local organic brands. LC/MS spectrometry determined that four brands — Whole Foods, Organic Valley, and two samples from a local dairy, Straus Family — all fell below the lab’s LOQ (o.03 micrograms/kilogram). We chose Organic Valley for milk and cheese because the brand is sold widely beyond Whole Foods.
Water
The quality of municipal water supplies varies greatly from place to place. Most contain measurable amounts of many chemicals of emerging concerns (such as Bisphenols, phthalates, and PFASs), as well as pharmaceuticals, and illegal drugs <<cite?>>
While research labs often have supplies of highly filtered water to remove chemical impurities, the kitchens of catering companies and institutional food facilities are usually filtered for taste and clarity only.
Unless a food preparation facility has a demand for multiple dietary studies that require expensive institutional filters, the most cost-effective approach is to install a residential-type, point-of-use filter.
Among many citations, a February 2020 study noted that: “All under-sink dual-stage and reverse osmosis filters tested showed near complete removal for all PFASs evaluated….In contrast, all other filters containing activated carbon exhibited variable PFAS removal. In these filters, PFAS removal efficiency was dependent on chain length.”
The most effective, according to the study, were the most expensive. Prices were not divulged in the study or supplemental material.
That study also did not assess the removal of bisphenol A or other chemicals. However, it did test a wide variety of filters, and including brand model names in the supplemental material. It is additionally significant because it explored the effects of molecular size on filtering efficiency. A further examination of molecular polarity — combined with size — may offer guidance on the ability to filter other substances.
An alternative selection method is to explore filters that have been NSF/ANSI certified for the removal of specific chemicals involved in a study. As one example, our proof-of-concept study focused on BPA, and as a result, selected a Multi-Pure/Aquaversa under-sink, carbon-filter model which is NSF/ANSI 401 certified for “reduction of the compounds atenolol, bisphenol A, carbamazepine, DEET, estrone, ibuprofen, linuron, meprobamate, metolachlor, naproxen, nonylphenol, phenytoin, TECP, TCPP, and trimethoprim.”
The Aquaversa system for our proof-of-concept study cost approximately $650 to install.
Reagents as Ingredients
Sometimes laboratory reagents make for the best ingredients. Salt, baking powder, and baking soda are of unknown provenance. All contain undisclosed non-clumping chemicals and other substances of unknown identity, purity, and concentration.
We opted for laboratory reagents and decided to use baking powder instead of yeast in bread and similar recipes because of plastic packaging and because we could not determine what possible contaminants may have been introduced in production and packaging.
Our baking powder was compounded 2:1 mixture (by weight) of cream of tartar and baking soda.
<<photo>>
Supplemental Material
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