Introduction
The resin which is biosynthesized mostly in the female inflorescence, is the most secondary metabolite condensated part of hemp \cite{marks2009identification,kleinhenz2020nutrient} and is primarily used for producing supplemental products, which have been researched and produced with accelerating speed but the industry lacks clear regulation to the contaminant contents of the end products. One important class of contamination is heavy metals, which are innate ingredients of the earth’s crust and are classified as essential heavy metals, which include Fe, Mn, Co, Cr and Mo, which are important for biological processes \cite{Bhattacharya2016} and non-essentials include Hg, Pu, Cd and Pb \cite{Bhattacharya2016,Kumar_2017,Tchounwou2012}, which are harmful even at low concentrations \cite{Bhattacharya2016,Tchounwou2012}.
Other than natural processes \cite{ismail2013phytoremediation,jadia2008phytoremediation}, the concentration of heavy metals are increased in the soil by human activities such as using heavy metal containing fertilizers, pesticides \cite{Asgari2017,Feng_2017} and ash \cite{Nieminen_2005}. One of the biggest sources of heavy metal accumulation in the soil is phosphate fertilizers \cite{Bolan_2013,McPartland_2017}. Phosphate ions are considered to be the main carrier of heavy metal contaminants and specifically hydroponic fertilizers have been found to have the highest tendency for contamination \cite{karadjovn.d.}. Additionally different manures have been proven to be a source of heavy metal contamination \cite{Singani_2012}.
Over one-third of the global arable lands are contaminated with heavy metals \cite{Tripathi_2016}. For example in Austria, France, Hungary, Slovakia and Sweden there are some 200,000 heavy metal contaminated areas \cite{yadav2018mechanistic}.
The bio-availability of heavy metals in the soil can be affected by using different kinds of fertilizers \cite{Barrutia_2009,nwaichi2010phytoextraction,li2012effect}. This has been documented in studies with oat (Avena sativa L.), ryegrass (Lolium multiflorum L.), carrot (Daucus carota L.), spinach (Spinacia oleracea L.) \cite{he1994crop,he1994cropa} and barley (Hordeum vulgare L.) \cite{singh1998cadmium}.
The ability to accumulate heavy metals can be enhanced by adding endophytes \cite{Citterio2005,Luo2012,Rajkumar_2008,Wu_2010} and plant growth hormones \cite{Hadi_2010,hernandez1996morphogenesis,lee1999effect,yasin1989response}, which is confirmed to increase the accumulation in hemp \cite{ahmad2015effective}. Heavy metal tolerance related genes are being researched for future genetic applications \cite{Ahmad_2015}.
When plant accumulates heavy metals or other toxins into their tissues in higher density than the growing medium, it can be called hyperaccumulator \cite{baker1989terrestrial}. The hyperaccumulator potential of hemp began to be discovered in Institute of Bast Corps of Ukraine in 1998, where it was planted after Chernobyl nuclear power plant accident to remove contamination from the soil \cite{Ahmad_2015}.
The translocation of heavy metals is important to understand as they can concentrate into edible products made from the plant \cite{M_Abdelsalam_2019}. Most of the heavy metals are accumulated into the roots \cite{Ahmad_2014,Ahmad_2015,citterio2003heavy,giovanardi2002possibilita,Shi_2011,Shi_2009}, though Cd and Zn are accumulated mostly into the shoots \cite{L_ser_2002} or more specifically into the leaves \cite{Arru_2004,__2005,giovanardi2002possibilita,Linger_2002}. In contrast the fiber does not accumulate heavy metals \cite{Linger_2002}. It has been shown that the translocation of heavy metals from roots to shoots increases with higher heavy metal concentrations in soil \cite{Ahmad_2015}. The heavy metal concentrations are very different with different hemp cultivars \cite{Shi_2011}.
In contrast to accumulation of heavy metals, hemp could be considered to be a Cd excluder \cite{Shi_2009a,Shi_2009} as its translocation factor (TF), which is the ratio of shoot and root heavy metal concentration \(TF=\frac{Cd_{shoot}}{Cd_{root}}<1\), i.e. Cd accumulates mostly into roots, which is suggested to be a property of excluder plants \cite{Baker_2002}. This means a lower risk for Cd to concentrate into products for human consumption.
Heavy metal accumulation in the body of the end user is a health concern as they can be toxic and carcinogenic. Especially cadmium, mercury, lead, arsenic and nickel are dangerous heavy metals. The bioavailability of heavy metals depends on the route of administration. For example pyrolysis turns contaminants into toxic compounds \cite{Sullivan_2013}. Respiratory administration is more bioavailable than digestion for mercury. When constantly accumulated these toxic metals cause oxidative stress which produces ROS (Reactive oxygen species), cancers, teratogenesis, mutagenesis, immunological syndromes \cite{H_diji_2015}, anemia, cognitive decline, cardiovascular diseases \cite{Iqbal2012} as well as brain, nervous system \cite{sastre2015metal}, bone, skin and teeth conditions \cite{Luo2012}.
The heavy metal and other contamination concentration of hemp products vary significantly for reasons mentioned above - there are multiple cases of products which have found to contain high concentrations of heavy metals. For example already in 1988 it was discovered that hemp grown in vulcanic soil in Hawai contained Hg per gram of inflorescence concentration of 440 ng/g \cite{Siegel_1988}. And more recently an analysis was done in european markets revealing that many of them had traces of heavy metals and other contaminants \cite{duclos2019}. Furthermore some medical products have been found to contain possible As contamination \cite{Combemale2005,No_l_2001}.
This preliminary review and study defines the amount of Cd, Hg, and Pb accumulation in NatureDry© lyophilized FINOLA® hemp inflorescence and leaf juice, grown in fine-sandy moraine soil type in Central Finland and to clarify the different ratios of biomaterials extractable from the flowers. In order to better plan industrial production of hemp inflorescence juice products.
Cadmium (Cd), mercury (Hg) and lead (Pb) was chosen for the metal analyses because as they are some of the most commonly occuring heavy metals in soil.
Methods
The field experiment was done with hemp cultivar FINOLA® in open pollinated field, with males and females. The soil type was fine-sandy moraine with organic matter class being medium and pH 6.7. Soil cultivation included plowing, harrowing, adding Belor Standard NPKS 26-5-5(-2) 2019 fertilizer 280 kg/ha. Location was in 61°51'57.1"N 25°43'48.8"E in Jyväskylä, Finland. Sowing date for the field experiment was 20.5.2019. Harvest date for the samples were in 3.9.2019, plants had around 60% mature seed, male flowers were already dead and seed harvest was approaching in few weeks. Samples were randomly gathered from the field by hand with scissors and were frozen in regular freezer to -19°C in vacuum sealed bags, with vacuum machine (OBH Nordica 7949 Supreme Food Sealer). Inflorescenses and leaves were juiced straight from freezer using a twin gear slow juicer Angel Juicer 7500 \cite{juicers} with fine strainer filter. Juice was frozen again in vacuum sealed bags to -19°C. Finally the juice was lyophilized with NatureDry© method at Lyotech Oy with in Helsinki. Laboratory analyses for heavy metal concentrations were done by Eurofins WEJ Contaminants GmbH in Hamburg Germany laboratory. Cd and Pb analyses was made with mass spectrometry and ionisation by inductively coupled plasma (ICP-MS). For Hg analysis, reduction of Hg compounds to Hg with Tin(II)-Chloride, then analysis of Hg vapour with atomic absorption spectroscopy (AAS).