Geographical Distribution Of Dioxins, Cadmium And Mercury Concentrations In Reindeer Liver, Kidneys And Meat In The Russian Far North

Reindeer herding is a vitally important agricultural sector in the Russian Far North. It is believed, that Nothern ecosystems are prone to accumulation of persistent pollutants for the reasons of trophical chains, features and climate. Reindeers graze on vast areas, having seasonal migrations on distances up to hundreds kilometers in one side in North-South direction, that increases likehood to cross a locally polluted area. Here we present results of a large-scale countrywide study of reindeer liver, kidneys and meat pollution by dioxins, cadmium and mercury. Samples were taken in 2015-2020 years from 41 locations in 8 reindeer-herding regions of Russia. Dioxins were determined in 383 samples of liver and 13 of meat, cadmium and mercury – in 505 samples of liver, 315 of kidneys and 22 of meat. Dioxin pollution has shown a clear geographical trend: liver concentrations gradually decrease from the Western to the Eastern parts on the country, with the highest concentration of 76.5 pg/g of fat WHO-TEQ. The following factors are likely to explain the discovered trend: localization of chemical enterprises and density of reindeer population. The highest concentrations of metals were found in kidneys (7.3 mg/kg of cadmium and 1.1 mg/kg of mercury). The contribution of local sources to cadmium and mercury pollution was found to be less, than expected. We also speculate, that reindeer liver may serve as a good additional indicator of environmental pollution by the investigated contaminants.

During all the year, lichens and plant, especially reindeer moss are the main feed for the animals. During winter period lichens account to more than 50% of feed. Accumulation of heavy metals in lichens linked with atmospheric contamination and active lichen supplementation during the winter period are believed to be the key factors determining the heavy metals content in reindeer [Hassan et al. 2012]. Among toxic elements (cadmium, mercury, lead and arsenic), reindeer liver and kidneys accumulate predominantly cadmium and mercury in high concentrations [Wel nger et al. 2011].
Deposition of persistent pollutants, including dioxins and heavy metals on the territory of the Russian Far North is mainly determined by transboundary atmospheric and hydrospheric carry-over from other regions of Russia and countries. For example, sources of mercury emission, located on the territory of Kola Peninsula account for only 13% of mercury deposition content, while sources located in other Russian regions account for 22%, and sources in Europe, China, USA, Central Asia and others -for the rest. However local sources, e.g. industrial objects are believed to make important contribution in dioxins and heavy metals deposition [AMAP 2004].
Within State Monitoring program we have analyzed reindeer meat, liver and kidney analyses for several persistent pollutants, including dioxins and dioxin-like polychlorinated biphenyls (dl-PCBs), organochlorine pesticides, indicator PCBs, polyaromatic hydrocarbons and heavy metals: arsenic, lead, cadmium and mercury. Dioxins, dl-PCBs in liver, and cadmium and mercury in liver and kidneys of reindeers were found in concentration generally much higher than in other's food producing animals (cows, pigs, poultry) tissues (National monitoring of undesirable substances in food and feed, data not shown). Providing that the 4 abovementioned contaminants exhibit well-known harmful effects on health, we have made the risk assessment [Makarov et al. 2018]. It was shown that cadmium poses the highest health risk comparing to sum of dioxins, dl-PCB and mercury. Consumption of reindeer offal in several regions may lead to cadmium intake exceeding the tolerable intake level more than threefold. In the meantime, consumption of reindeer meat poses no health risk related to any of the investigated contaminants [Makarov et al. 2018].
The main aim of our study on reindeer offal and meat monitoring for dioxins, cadmium and mercury, was the detailed investigation of geographical distribution of the contaminants and assessment of reindeer offal as a suitable indicator of environmental pollution. Dl-PCBs were not included in the program as these compounds are not yet regulated by Russian Food Legislation.

Materials And Methods
Reindeer (Rangifer tarandus) tissue samples were collected by regional authorities in 2014-2020 years within the National Program of Undesired Substances Monitoring in Food and Feed. In total, 842 samples of liver, kidneys and meat were taken from semi-domestic reindeer in 8 reindeer-herding regions: Kola Peninsula (other name is Murmansk Oblast), Nenets Autonomous Okrug (AO), Komi Republic, Yamalo-Nenets Autonomous Okrug (AO), Taymir Peninsula (part of Krasnoyarsk Krai), Republic of Sakha (Yakutia), Chukotka and Kamchatka (see Fig. 1). The animals were raised on natural food resources (lichens, plants etc.) without any feed additives or supplements. The age of the animals of both sexes varied from 0.5 to 5 + years, generally being more than one year. Detailed data on age and sex was available only for one region -Yamalo-Nenets Autonomous Okrug. Liver, kidneys and meat samples, weighting 100-300g, from individual animals were taken at slaughterhouses in the winter period (from the end of November to the beginning of April) and stored at -10°C prior to analysis.
Dioxins (sum of PCDDs and PCDFs) were determined by gas-chromatography/high resolution mass-spectrometry (GC-HR MS) according to GOST (Customs union o cial standard) 34449 − 2018 «Food products and feed materials. Determination of dioxins by GC-HR MS». Brie y, thawed samples (50-100g) were homogenized and internal standards (solutions of isotopelabelled dioxins, «Wellington labs», Canada) were added. Homogenized samples were thoroughly mixed with the Prep DE sorbent («Dionex», USA). Dioxins were extracted by hexane:dichloromethane 1:1 v/v mix under pressure using ASE 350 extractor («Dionex», USA). The extract was defatted (by H 2 SO 4 /silicagel), cleaned up on columns with 10% activated charcoal on zeolite and evaporated. Resuspended extract was analysed using GC/HRMS instrument Autospec Premier («Waters Corp.») in selected ion monitoring mode at resolution not lower than 10.000. Capillary column VF-Xms (60 m ½ 0,25 µm, Agilent, USA). Dioxins concentrations (sum of PCDDs and PCDFs) were expressed as pg WHO-TEQ(2005)/g of fat. Fat content in liver and meat was determined by gravimetry (GOST 23042-86 «Meat and meat products. Determination of fat content»).The percent of fat in reindeer liver samples varied from 3.9 to 7.5%, for most part of the samples being in the range of 5-7%.The fat content in meat was around 5%.
Cadmium and mercury concentrations were determined by mass-spectrometry with inductively-coupled plasma according to GOST 31414 − 2017 «Food products and feed. Determination in arsenic, cadmium, mercury and lead by ICP-MS». Measurements were performed using an inductively coupled plasma mass spectrometer Agilent 7900MS model ICP-MS system (Agilent, USA) equipped with an autosampler, a Babington nebulizer, nickel cones, and a peristaltic sample delivery pump. Analysis of each sample was done in duplicate. High purity argon gas was used to form the plasma in the ICP-MS. The pulse to analog factor was determined on the day of analysis. Agilent ICP-MS tuning solution of 10 µg/L (Ce, Co, Li, Tl, and Y) was used to tune the instrument. Data acquisition was done in spectrum analysis and full quantitative mode. The following isotopes of trace elements were considered: 111 Cd and 200 Hg. A microwave system Ethos up (Milestone, Italy) equipped with a microwave acid digestion bomb made from Te on was used for microwave digestion. All solutions were prepared using ultrahigh purity water (18.2 MΩ cm − 1, Ultrapure Water System (Sartorius arium mini plus). All the reagents used were of analytical reagent grade. High purity ICP-MS multi element standard solution obtained from Merck (Darmstadt, Germany) was used for the preparation of calibration curves in the quantitative analysis of the elements. This solution is a mixture of 10 mg/L of elements. Internal standards (Indium and Bismuth) were used for control of mass shift and signal suppression.
Selected round of internationally recognized Pro ciency Testing Schemes of cadmium, mercury and dioxins determination are shown in Table 1. Z-index ≤ 2 means that the round was successfully passed.

Results And Discussion
Dioxins Dioxins mean concentrations in reindeer liver and meat depending on sampling place location are presented in Table 2. Mean concentrations depending on region, including values of standard deviation and concentrations ranges in 95% Con dence Intervals are shown in Table 3.  A clear trend in geographical distribution in dioxins concentration in liver is shown with the highest concentration in the western part of the country and than gradually decreasing as one proceeds to the east. Heatmap of dioxins concentrations is presented in Fig. 2. Diagram of dioxins concentrations in liver depending of latitude is shown in Fig. 3. Coe cients of correlation between dioxins concentrations in liver and latitude are presented in Supplementary Table 1.
Providing that dioxin concentrations are generally higher in calves than in adult hinds, [4] and that we had no detailed information on age and sex of the animals for most part of the samples, the absence of data strati cation by age group could be a source of bias.
Several factors, that may contribute to discovered trend in geographical distribution of reindeer liver dioxin contamination, are discussed below. Other important factor, that may contribute to dioxin pollution geographical trend, is the density of reindeers at pastures. The more animals graze on one area, the higher is the probability that due to lack to reindeer moss and other lichens and plants, more particles of soil will be ingested by the animals. Soil is a well-known reservoir of dioxins  Table 6. Cadmium concentrations were found to be generally much higher than mercury concentrations. The concentrations of metals in kidneys were much higher than in liver. In meat only low concentration of cadmium and mercury were found, mostly below the limit of detection. The highest concentrations of cadmium in liver (more than 1 mg/kg) were found in 3 sampling places Heatmap of cadmium and mercury concentrations in kidneys are presented in pictures 6 and 7, respectively. Unlike dioxins, no signs of concentration dependence on latitude may be seen from our data. Coe cients of correlation between heavy metals concentrations and latitude are presented in Supplementary Table 1.
Limited data on age and sex of animals did not allow to plot data by these parameters. However, these data was provided for samples from Yamalo-Nenets AO. Data on cadmium and mercury concentrations depending on sex are presented in Supplemental Table 2. For mercury and cadmium, the difference in concentrations between two sexes did not exceed 10% for both liver and kidneys. Data on metals concentrations, depending on age of the animals, is presented in Supplemental table 3.
Coe cients of correlation between heavy metals concentrations and age groups are presented in Supplementary Table 1  Reindeer and caribou graze on the whole path of their migration, and have a higher, than other animals, likehood to cross the contaminated local areas. So, their tissues may be representative for dioxins and metals environmental concentrations for the whole grazing area, and thus may serve for effective comparison of pollution between the regions with different latitudes [EFSA 2011; Kachova 2015].
-Most reindeers are semi-domestic animals, feeding with natural feeds e.g. lichens, mosses, plants, mushrooms etc. Supplementary feeding is used only in certain conditions [Horstkotte et al. 2020], so the impact of arti cial feeds on reindeer dioxin and heavy metals contamination is minimal. At the other hand, taking samples from reindeer may be easily made at slaughterhouses and presents less di culties, than sampling of wild animals.

Conclusions
Here for the rst time we present the results of a large-scale investigation of reindeer liver, kidneys and meat pollution by dioxins, cadmium and mercury in the Russian Far North. Dioxins geographical distribution show a clear trend with highest concentrations in the West with gradual decrease as one proceeds to the East. The most important factors, contributing to the trend discovered, are likely to be: the geographical localization of chemical industry enterprises (sources of dioxins pollution); and greater density of reindeer population in the Western part of the country, leading to increased ingestion of soil particles that accumulate dioxins. We have founds no trend in geographical distributions of cadmium and mercury. Our data indicates, that objects of mining industry, that are local sources of cadmium and mercury environmental pollution, may not play a crucial role of cadmium and liver accumulation in reindeers at the distances, greater than 60 km. We also speculate, that liver of reindeer and caribou may serve as a good additional indicator of environmental pollution by the investigated contaminants.

Figure 1
Regions of the Russian Far North whence reindeer samples were taken. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
Heatmap of dioxins concentrations in reindeer liver from different sampling places. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 3
Diagram of dioxins concentrations in reindeer liver depending on the latitude of the sampling place.

Figure 4
Location of the chemical and oil re ning industry objects of the Soviet Union (1980s). Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 5
Density of reindeer population on the territory of the Russian Federation (2000s). One purple point corresponds to 10.000 of animals. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 6
Heatmap of concentrations in reindeer kidneys from different sampling places. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.