Implicit, Intrinsic, Extrinsic (or Environmental), and Host Factors Attributing the Covid-19 Pandemic. Part 2-Implicit Factor Pesticide Use: A Systematic Analysis

Advances in our understanding of complex COVID-19 pandemic would allow us to effectively eliminate and eradicate SARS- COV2 virus. Although tremendous amount of research devoted to the robustness across its biology, diagnostics, vaccines and treatment has exploded in the past two years. However, still science do not have robust answers for causes, for example (i) What are the reasons of non-uniform global distribution of COVID-19? (ii) Why the United States, India, and Brazil, are the first-three most affected nations?, (iii) How did Bhutan, a nation sharing a boundary with China manage nearly 0.34% infections and 3 deaths from COVID-19? Nonetheless, the biomass bistribution of biosphere report suggest more than 1550- fold larger microbial biomass involving bacteria, fungi, archaea, protists and viruses is exist in comparision to all global human population in the biosphere. The rich microbiota act a first line of defence to invade pathogens and affect us both through the environment and microbiome. Unfortunately, a role of pathogen-transmission factors viz . implicit factors (competitive microflora) is still under represented. This study is an attempt from a gold standard correlation methodology using a large pesticide use global data. The non-specific pesticides kill both pests as well as protective microbiota, resulting a loss in ri ch biodiversity and allow easy pathogen entry to human. Entire predictions were found consistent with the recently observed evidences. These insights enhanced scientific ability to interrogate viral epidemiology and recommended to limit pesticide use for future pandemic prevention.

The origin of life on earth is a natural process ranging from non-living microorganisms (viruses and viroids) to living micro-/macro- organisms (bacteria, archaea, fungi, protists, plants, animals and humans). The microorganisms are found almost in every habitat present in nature and their coexistence with the environment determines the health of the entire ecological system of earth [1]. Soil is an important habitat for both bacteria and cyanobacteria due to an abundance of organic carbon on earth. The robustness of prokaryotic life in the deeper soil subsurface below 8 m, is also enormous (10 3–106 cells/cm3), and biomass of prokaryotes are detected even at higher altitudes 57–77 km. Airborne prokaryotes also represent a large fraction with 5 × 10 19 cfu[2]. The average microbial density on earth is approximately 10 8 microbes/ml, which showed inseparable interactions between humans and microbes everywhere on the planet. Particularly, the human itself contains trillions microbiome in a surprising ratio of 10:1 with human cells. A huge number of a total of 10 30 prokaryotes exist on earth, out of about 1400 (negligible 1.4×10-25%) microbial species are recognized as human pathogens[3]. Interestingly, a report of the biomass distribution of large Biosphere suggests though large, human population 0.06 gigatons of carbon (Gt C), is still negligible in comparison to bacteria 70 Gt C, fungi 12 Gt C, archaea 7 Gt C, protists 4 Gt C and viruses 0.2 Gt C [4]. Hence, a large number of ecological communities of microorganisms, so-called microbiota with a high level of adaptation and evolution, exist in the environment which protects humans from pathogenic microorganisms and toxins. Unfortunately, human activities over the years via antibiotics, agri-/industrial chemicals, and lifestyle have significantly altered biodiversity[5] . In fact, several times warnings to humanity from leading global scientists including Nobel laureates are well documented[6-9]. The scientific report also highlighted 58% of the earth's land coverage already falls below safe level[10] and merely ten months old UN report stipulates up to 1million species are at risk of annihilation[11]. Thus, a well-balanced ecosystem products/services usually reduce pathogen transmission, and several studies suggest new virus infections may be a result of loss in biodiversity[12-14]. The modelling of infectious disease dynamics remains the ideal research design and it is essential to identify the key factors responsible for any communicable disease transmission in population level. The epidemiological model usually improves the scientific ability by two-fold: (i) to interrogate viral epidemiology, and (ii) to run counter operations for movi ng forward all with more accuracy, precision, and reliab ility. Previous studies also suggest the importance of four crucial transmission factors for any microbial infections, such as (i) intrinsic factors (related to direct food supply to microorganis m, for example, nutrient content); (ii) extrinsic or environment factors (external to the food, for example, temperature); ( iii) implicit factors (competitive microflora); and (iv) host factors (for the spread of disease, for example, malnutrition)[15,16]. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for thisthis version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.09.21263347doi: medRxiv preprint The rising population has significantly impacted the human activities, and we assume fert ilizer consumption/nitrous oxide emissions- which itself supplies nutrients, and non-specific pesticides- itself inhibits microflora, could be a part of intrinsic-and implicit- factors respectively. The COVID-19 disease caused by novel corona (SARS-COV2) virus has posed serious health challenges with enormous morbidity and mortality across the world. Till date of writing this report COVID-19 has claimed a total of 185.29153 million confirmed cases and the mortality is again on constant surge with each passing day. With e nough scientific evidences reported till date top ten COVID-19 affected countries with cumulative total cases, (i) United States of America (33451965), (ii) India (30752950), (iii) Brazil (18909037), (iv) Russian Federation (5733218), (v) France (5686066), (vi) Turkey (5465094), (vii) The United Kingdom (5022897), (v iii) Argentina ( 4593763), (ix) Colombia (4426811) and (x) Italy (4267105) [17]. The knowledge gap insists us to explore role of key factors over COVID-19 progression as an ideal model system. Hence, a wider sampling and subsequent data analysis of current COVID-19 infectious cases with Pesticides including Fungicides and Bactericides, Insecticides, and Rodenticides use countrywise were envisaged in understanding the epidemiology of COVID-19 and create an unprecedented opportunity for COVID-19 prevention in restoring good quality of life.

All COVID-19 infectious cases were collected from five WHO situation reports- dated from 11-Jul-2021 to 28-Mar- 2020[17]. The data pertaining to pesticide use countries/ territories- wise was free to use and supplied by the Food and Agriculture Organization (FAO) [18]. Here, the Countries/ Territories with missing entries were omitted, and 98 countries randomly fitted to top variable indices of correlation coefficient for unbiased cause ‐ effect relationships.

and Discussion There is considerable evidence that intrinsic, extrinsic (or environment), implicit, and host factors are key regulators for pathogen transmissions. In this study, we have observed a total pesticide use was optimally correlated with the number of COVID-19 infected cases, as appeared on March 13th (r 2=0.93), 19th (r2=0.86), and 28th (r 2=0.57), 2020 (Table 1). Later on consistant correlation (0.51 ≤ r2 value ≤ 0.54) was seen in case of a total fungicides and bactericides from last one year i.e. March 28th, 2020 to July 09th, 2021 with a very weak standard error value 0.02. From December 22th, 2020 a high correlation was witnessed in case of a total insecticides use with r 2 value ranges from 0.80 to 0.83. The United States of America, Brazil, Thailand, and India are among top four insecticides consumer countries and currently United States of America, India, and Brazil are top three COVID-19 affected nations. Other hand, as per FAO report Bhutan do not ut ilize insecticides and Brunei Darussalam, consumed very limited insecticides, a total of 1 and 3 tonnes only. Both Bhutan, and Brunei Darussalam are among top two least COVID-19 affected nations. These real evidences are advised the direct role of pesticides use on increasing COVID-19 infected cases. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for thisthis version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.09.21263347doi: medRxiv preprint Table 1. Cumulative COVID-19 Infected Cases and Pesticides Consumption Worldwide Country/ Territory/Area CUMULATIVE COVID-19 INFECTED CASES Total Pesticides (Tonnes) Total Fungicides and Bactericides (Tonnes) Total Insecticides (Tonnes) Total Rodenticides (Tonnes) 09-Jul- 21 22-Dec- 20 13-Mar- 20 19-Mar- 20 28-Mar- 20 1 Albania 132565 52542 23 59 197 442 199 119 36 2 Algeria 143652 94781 25 72 367 6067 1577 367 3 Argentina 4593763 1531374 31 79 589 172928 3427 3747 0 4 Armenia 226135 153825 1 84 372 581 302 143 17 5 Australia 30905 28128 140 510 3635 63416 4544 14196 6 Austria 647111 334629 361 1646 7697 5295 2272 1596 0 7 Azerbaijan 336788 199127 11 34 147 543 276 169 7 8 Bahrain 266797 90062 195 256 473 9 1 5 1 9 Bangladesh 1000543 499560 3 10 48 15144 11758 2184 7 10 Belarus 424554 171579 12 46 94 3786 954 91 11 Belgium 1092477 625928 314 1486 7284 6637 2458 478 0 12 Bhutan 2258 446 1 1 3 4 3 0 0 13 Bolivia 449687 149149 3 12 61 14758 3364 2619 120 14 Brazil 18909037 7162978 77 291 2915 377176 59124 60607 15 Brunei Darussalam 266 152 12 56 115 21 2 1 0 16 Bulgaria 422353 191029 7 92 293 5067 1817 601 0 17 Burkina Faso 13505 4954 2 26 146 843 0 186 0 18 Cameroon 80858 25849 2 10 75 1373 705 243 1 19 Canada 1418632 495346 138 569 4018 90839 9066 5116 20 Chile 1579591 583354 33 238 1610 9831 4424 1436 0 21 China 119141 95716 80991 81174 82230 1773689 22 Colombia 4426811 1482072 9 93 491 37773 7214 5188 0 23 Costa Rica 377091 157472 22 50 231 12811 5871 2101 0 24 Cote d Ivoire 48693 21772 1 9 92 93 8 75 0 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for thisthis version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.09.21263347doi: medRxiv preprint 25 Croatia 360680 194962 16 81 586 1676 752 127 0 26 Cyprus 81581 17476 6 58 162 1141 824 155 0 27 Czechia 1668891 624140 116 522 2279 4092 1319 236 4 28 Denmark 297543 131606 674 1044 2046 2629 438 46 0 29 Dominican Republic 331826 159064 5 21 581 7070 2525 972 80 30 Ecuador 465878 205920 17 155 1595 60733 21329 10346 0 31 Egypt 282582 124891 67 196 536 8044 3599 3199 0 32 Estonia 131396 21794 13 258 575 636 107 29 33 Finland 97651 32582 109 359 1025 1073 0 22 34 France 5686066 2418439 2860 9043 32542 85072 39112 6180 0 35 French Polynesia 19026 16182 1 3 30 27 3 6 0 36 Georgia 374836 208638 25 38 85 2237 1516 305 20 37 Germany 3734468 1494009 2369 8198 48582 44948 11687 16125 5 38 Greece 433021 130485 98 418 966 9932 2014 2258 1327 39 Guyana 20645 6076 1 4 8 359 13 52 0 40 Honduras 270689 116212 2 9 67 7195 758 102 0 41 Hungary 808437 302989 16 58 343 8539 3535 793 0 42 Iceland 6675 5621 61 250 890 2 0 0 0 43 India 30752950 10031223 74 151 724 58160 13055 20619 0 44 Indonesia 2455912 657948 34 227 1046 1597 224 929 53 45 Iran 3327526 1152072 10075 17361 32332 6841 1100 1756 5 46 Iraq 1406289 583118 70 164 458 215 12 85 3 47 Ireland 276104 78776 70 292 2121 2625 602 29 0 48 Israel 845123 371373 75 427 3460 6093 2843 525 0 49 Italy 4267105 1938083 15113 35713 86498 54153 31398 10464 454 50 Jamaica 50497 12135 1 13 26 621 177 204 15 51 Japan 813976 195880 675 873 1499 52332 21461 17125 3 52 Jordan 754927 272797 1 52 235 675 216 391 0 53 Kuwait 369227 147775 80 142 235 32 4 14 12 54 Latvia 137797 30297 16 71 280 1568 213 36 0 55 Lebanon 546366 156570 66 133 391 1816 988 255 39 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for thisthis version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.09.21263347doi: medRxiv preprint 56 Lithuania 279024 112359 3 26 358 2049 677 57 0 57 Luxembourg 71879 44067 17 210 1605 148 76 24 0 58 Malaysia 808658 91969 129 673 2161 44115 3021 3547 94 59 Maldives 74848 13474 8 13 13 363 51 279 30 60 Malta 30851 11621 9 48 139 89 83 3 0 61 Mexico 2558369 1301546 12 93 589 53144 28601 12991 7 62 Mongolia 131962 953 1 5 12 116 10 5 63 Morocco 537253 415226 6 49 358 13697 3965 7775 295 64 Nepal 652859 253184 1 1 3 574 247 181 11 65 Netherlands 1701911 676589 614 2051 8603 9309 4288 254 0 66 New Zealand 2409 1760 5 20 416 5086 1296 303 0 67 North Macedonia 155760 77949 7 36 219 98 60 27 68 Norway 132572 42775 489 1423 3581 614 83 13 22 69 Oman 281688 127019 18 33 152 204 29 112 0 70 Pakistan 967633 454673 20 241 1235 1 0 0 71 Panama 411226 206310 14 86 674 2403 551 160 0 72 Paraguay 432801 98296 5 11 52 19662 4539 2834 0 73 Peru 2071637 993760 22 145 580 4800 1189 705 15 74 Philippines 1455585 458044 52 187 803 12595 4463 1100 14 75 Poland 2880670 1202700 49 287 1389 22742 7992 1770 0 76 Portugal 899295 370787 41 642 4268 8172 4183 2084 0 77 Qatar 222918 141858 262 442 562 68 8 60 78 Republic of Korea 165344 49665 7979 8413 9478 18716 5576 5674 79 Republic of Moldova 257272 134578 4 36 199 2853 1317 391 1 80 Réunion 31845 8704 3 12 135 5141 1760 641 81 Russian Federation 5733218 2848377 34 147 1264 76369 26164 10198 253 82 Saudi Arabia 497773 360848 21 238 1104 5413 2011 3131 0 83 Senegal 44790 17670 10 36 119 479 20 85 2 84 Slovakia 391813 151336 21 105 295 1786 469 137 1 85 Slovenia 257619 105013 57 286 632 1172 849 55 0 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for thisthis version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.09.21263347doi: medRxiv preprint 86 South Africa 2135246 912477 17 116 1170 26857 8928 6158 87 Spain 3915313 1797236 2965 13716 64059 61343 38067 6488 88 Sri Lanka 269946 36049 3 42 106 2260 818 255 0 89 Sweden 1092308 367120 620 1279 3046 1459 209 46 90 Switzerland 701473 402264 858 3010 12104 2077 1012 303 0 91 Thailand 317506 4331 75 212 1136 35287 0 21601 0 92 Togo 14176 3396 1 1 25 1293 19 522 7 93 Tunisia 464914 119151 7 29 227 1003 645 236 9 94 Turkey 5465094 1189947 1 191 5698 60020 23047 16069 309 95 Ukraine 2240246 964448 3 16 311 25341 4802 1806 13 96 United Kingdom 5022897 2004223 594 2630 14547 19301 6506 376 0 97 United States of America 33451965 17314834 1264 7087 85228 407779 24040 65771 0 98 Vietnam 24810 1411 39 66 169 19154 5391 9661 8 Correlation Total Pesticides 0.21 0.23 0.93 0.86 0.57 Total Fungicides and Bactericides 0.54 0.52 0.30 0.42 0.51 Total Insecticides 0.80 0.83 0.11 0.18 0.47 Total Rodenticides 0.00 0.00 0.21 0.22 0.14 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for thisthis version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.09.21263347doi: medRxiv preprint Pests, pathogens, and weeds pose a major challenge to agricultural production, and pesticides are widely used to ensure food security across the world. However, the toxicological effects of pesticides on the human health is subjected to many reviews via multiple pathways involving food, drinking water, residential, occupational [19-21]. At this moment COVID-19 outbreak is resulting more than 250 plus global fatality every hour in human affairs and its counter response is weak in many countries. Outcome from our unbiased scientific data analysis reveal the use of pesticides has appeared as the third most contributor (10.80%) in case of COVID-19 incidences. A report by the Food and Agriculture Organization of the United Nations (FAO) suggests that China is the world’s largest consumer of agricultural chemicals and country, which alone uses more than 30% of global fertilizers/ pesticides in 9% of global cropland to fulfill the high food demands. Second pesticides consumer United States, and third, Brazil, today as on 09-July-2021 are with first-three largest COVID-19 infected cases 33451965, and 18909037 respectively. Further 4th pesticide consumer country, Argentina supports for a high 4593763 cases, in our opinion country Argentina is at a high COVID-19 risk during the writing of this paper. Another leading pesticide consumers Canada(5th), Ukraine(6th), France (7th) etc. are currently with high outbreaks of COVID-19. On the other hand, wholly organic Bhutan reports just 2258 COVID-19 infected individuals and most of them are imported cases only. In India, the largest pesticide consumer state Maharashtra as of July 11th, 2021 is with the highest 117270 total active COVID-19 cases, whereas wholly organic Indian state Sikkim is with a low number of 2244 cases. Countries with high population density including Pakistan, Bangladesh, Nigeria, etc. are not that much affected as leading pesticide consumers, provides further evidence to redefine multifactorial approach in COVID-19. A handful of healthy soil comprised of millions of ecological communities of microbes (microbiota), also regarded as a protective shield against pathogen invasion. Figure 1. Mechanism of Pesticide Use over COVID-19 progression Pesticides are non-specific=> inevitably use kills both pests as well as protective microbiota in the surrounding soil, and water sources => a loss in biodiversity (Fig. 1). Thus, a lower amount of pesticide consumption-led drop in COVID-19 cases and vice-versa. Nonetheless, pesticides sold in Africa is less than 4% of global pesticide trade and African continent is noticeable with low COVID-19 infected cases all over. Overall, a scientific dose-response relationship is fitting well for pesticide consumption and active COVID-19 cases. Therefore, it could be easily postulated that the generous use of pesticides might be responsible to trigger the high transmissibility of the SARS-CoV-2 virus. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for thisthis version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.09.21263347doi: medRxiv preprint In fact, two researchers, Lusk and Chandra highlighted during the beginning of COVID-19 (from March 1, 2020, to March 31, 2021), a reduction in agricultural output by about $309 million in the United States, all due to a reduction in labor availability[22]. The study also found that the average incidence and death incidence rate in farmers/farmworkers amounted to 9.32 % and 0.17% respectively. Both average incidence and death incidence rates were raised as high as about 3-fold in farmers/ farm workers in comparison to other normal individuals. Unfortunately, the cause of high COVID-19 outbreaks in farmers/farmworkers remains unpredictive. This evidence clearly suggests that farmers/ farm workers working near pesticide agricultural places are potentially at a very high risk for contracting COVID-19. The use of household insecticides in form of spray-cans, oil-sprays, mosquito coils and vaporizing systems are widely used to kill cockroaches, mosquitoes, bedbugs, beetles, ants etc. With increased urbanization and awareness of home pest control methods all over, for instance, New York US, Bergamo Italy, Community of Madrid Spain, New Delhi India, Sao Paulo Brazil etc. have become favorable for insecticide consumption. These places have already become epicenter of COVID- 19 outbreaks. The SARS-COV2 virus is continually mutating and this natural evolution led increased transmissibility, virulence, and resistance to antibody neutralization poses. Recently as on 14-Jun-2021, eight variants of interest viz. α , β , γ , δ , η , ι , κ , and λ are listed by the WHO successfully [23]. SARS-CoV-2 virus contains the genetic element s2m and as reported by the Tengs andcoworkers, recently xenologue of s2m was found in a large number of insect species [24]. As a result, the earlier record of a total pesticides is recently shifted to insecticide use in this report. Undoubtedly, microbiota represents the first line of defense and protection against pathogens. For instance, presence of commensal microbiome in human skin offers temperature regulation, ultraviolet (UV) radiation protection, vitamin D production, and most importantly, keeping pathogenic microbes outside the body. The pesticides are non-specific and its indiscriminate use leads to the degradation of useful microbiota. Which may likely to increase the harmful pathogen entry in human. Recent finding with high correlation values are in full agreement that local insecticide consumer sites may likely to increase the high transmissibility of the SARS-CoV-2 virus. Formal education and counseling of farmers and other stake-holders on pesticide use are urgently needed. Conscious monitoring of pesticide use worldwide (For harmonious coexistence of micro-/macro- organisms in nature). In line with modern science multi-factorial approach, this study is endorsing nature's role in direct pathogen invasion and to limit the transmission of SARS-COV-2 pathogen. Thus, similar approach help researchers think undoubtedly about the causation of COVID-19 disease. We hope these insights presented above along with disease reduction framework will be a game-changer in fighting against the COVID-19 pandemic.

The authors are thankful to the Food and Agriculture Organization (FAO) for keeping data free to use. The authors are also thankful to the GEHU India for providing necessary facilities and technical support. Disclosure statement The authors declare no competing financial interest. Author contributions AA and RR collected and verified data. PG designed and GJ analyzed the data. PG drafted the manuscript. All authors critically revised the manuscript and approved the final version for submission. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for thisthis version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.09.21263347doi: medRxiv preprint

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