cause DNA fragmentation [79]. This condition causes mitochondrial damage, which can lead to a release of Cytochrome C, Caspase activation and finally cell death [80].
Low indoor air quality in classrooms is well known to be associated with a negative impact on the learning capacity of school children [15,16,76]. To establish whether this only indicates a short-term effect or possible substantial damage to brain function, a study in mice was performed and published [81]. Adolescent mice were exposed 24 hours a day for 7 weeks to a level of 0.3% carbon dioxide, but with normal atmospheric levels of oxygen [81]. At the end of the study a so-called water maze exercise was performed. Here the mice have to find a life-saving platform in a water basin. This test distinguishes between impact on physical function and on mental function. Mice were tested on four consecutive days. On the first test day mice in all groups (carbon dioxide exposed and normal air exposed) typically needed around 40 seconds to find the platform. Healthy mice exposed to normal air learned to find the platform more quickly and after four days the healthy mice finally only needed 20 seconds to find the platform, whereas the carbon dioxide exposed mice were unable to learn the shortest way to the platform. Although the carbon dioxide exposed mice were able to swim as quickly as their healthy controls, they were not able to learn the shortest route. They swam around in a very disoriented manner day after day of the four test days. Histology tests demonstrated apoptosis of brainstem neurons in those 0.3% carbon dioxide exposed mice [81]. This is a very disturbing finding because this CO2-induced loss of neurons is irreversible.
When exposure to low level CO2 is prolonged (several hours to one week) the organism depletes its buffer systems [81-84]. The number of cells in the brain of adolescents is a result of the equilibrium of cell proliferation and apoptosis. External factors can affect both cell proliferation and death. In the case of prolonged low-level CO2-exposure the latter occurs, especially under exercise or stress [85-88]. Blood carbon dioxide concentration exerts an important influence on intra- and extracellular pH, CO2 passes quickly through the cell membranes to form carbonic acid with H2O, which releases H+ ions and, in excess, causes acidosis [89-91]. Acidosis decreases transmembrane Ca+2 conductivity and decreases the excitability of neurons [92,93]. Calcium overload causes excitotoxicity and apoptosis during hypoxia [94].
3.2.3. Male reproductive toxicity
As a rise in carbon dioxide when wearing a mask is scientifically proven (Tables 1 and 2) [18-27,29-44], further information about the phenomenon of the toxicological influence of elevated carbon dioxide of inhaled air on male fertility needs to be discussed. The toxic effects of low level carbon dioxide exposure on male fertility have been studied extensively in animal experiments. The exposure of adolescent rats to a carbon dioxide level of 2.5% once for four hours induced pathological signs of diminished fertility in rat testes [95]. A correct estimation of an exposure limit from animal toxicity studies to humans requires implementation of a safety factor [59,60,96]. One has to consider that small laboratory animals, evolutionarily adapted to living in burrows and caves, are limited as indicators for carbon dioxide, since they are much less sensitive to it than humans [57]. As aforementioned, the US Navy was using a safety factor of 3 from a level with no adverse effects on rat pregnancies [59,60]. In the study referred to on rat testicular function of carbon dioxide no so-called NOAEL (No-Observed-Adverse-Effect-Level) was observed [95]. Using the 2.5% level with marked damage to testes function and a minimum safety factor of 5, an exposure limit for adolescent males needs to be set at 0.5% for a maximum of 4 hours a day [59,60,95,96].
The damaging mechanism of CO2 affecting testicular tissues is based on the conditions of oxidative stress and acidosis with increased inflammation and apoptosis as described above [72,73-76,78,79]. Testes metabolism and cell respiration have been shown to be increasingly inhibited by rising levels of CO2 [95]. It has to be pointed out here that this data on the toxicity of carbon dioxide on reproduction has been known for 60 years. Exposure limits have therefore typically been set at 0.5% CO2 in working environments, e.g. according to a Safety Data Sheet by Linde Company on Exposure Limits [97]. These limits are based on EU Indicative Exposure Limit Values in Directives 91/322/EEC, 2000/39/EC, 2006/15/EC, 2009/161/EU, 2017/164/EU. An 8-hour exposure limit of 0.5% CO2 has been defined in the NIOSH regulations [17]. Looking at the potential damage to the reproduction function by subacute or chronic carbon dioxide exposure proven in animal experiments makes it very clear why these limits exist.
Table 3 sums up the significant toxicity of inhaled carbon dioxide at low levels in animal studies.