**4. Conclusions**

*Advances in Microfluidics and Nanofluids*

Regarding the ecosystem quality, **Figure 4D** and **E** present impacts on the ecotoxicity freshwater category and the freshwater and terrestrial acidification category. According to Aurisano et al. [27], assessing ecotoxicological impacts on freshwater ecosystems after chemical exposure is an important component of various environmental and chemical management frameworks. These impact categories were considered here because we needed to determine impacts associated with compounds from our process that potentially come into contact with aquatic organisms and human beings [28]. Results showed that ABTS had the highest impact contribution on these categories due to its potential impact on aquatic ecosystems. Many authors have agreed that freshwater acidification is mainly caused by protons resulting from the mineralization of nitrogen and sulfur deposition, while carbon dioxide is the main cause of (coastal) marine acidification [29, 30]. These environmental impacts directly compromised the operation stage of micromixers in wastewater treatment. **Figure 4F** shows impacts on the climate change category. Emissions of CO2 and other greenhouse gases (GHGs), aerosols, and ozone precursors are thought to be responsible for detrimental climate impact [31]. In this study, energy use in operation processes of micromixers had the highest contribution to this impact category, which agrees well with previous studies [32]. This energy along with the energy used during the life span of a micromixer comprise the life-cycle energy and emissions footprint. According to Yousefi et al. [33], in addition to the energy consumption issue, greenhouse gas (GHG) emission issues and an understanding of emissions in a production process based on the kilogram of carbon equivalent (CO2eq) are also critical in any production process. Several studies have reported some greenhouse gas removal technologies that will be needed to balance residual emissions and meet the emission targets [34]. Overall, most of these technologies proposed involve carbon dioxide removal or conversion of a higher global warming potential (GWP) gas to a lower GWP gas [35]. However, some removal technologies require significant amounts of energy for both installation and operation. Therefore, it is necessary to continue investigating in this field to assess potential environmental tradeoffs, including those related to energy use and climate change. Finally, **Figure 4G** and **H** show the impact assessment results for the resource depletion of minerals and metals category and resource depletion of dissipated water category, respectively. Results in these categories are mainly associated with the energy consumption due to the use of non-renewables such as fossil fuels. According to Klinglmair et al. [36], resources could be evaluated according to their depletion (consumption related to geological or natural reserve), scarcity (economic availability) and their criticality (a resource that is scarce and crucial for society). Hence, depletion refers to the decrease of the physical amount of a resource that is available for future human use [37]. Minerals and metals depletion are considered within the abiotic depletion potential (ADP) method, which is recommended by the ILCD handbook and the Product Environmental Footprint (PEF) as the best available practice for assessing resource depletion on a midpoint level [37, 38]. Therefore, here we considered this impact category to determine the potential impacts associated with resource use when operating wastewater treatment processes enabled by the developed micromixers. However, both environmental and human health impacts related to extraction or use, such as toxic emissions, are kept as separate environmental impact categories, and resource depletion directly

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**3.3 Total impact assessment**

impacting ecosystem health was disregarded in importance.

**Figure 5** shows the impact assessment results of manufacturing and operation stages for each micromixer. The manufacturing stage had the highest contribution

Results from this study showed that six prototypes of micromixers for wastewater treatment can be analyzed in terms of impacts to human health and environment using the LCA methodology. This tool confirmed to be useful for this early research stage as it allows to identify potential impacts during the different phases required to implement these technologies.

According to the four general impacts categories considered in this study, we successfully identified the main flows that contributed to each one. The ABTS chemical for enzyme activity assays significantly contributed to human health and ecosystem quality categories. Assessment of potential toxicological effects of this compound on human health were determined in several impact categories including human toxicity, cancer and non-cancer effects, and photochemical ozone production. Also, in terms of ecosystem quality, impacts on the ecotoxicity freshwater and the freshwater and terrestrial acidification categories were considered. Toxic effects of the ABTS were the highest compared to other raw materials during the operation stage mainly due to its release to aquatic ecosystems where it might eventually reach organisms and human beings. Moreover, energy use contributed to climate change and resource depletion categories. Emissions of CO2 and other greenhouse gases were considered in the climate change category. Regarding the resource depletion category, results showed that the use of non-renewables such as fossil fuels to produce electricity was the major contributor to this category.

Multiple layers micromixers showed the highest impact while the one-layer ones the lowest. These results were associated directly with the manufacturing stage, where PMMA and energy used had the highest contribution to impacts on environmental and human health categories, respectively. Therefore, the manufacturing stage had the highest contribution to the total impact of each micromixer in all impact categories. Also, the operation stage depended directly on the retention of the active bionanonanocompounds within each micromixer, in addition to others raw materials necessary for wastewater treatment. Finally, impact assessment results of the manufacturing and operation stages determined the total impact of a micromixer during its work cycle.

This study represents a first step for the impact assessment on the environment and human health of the wastewater bioremediation treatment enables by low and high efficiency micromixers. Moreover, this work sets a starting point to further explore the potential of micromixers and the possible environmental concerns arising from their implementation in large-scale operations.
