Living Machines as a Possible Treatment for Ibuprofen in Wastewater

Author Summary

Historically, wastewater treatment has focused on disinfection and reducing nutrient concentrations, but there is now growing concern over the increased concentrations of pharmaceutical compounds in agricultural and human wastewater. More comprehensive conventional wastewater treatment, however, leads to increased resource and energy consumption and greenhouse gas emissions. Living Machines are an alternative, decentralized ecological wastewater system that have been successful at disinfecting and reducing nutrient loads in wastewater with lower capital and operating costs and improved energy efficiency. We conducted preliminary experiments using small-scale Living Machines to test their effectiveness at reducing a common pharmaceutical, ibuprofen, which is increasingly used in the agricultural sector and for human health. We found that these Living Machines were efficient and effective in reducing concentrations of ibuprofen in wastewater, even at input levels much higher than reported in the literature, and hence show promise for a range of possible applications. 

Introduction

Effective treatment of wastewater, whether from municipal, industrial, or agricultural sources, is vital for human and ecosystem health. While historically wastewater treatment has focused on disinfection and reducing nutrient concentrations, there is now growing concern over the increased concentrations of pharmaceutical compounds found in wastewater and their multi-faceted impacts.^1-3 More comprehensive wastewater treatment, however, comes at a cost. Full life cycle analyses of conventional systems in more industrialized regions of the world show increased infrastructure resources, chemical consumption, operational energy, and direct greenhouse gas emissions with increased treatment.⁴ In addition, the highly centralized nature of these conventional systems, compared to more decentralized systems, show increased impacts using a range of environmental indicators, mainly due to increased infrastructure and energy consumption.⁵

Since the 1970s, Living Machines, an alternative ecological wastewater system, have addressed a variety of water treatment needs around the world. These systems typically consist of series of anaerobic and aerobic cells populated with flora and fauna that treat wastewater for individual buildings to small communities, and various designs have also been used to treat industrial and livestock wastewater. Living Machines have been successful in reducing key targets of wastewater treatment, including total suspended solids, biochemical oxygen demand, nutrient levels, and fecal coliform bacteria,⁶ and offer other potential benefits including lower capital and operating costs, improved energy efficiency, and potential commercial food and/or flower production.⁷ To our knowledge, Living Machines have not been evaluated for their treatment of pharmaceuticals, and, given their more localized and varied applications, they may offer a means of circumventing the broader environmental impacts often associated with higher levels of wastewater treatment and centralized systems.

Ibuprofen is a non-steroid anti-inflammatory pharmaceutical compound that is used for both humans and livestock with increased frequency. In fact, the global ibuprofen market was valued at USD 89 million in 2020 and is expected to reach USD 109 million by the end of 2027.⁸ While ibuprofen is largely metabolized, concentrations of ibuprofen, and the various active substances made as the body breaks down ibuprofen, are measurable in both the input (influent) and output (effluent) of wastewater treatment plants in significant concentrations (up to 36 μg/L of ibuprofen in influent and up to 4 μg/L in effluent) and surprisingly high concentrations have also been found in river samples (up to 2.4 μg/L).⁹

The overall health impacts of ibuprofen in wastewater effluent are not well understood, although in examining a variety of non-steroid anti-inflammatory pharmaceuticals, the World Health Organization indicated that these drugs do not persist in wastewater effluent at high enough concentrations to pose an immediate threat to human health. Questions remain, however, regarding the impact of long-term exposure.¹⁰ The effects of ibuprofen on aquatic environments are also not well studied, although ibuprofen compounds have been found in the bile of bream (Abramis brama) and roach (Rutilus rutilus) downstream of wastewater treatment plants and low concentrations of ibuprofen (e.g., 0.1-10 μg/L) have been found to effect South American catfish (Rhamdia quelen) by decreasing white blood cell counts, causing rapid deterioration of kidney function and suppressing immune responses.¹¹

Given the increasing use and unknown impacts of ibuprofen in wastewater and the potential role of Living Machines in wastewater treatment, we conducted a preliminary study to evaluate the effectiveness of Living Machines to treat ibuprofen. 

Methods

We constructed four small-scale Living Machines for this experiment, each consisting of three 17 L cells (one anaerobic cell and two aerobic cells). The cells were mounted vertically on a pole so water could flow with gravity through spigots from the anaerobic cell and then sequentially through the two aerobic cells (Figure 1). Each cell included pieces of ceramic pots and rocks (primarily dolostone) to increase surface area and soil from a wetland in Saratoga Springs, NY to provide an initial bacterial community. The aerobic cells included flora and fauna to mimic local wetlands (e.g., several species of snails, cattails (Typha spp.), duckweed (Lemnoideae spp.), etc.) and aquarium aerators for oxygenation. The Living Machines were given seven days to establish prior to adding any influent wastewater and were maintained for the duration of the experiments in a greenhouse at 25.5-26.5 ℃ and with a 12/12 light/dark cycle.

Figure 1. The four prototype Living Machines we used in this experiment, each consisting of three cells mounted vertically so water could flow through the top anaerobic cell and sequentially through the two lower aerobic cells.

The influent was added to the anaerobic cells in 2 L increments and consisted of a filtered (590 μm mesh) slurry of 50 g/L of horse manure and 0.5 mg/L of ibuprofen (we used horse manure as opposed to human waste due to biosafety concerns and because Living Machines are used to treat agricultural waste as well as human and industrial waste). Water was moved from the anaerobic cell and sequentially through the two aerobic cells in 2 L volumes, after being held in the cells for either 24 or 48 hours. The duration water is held in a cell is referred to as retention time, and it is an important variable for wastewater treatment since it influences the efficiency of systems or how quickly wastewater can be treated. Twenty-four and 48-hour retention times are common in conventional wastewater systems, and hence we tested two of our Living Machines at a 24-hour cell retention time and two at a 48-hour retention time. Influent was added and water was moved from one cell to the next for 14 days to allow the machines to further establish, and then 100 ml samples were taken from the input slurry and effluent of each of the cells for all Living Machines on several sampling days. Samples were frozen until analyzed using liquid chromatography-mass spectrometry (LC-MS).  

Samples were prepared for the LC-MS by adding 10 mL of ethanol to thawed samples, shaking vigorously by hand for one minute, and filtering using a 0.22 μm syringe filter (Millipore GVHP Durapore Hydrophobic PVDF). Additional details regarding our LC-MS methods can be found in note #1.

Results and Discussion

Additional details regarding our statistical analyses can be found in note #2, but overall, we found statistically significant differences in ibuprofen concentrations between all groupings of influent and cell effluent (p < 0.001) except between cells 2 and 3 (p = 0.623), and we found no significant difference between the ibuprofen concentrations in the 24 and 48-hour retention times for each cell (p > 0.05) (Figure 2).

Figure 2. Mean ibuprofen concentrations in the influent wastewater and the effluent of the three cells of the Living Machines, separated by machines with a 24-hour cell retention time and a 48-hour retention time (error bars = SDs). Statistically significant differences (p < 0.05) among groupings are indicated with *, and there were no significant differences between the 24-hour and 48-hour retention times for each cell.

These results indicate that our small-scale Living Machines were effective in reducing concentrations of ibuprofen in wastewater, even at influent levels much higher than reported in the literature (5 mg/L compared to the maximum of 36μg/L reported in Ferrando-Climent et al.⁹) and to effluent levels lower than reported in some river systems (< 2 μg/Lcompared to the maximum of 2.4 μg/L reported in Ferrando-Climent et al.⁹). In addition, they were efficient at achieving these impressive ibuprofen reductions given that the 24-hour cell retention times were as effective as 48-hour retention times. While our data are limited, these experiments point to the potential of Living Machines to address increasingly prominent pharmaceuticals in wastewater efficiently and in a way that potentially avoids the increased environmental costs (e.g., increased greenhouse gas emissions⁵) associated with higher levels of treatment in our conventional, centralized wastewater treatment systems.

In addition, lower capital costs, lower operating costs, and improved energy efficiency of Living Machines makes them an interesting option for a wide range of wastewater applications where we need to address not only nutrient loads and disinfection, but also the growing use of pharmaceuticals such as ibuprofen. Living Machines might have a particularly important roles to play in human wastewater treatment in small communities in less developed regions, where relatively little wastewater treatment is already in place, and as a more localized supplement to existing centralized treatment in more developed regions. In addition, given the increased use of ibuprofen and other pharmaceuticals in agriculture, there is significant potential in using Living Machines to treat wastewater from livestock on a localized scale. 

Additional research could strengthen our preliminary findings by increasing replicates, reducing cell retention times to assess even higher levels of efficiency, evaluating other common pharmaceuticals, exploring the effectiveness of different Living Machine design, and the specific applications where Living Machines could play the most significant role in wastewater treatment. The emergent literature on effective plant-bacteria synergies to reduce pharmaceuticals could also guide Living Machine design for more holistic wastewater treatment.¹²

References

1. Ojemaye, CY & Petrik, L. Pharmaceuticals in the marine environment: A review. Environmental Reviews 27:2 (2018) (doi:10.1139/er-2018-0054).
2. Brodin, T et al. Ecological effects of pharmaceuticals in aquatic systems – impacts through behavioural alterations. Philosophical Transactions of the Royal Society B: Biological Sciences 369, 1656 (2014) (doi:10.1098/rstb.2013.0580). 
3. Arnold, KE et al. Assessing the exposure risk and impacts of pharmaceuticals in the environment on individuals and ecosystems. Biological Letters 9(4):20130492 (2013) (doi:10.1098/rsbl.2013.0492).
4. Foley, J et al. Comprehensive life cycle inventories of alternative wastewater treatment systems. Water Research44, 1654-1666 (2010).
5. Opher, T & Friedler, E. Comparative LCA of decentralized wastewater treatment alternatives for non-potable urban reuse. Journal of Environmental Management 182, 464-476 (2016).
6. Todd, J, Brown, EJG. & Wells, E. Ecological design applied. Ecological Engineering 20, 421-440 (2003).
7. Engineering for Change [online]. https://www.engineeringforchange.org/.
8. Globe News Wire [online]. https://www.globenewswire.com/.
9. Ferrando-Climent, L et al. Comprehensive study of ibuprofen and its metabolites in activated sludge batch experiments and aquatic environment. Science of the Total Environment 438, 404-413 (2012).
10. World Health Organization. World Health Organization. Pharmaceuticals in Drinking Water. World Health Organization (2012) (9789241502085_eng.pdf).
11. Mathias, FT et al. Effects of low concentrations of ibuprofen on freshwater fish Rhamdia quelen. Environmental Toxicology and Pharmacology 59, 105-113 (2018).

Notes

1. Concentrations of ibuprofen in the samples were calculated using a range of standards (0.005 mg/L – 0.5 mg/L) and the resulting regression line had an R² = 0.99. LC-MS analysis was performed using a Thermo Scientific Vanquish liquid chromatography system equipped with a VF-D40A variable wavelength detector, an ISQ-EC mass spectrometer, and a Restek Ultra C₁₈ 150-mm column. Although our system is equipped with a mass spectrometer, this experiment can be performed using a single wavelength UV detector at 221 nm. The mobile phase consisted of an 80:20 methanol/formic acid (0.1%) mixture at a flow rate of 0.8 mL/min.   
2. Our data set satisfied the requirements for parametric analysis (Shapiro–Wilk; p > 0.05) and homogeneous variances (Levene; p > 0.05), and a one-way ANOVA revealed a statistically significant difference in ibuprofen concentrations between Living Machine influent and cell effluent (F = 1150.411, df = 31, p < 0.001; Figure 2). Tukey HSD test for multiple comparisons showed statistically significant differences between all groupings of influent and cell effluent (p < 0.001) except between cells 2 and 3 (p = 0.623). T-tests indicated no significant difference between the ibuprofen concentrations in the 24-hour and 48-hour retention times for each cell (p > 0.05).