The wastewater used for experimental research is typically collected from a wastewater treatment plant or prepared as a synthetic solution in the lab. These options represent transportation and cost challenges, respectively, particularly for experiments requiring large volumes of wastewater. Here, we describe a method for creating inexpensive synthetic wastewater from readily available household products. The base solution, synthesized by soaking dog food pellets for 24 hours and straining the solution, had average nutrient values of 9.7 mg/L ammonia as N, 12.2 mg/L nitrate as N, 227 mg/L total nitrogen, and 4870 mg/L chemical oxygen demand (COD). Degradation tests demonstrated that soluble COD was biodegradable. The base solution was then used to prepare synthetic wastewater that met the requirements for two experimental applications; (1) anaerobic treatment of primary effluent and (2) land-application treatment of secondary effluent. Cost analysis indicated that the single-ingredient synthetic wastewater cost 92% less to produce than synthetic wastewater recipes that used laboratory chemicals, and reduced preparation time. These results demonstrated that use of commercial products can simplify the wastewater synthesis process and reduce experimental costs for large-volume research applications while still maintaining consistent wastewater characterization.
Citation: Kargol AK, Burrell SR, Chakraborty I, Gough HL (2023) Synthetic wastewater prepared from readily available materials: Characteristics and economics. PLOS Water 2(9): e0000178. https://doi.org/10.1371/journal.pwat.0000178
Editor: Van-Huy Nguyen, Shoolini University, INDIA
Received: February 10, 2023; Accepted: August 14, 2023; Published: September 20, 2023
Copyright: © 2023 Kargol et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data related to this study are available in the main text or supplemental information of this manuscript.
Funding: This work was supported in part by the McIntire-Stennis Cooperative Forestry Program grant no. NI20MSCFRXXXG040/project accession no. 1017343 from the USDA National Institute of Food and Agriculture (HLG). AKK was funded through the University of Washington, College of the Environment Graduate Research Opportunity Enhancement (GROE) Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Synthetic wastewater (SWW) has long been used in wastewater research . It is used when a predictable wastewater composition is required [2–4]. SWW is also used when access to a wastewater treatment facility is limited  or year-round access and transport are not realistic . Most SWW recipes include many chemical components and can be time-consuming and costly to prepare [7, 8]. Recipes commonly include peptone, meat and yeast extracts, cellulose, casamino acids, urea, and various trace elements to simulate the complex mixture of carbon and nutrients found in real wastewater [6, 9]. For studies that require large volumes of wastewater [8, 10–12], producing a steady supply of synthetic wastewater could become a challenge.
Published SWW recipes vary in their characteristics and limitations. For example, the Organization for Economic Co-operation and Development (OECD)  has published a standard method for preparation of SWW that includes peptone, meat extract, urea, and trace elements , which they recognize to contain higher nitrogen, and lower carbon (as chemical oxygen demand, COD) content than typical wastewater. The recipe for another SWW (“SYNTHO”) uses similar components but in different proportions, resulting in a COD to total N ratio of 7.3:1 . A review by Prieto et al.  of 24 other SWW recipes found that most are either based on one of the recipes above or are unique to individual studies. These recipes are generally prepared with a large number of ingredients to create well-defined synthetic wastewater. The use of unique recipes or significant modification of existing formulas suggests that the standard recipes are not able to meet the experimental needs for all wastewater studies.
Preparation cost is also a barrier, which is stated in the ASTM SWW recipe protocol . Some recipes replace laboratory chemicals with less expensive commercial products such as whey, milk powder, soybean oil [6, 14] or molasses . Others have utilized waste products, including watermelon peels , cheese whey water, and sweet potato extract  to reduce costs. Additionally, when organic material is added to synthetic wastewater as simple carbon sources such as glucose, sucrose, or acetate , it does not replicate the complex mixture of compounds in real wastewater. Components such as trace organics and metals may also be omitted . In all cases, the SWW requires multiple chemical components, which additionally introduces a preparation time barrier.
In this paper, we describe the development of a SWW recipe using dry dog food as the base ingredient. Commercially available dog food has previously been explored as a way to simulate biosolids  and food waste , and to increase particulate concentration in colloidal wastewater , but has not been used as the main carbon and nitrogen source in a synthetic wastewater. The formulation of dog food combining protein, fats, and simple carbohydrates makes this an ideal candidate for SWW. Using the dog food SWW (DSW) base, we evaluated the addition of supplemental household and laboratory chemicals to achieve the characteristics needed to satisfy different experimental requirements. Cost analysis was conducted to compare the new DSW formula with the OECD standard method for synthetic wastewater preparation. This simplified synthetic wastewater recipe offers a novel approach for cost and preparation time challenges that will advance research capabilities for large volume bioreactor testing and for easier transportation to remote field-scale biological treatment testing sites.
2.1 Selection of dog food brand
Four brands of dry dog food were compared. The formulations tested were Pedigree Small Dog Roasted Chicken, Rice, and Vegetable Flavor (Pedigree), Rachel Ray Super Medleys Superfoods and Beef Recipe (Rachel Ray), Nature’s Recipe Prime Blends Salmon, Barley, and Chicken recipe (Nature’s Recipe), and Blue Wilderness Adult Small Breed Mix with Chicken (Blue Wilderness). Brands were selected based on price, nutritional information on the packaging for protein and fat composition (Table 1), and their routine availability within the United States. Costs for each brand were obtained in June 2023 from chewy.com. Tests were conducted within four weeks of purchase date and within two weeks after opening the package.
2.2 Wastewater characterization
The following methods were used to determine the characteristics of SWW. pH was measured with an Orion Dual Star pH/ISE meter (Thermo Scientific, Waltham, USA). Total nitrogen was measured using Total Nitrogen Acid and Hydroxide (Hach Company Reagent sets 2672145 and 2714045, Loveland, USA) reagents or Hach Test-n-Tube (TNT) Plus Total Nitrogen kits (Method: TNT 826). Ammonia-N was measured using the Hach AmVer High Range Ammonia (Reagent set 2606945) and TNT Plus Ammonia (Method 830) kits. Nitrate-N was quantified using Hach NitraVer X Test-N-Tube kits (Reagent set 2605345). Chemical oxygen demand (COD) was measured using Hach high-range and low-range COD digestion vials (Hach product numbers 2125915 and 2125815, respectively). Total suspended solids (TSS) and volatile suspended solids (VSS) were determined using Standard Method 2540D/E  using a 0.45 μm glass fiber filter (VWR International, Radnor, USA).
2.3 Synthetic wastewater preparation
Fig 1 illustrates the steps used to develop the preparation protocol. Deionized (DI) water was obtained from a Millipore System (MilliPak 0.22μm filter). Pedigree dog food was added to DI water at 60 g/L dry and either autoclaved at 121°C for 20 minutes or incubated at room temperature for 24 or 72 hours.
Wastewater was prepared with Pedigree brand dog food.
The impact of filtering on nutrient concentration was also tested. Dog food was soaked for 24 hours and filtered through 0.2 μm cellulose acetate filters (VWR International, Radnor, USA) to remove particulate matter or strained (~2mm mesh bag) to remove only large particles. Filtered and strained wastewater were tested for ammonia, nitrate, and total nitrogen.
For comparison of dog food brands, the following preparation was used: 60 mg/L of dog food in DI water, incubated at room temperature for 24 hours. Liquid was strained and spent pellets were discarded. For final characterization of the DSW base solution, Pedigree brand dog food was used and 3 replicate batches were compared.
2.4 Dog food synthetic wastewater biodegradability
To test the biodegradability of DSW, COD was measured in batch inoculations over time following OECD standard method #307 . Briefly, 25g dry equivalent of soil from a wastewater discharge infiltration gallery was used as a microbial inoculum. The inoculum was suspended in 200 mL of DSW base solution. The solutions were incubated at room temperature and aerated with filtered ambient air (Rezist 0.2μm PTFE filter). Samples were collected over 48 hours, filtered through 0.2 μm cellulose acetate filters, and tested for soluble COD. A sample of the initial feed was also filtered.
2.5 Preparation of DSW base for application
DSW base was used to prepare SWW with consistent characteristics of (1) synthetic primary effluent (DSPE), and (2) synthetic secondary effluent (DSSE), each for a different laboratory study.
The DSPE was prepared for use in rapid-upflow anaerobic reactors (rapid UASB) designed to simulate an operation being tested by partners in another region. The DSW base was diluted 1:10 to reach the targeted influent COD concentration. The base was supplemented with whey powder (Nature’s Best Isopure whey protein isolate, unflavored, 3 g/L) to decrease the C:N ratio and food-grade baking soda (NaHCO3, 5 g/L) to increase alkalinity to match the characteristics measured by the collaborative partner.
For DSSE, the base DSW was supplemented with NH4Cl and NaNO3 in lab to introduce the non-organic forms of nitrogen typical for secondary effluent. The supplemented DSW base was transported to a field site for experimental use in vegetated infiltration galleries. On site, the DSW was diluted 1:100 using municipal tap water. Following on-site dilution, the DSSE was characterized for ammonia, nitrate, total nitrogen, and COD. To test reproducibility, nine preparations were compared over three months.
2.6 Cost analysis
The cost to synthesize the DSW base was compared to the price of preparing SWW following the OECD protocol . Costs were normalized to the COD concentration. Chemical costs were obtained from the Fisher Scientific on-line catalog, using the least expensive formulation of each product. COD of the OECD SWW was calculated based on COD values of individual ingredients (peptone, meat extract, and urea).
3 Results and discussion
3.1 Comparison of preparation methods
Fig 1 illustrates the decision tree used to compare the preparation methods. In the autoclaved preparation, dog food particulates dissolved into the solution, which was bright orange in color. Total nitrogen was 3150 mg/l, likely due to the complete dissolution of the particulates and release of large quantities of organic nitrogen into the solution. The dissolved dog food made the SWW difficult to work with, and particulates could not be removed. Autoclaving was eliminated as an option because the solids content in the SWW could not be used for pump-fed reactors, as it would clog the tubing. The DSW soaked for 72 hours had a strong odor and was eliminated from consideration.
Pellets soaked for 24 hours absorbed significant water volume and released particulate matter into the solution. Water volume decreased by approximately 10 percent, so to recover 1L of solution, 1.1 L should be prepared. Ammonia concentration in the filtered 24-hour batch was too low for wastewater applications (<0.015 mg/L); filtering large volumes was also considered impractical for producing large volumes of DSW. Therefore, filtration was eliminated. When strained to remove large food pieces, the final SWW had an ammonia concentration of 9.7 mg/L, which falls within the typical secondary effluent range of 0.1–10 mg/l . The finalized approach selected for DSW preparation was soaking for 24 hours and straining through a mesh bag. The prepared DSW was frozen within a day of preparation for storage until use.
3.2 Wastewater characteristics
To evaluate if manufacturer-reported nutrient content would influence the final DSW characteristics, four brands were compared. Table 1 shows the manufacturer-reported nutrient content and the resulting DSW characteristics. pH was consistent among brands while total nitrogen varied by 20%. Ammonia and nitrate content had the highest variability among the measured parameters and were not predicted by the manufacturer protein content; Pedigree and Rachel Ray had higher nutrient values. Three brands had similar COD values while Nature’s Recipe was 2,000 mg/L higher than the others. Three brands had no more than a 35 mg/L difference between TSS and VSS, while Rachel Ray had a nearly 500 mg/L difference.
Pedigree and Rachel Ray were found to produce DSW with typical wastewater characteristics, although both fall near the lower end of the range for ammonia and nitrate concentrations. Pedigree brand was chosen for use in the synthetic wastewater recipe. The results suggested that a variety of dog food brands could be suitable for DSW synthesis. Suitability should be confirmed by testing key characteristics.
Dry dog food stored longer than 12 weeks produced wastewater with reduced carbon content, with COD decreasing by 67%. The food was stored in a covered but not airtight container. This storage method may have caused moisture losses over time, compacting particle structure and decreasing the available surface area for nutrient release. Therefore, it is recommended to purchase dog food in smaller bags to reduce moisture loss over time. Storage in an airtight container was not tested but may increase the stability.
3.3 Comparison to other wastewater recipes
Table 2 shows the characteristics of DSW and two other synthetic wastewater recipes. Concentrated DSW had a COD content about 10 times higher than most synthetic wastewater recipes , 6 times higher than SYNTHO  and 8 times higher than OECD SWW . DSW was advantageous over the other recipes because the dog food base added complex COD, nitrogen, and trace elements to the SWW in a single step. Total nitrogen in other wastewater recipes ranged from 5.2 mg/l to 165 mg/l with supplementation, while the DSW base reached the higher end of that range without the addition of supplemental nitrogen. The testing method used in this study measures free ammonia. Thus, it is likely that most nitrogen in the dog food exists as organo-complexes (e.g., proteins).
3.4 Biodegradability of DSW
Fig 2 shows the decrease in COD over time. Over two days, COD decreased on average by 69% ± 7.2%. This showed that the carbon in the SWW was readily available for biodegradation by microorganisms. Therefore, it can be used as feed for wastewater bioreactors to provide an organic carbon and nitrogen source.
3.5 Preparation of SWW for experimental application
DSW prepared from Pedigree brand was used as the base ingredient for preparation of synthetic wastewater for two different research needs, one requiring primary effluent and one requiring secondary effluent (Table 3). For each, we demonstrated how addition of small amounts of supplemented materials were used to match the COD, nutrient, and alkalinity experimental needs.
DSSE was analyzed 9 times over 3 months of experiments in a planted infiltration gallery (S1 Table). Average COD content aligned with typical values for secondary effluent . Total nitrogen and ammonia were similar to typical secondary effluent, while nitrate was slightly below typical. When supplemented with nitrogen sources, nitrate-N and ammonia-N concentrations increased to 1.3 mg/L and 1.2 mg/L, respectively. Consistency was observed over 9 separate preparations, which suggested that, once methods for synthesis and baseline nutrient concentrations have been established, consistent concentrations can be expected. Thus, the recipe can be used long-term with only occasional quality control testing needed, which simplifies its use at field sites.
These two examples show how the base recipe is readily modifiable through addition of other simple products (whey powder, baking soda) or small masses of lab chemicals (nitrate). This allows the recipe to be adapted to match the characteristics of primary or secondary wastewater from both strong and dilute sources.
3.6 Cost analysis for DSW preparation
The costs to prepare DSW and OECD wastewater were compared in Table 4. Prices were obtained in June 2023 and reflect the costs to obtain materials at the study location. Costs might vary in other locations or by other factors such as institutional discount pricing. Costs were normalized to COD concentration. OECD wastewater was calculated to have a COD of 555 mg/L (S2 Table); thus, 10.7 L of OECD SWW was needed to achieve the COD mass equivalent of 1L of DSW. For the same COD equivalent, DSW can be synthesized for 8% of the cost of the OECD SWW. For an experiment requiring 20L of SWW base per week, this would result in a monthly cost of $10.40 compared to $126.40 for the OECD recipe. Note that this cost savings does not include the additional savings associated with reduced person work hours required to prepare a single-ingredient synthetic wastewater.
We have demonstrated the preparation of a single-ingredient synthetic wastewater starting with commercial dog food. The resulting COD and nitrogen content were similar to wastewater and (with dilution) treated wastewater effluent. Straining enabled removal of large particles that might clog tubing used with lab- or pilot-scale reactors. Cost comparisons showed that the DSW cost was 8% of the multi-component synthetic wastewater recipe published by OECD. With minor supplementation, the DSW was adapted to meet study needs for two different research applications. The difference in nutrient content among brands highlighted the importance of characterizing DSW before use. This approach can be expanded to add other nitrogen components or targeted compounds such as emerging contaminant chemicals to the DSW. Having cost effective means to produce large volumes of synthetic wastewater with reliable characteristics advances the ability to conduct reproducible research when access to materials from a wastewater treatment facility is not feasible. This is important as climate change and other factors require the development of creative new wastewater treatment solutions, all of which must be scaled up and tested before implementation.
S1 Table. Nutrient characteristics of synthetic secondary wastewater effluent prepared from Pedigree brand dog food including chemical oxygen demand, total nitrogen, ammonia, and nitrate.
- 1. Cokgor EU. Anaerobic treatment of synthetic domestic wastewater. Fresenius Environ Bull. 1998; 7 (12A):912–9.
- 2. de Kreuk MK, Heijnen JJ, van Loosdrecht MC. Simultaneous COD, nitrogen, and phosphate removal by aerobic granular sludge. Biotechnol Bioeng. 2005; 90 (6):761–9. pmid:15849693
- 3. VanderGheynst JS, Gossett JM, Walker LP. High-solids aerobic decomposition: pilot-scale reactor fevelopment and experimentation. Process Biochemistry. 1997; 32 (5):361–75.
- 4. Wu H, Fan J, Zhang J, Ngo HH, Guo W, Hu Z, et al. Decentralized domestic wastewater treatment using intermittently aerated vertical flow constructed wetlands: impact of influent strengths. Bioresour Technol. 2015; 176:163–8. pmid:25460998
- 5. O’Flaherty E, Gray NF. A comparative analysis of the characteristics of a range of real and synthetic wastewaters. Environ Sci Pollut Res Int. 2013; 20 (12):8813–30. pmid:23740303
- 6. Prieto AL, Criddle CS, Yeh DH. Complex organic particulate artificial sewage (COPAS) as surrogate wastewater in anaerobic assays. Environmental Science: Water Research & Technology. 2019; 5 (10):1661–71.
- 7. Boeije G, Corstanje R, Rottiers A, Schowanek D. Application of the CAS test system an synthetic sewage for biological nutrient removal: Part I: Development of a new synthetic sewage. Chemosphere. 1999; 38 (4):699–709.
- 8. Khurelbaatar G, Sullivan CM, van Afferden M, Rahman KZ, Fühner C, Gerel O, et al. Application of primary treated wastewater to short rotation coppice of willow and poplar in Mongolia: Influence of plants on treatment performance. Ecological Engineering. 2017; 98:82–90.
- 9. OECD. Test No. 303: Simulation Test—Aerobic Sewage Treatment—A: Activated Sludge Units; B: Biofilms2001. https://doi.org/10.1787/9789264070424-en
- 10. Martinez-Hernandez V, Leal M, Meffe R, de Miguel A, Alonso-Alonso C, de Bustamante I, et al. Removal of emerging organic contaminants in a poplar vegetation filter. Journal of Hazardous Materials. 2018; 342:482–91. pmid:28866407
- 11. Guidi Nissim W, Jerbi A, Lafleur B, Fluet R, Labrecque M. Willows for the treatment of municipal wastewater: Performance under different irrigation rates. Ecological Engineering. 2015; 81:395–404.
- 12. Amiot S, Jerbi A, Lachapelle-T X, Frédette C, Labrecque M, Comeau Y. Optimization of the wastewater treatment capacity of a short rotation willow coppice vegetation filter. Ecological Engineering. 2020; 158.
- 13. ASTM. Standard Practice for the Praparation of Substitute Wastewater. 2018.
- 14. de Sousa JT, Foresti E. Domestic Sewage Treatment in an Upflow Anaerobic Sludge Blanket- Sequencing Batch Reactor System. Water Sci Technol. 1996; 33 (3):73–84.
- 15. Duran M, Speece RE. Biodegradability of Residual Organics in the Effluent of Anerobic Processes. Environmental Technology. 1999; 20:597–605.
- 16. Hasanin MS, Hashem AH. Eco-friendly, economic fungal universal medium from watermelon peel waste. J Microbiol Methods. 2020; 168:105802. pmid:31809830
- 17. Ibrahim AA. Optimization of a New Medium of Whey Water and Sweet Potato Extract for Different Lactobacillus Species by Plackett-Burman Statistical Design Using Mini-Tab16 Software. Journal of Microbiology, Biotechnology and Food Sciences. 2021; 10.
- 18. Krismastuti FSH, Hamim N. Designing a formulation of synthetic wastewater as proficiency testing sample: a feasibility study on a laboratory scale. Accreditation and Quality Assurance. 2019; 24 (6):437–41.
- 19. Langenhoff A, Intrachandra N, Stuckey D. Treatment of Dilute Soluble and Colloidal Wastewater Using an Anaerobic Baffled Reactor: Influence of Hydraulic Retention Time. Water Res. 2000; 34 (4):1307–17.
- 20. AWWA. Standard Methods for the Examination of Water and Wastewater, 2540E: Fixed and Volatile Solids at 550C. Standard Methods for the Examination of Water and Wastewater. New York: American Water Works Association; 1998.
- 21. OECD. Test No. 307: Aerobic and Anaerobic Transformation in Soil 2002. https://doi.org/10.1787/9789264070509-en
- 22. Tchobanoglous G, Stensel HD, Tsuchihashi R, Burton F, Abu-Orf M, Bowden G, et al. Wastewater Engineering, Treatment and Resource Recovery. 5th ed: McGraw Hill Education; 2014.
- 23. Aiyuk S, Verstraete W. Sedimentological evolution in an UASB treating SYNTHES, a new representative synthetic sewage, at low loading rates. Bioresour Technol. 2004; 93 (3):269–78. pmid:15062822