Advances in freshwater risk assessment: improved accuracy of dissolved organic matter-metal speciation prediction and rapid biological validation.

Speciation modeling of bioavailability has increasingly been used for environmental risk assessment (ERA). Heavy metal pollution is the most prevalent environmental pollution issue globally, and metal bioavailability is strongly affected by its chemical speciation. Dissolved organic matter (DOM) in freshwater will bind heavy metals thereby reducing bioavailability. While speciation modeling has been shown to be quite effective and is validated for use in ERA, there is an increasing body of literature reporting problems with the accuracy of metal-DOM binding in speciation models. In this study, we address this issue for a regional-scale field area (Lake Tai, with 2,400 km2 surface area and a watershed of 36,000 km2) where speciation models in common use are not highly accurate, and we tested alternative approaches to predict metal-DOM speciation/bioavailability for lead (Pb) in this first trial work. We tested five site-specific approaches to quantify Pb-DOM binding that involve varying assumptions about conditional stability constants, binding capacities, and different components in DOM, and we compare these to what we call a one-size-fits-all approach that is commonly in use. We compare model results to results for bioavailable Pb measured using a whole-cell bioreporter, which has been validated against speciation models and is extremely rapid compared to many biological methods. The results show that all of the site-specific approaches we use provide more accurate estimates of bioavailability than the default model tested, however, the variation of the conditional stability constant on a site-specific basis is the most important consideration. By quantitative metrics, up to an order of magnitude improvement in model accuracy results from modeling active DOM as a single organic ligand type with site-specific variations in Pb-DOM conditional stability constants. Because the biological method is rapid and parameters for site-specific tailoring of the model may be obtained via high-throughput analysis, the approach that we report here in this first regional-scale freshwater demonstration shows excellent potential for practical use in streamlined ERA.

Structured expert judgement approach of the health impact of various chemicals and classes of chemicals.

INTRODUCTION: Chemical contamination and pollution are an ongoing threat to human health and the environment. The concern over the consequences of chemical exposures at the global level continues to grow. Because resources are constrained, there is a need to prioritize interventions focused on the greatest health impact. Data, especially related to chemical exposures, are rarely available for most substances of concern, and alternate methods to evaluate their impact are needed. STRUCTURED EXPERT JUDGMENT (SEJ) PROCESS: A Structured Expert Judgment (Research Outreach, 2021) process was performed to provide plausible estimates of health impacts for 16 commonly found pollutants: asbestos, arsenic, benzene, chromium, cadmium, dioxins, fluoride, highly hazardous pesticides (HHPs), lead, mercury, polycyclic-aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), Per- and Polyfluorinated Substances (PFAs), phthalates, endocrine disrupting chemicals (EDCs), and brominated flame retardants (BRFs). This process, undertaken by sector experts, weighed individual estimations of the probable global health scale health impacts of each pollutant using objective estimates of the expert opinions' statistical accuracy and informativeness. MAIN FINDINGS: The foremost substances, in terms of mean projected annual total deaths, were lead, asbestos, arsenic, and HHPs. Lead surpasses the others by a large margin, with an estimated median value of 1.7 million deaths annually. The three other substances averaged between 136,000 and 274,000 deaths per year. Of the 12 other chemicals evaluated, none reached an estimated annual death count exceeding 100,000. These findings underscore the importance of prioritizing available resources on reducing and remediating the impacts of these key pollutants. RANGE OF HEALTH IMPACTS: Based on the evidence available, experts concluded some of the more notorious chemical pollutants, such as PCBs and dioxin, do not result in high levels of human health impact from a global scale perspective. However, the chemical toxicity of some compounds released in recent decades, such as Endocrine Disrupters and PFAs, cannot be ignored, even if current impacts are limited. Moreover, the impact of some chemicals may be disproportionately large in some geographic areas. Continued research and monitoring are essential; and a preventative approach is needed for chemicals. FUTURE DIRECTIONS: These results, and potential similar analyses of other chemicals, are provided as inputs to ongoing discussions about priority setting for global chemicals and pollution management. Furthermore, we suggest that this SEJ process be repeated periodically as new information becomes available.

Maritime disasters and pollution: X-Press Pearl maritime debacle.

Ocean ecosystems and global well-being are connected and significant. Over the past few decades, shipping accidents have caused severe marine pollution all over the world, and after a lull during the hike of COVID pandemic, polluting events are again on the rise. Marine pollution caused by maritime accidents requires a clear understanding of the fate of spilled pollutants, post-disaster challenges, pollutant removal strategies, and mitigation strategies against environmental damage. Considering proactive prevention is always better than reactive response, while understanding accidents and ensuring corrective action is even more important. This Special Issue provides a broad overview of the marine and coastal pollution, not limited to, but focused on the 2021 X-Press Pearl containership disaster off the coast of Sri Lanka, and the impact on the marine environment. Topics address the most unprecedented nurdle and pyroplastic spill and subsequent oil spillage of the X-Press Pearl, causes and consequences of polluting ship disasters, novel oil pollution mitigation approaches, needfulness of post-disaster environmental assessment plans, future requirements for ecosystem restoration and environmental management of shipping, and other aspects of coastal pollution that are timely to consider unprecedented pressures, which marine environments are now subjected to.

We need to plan streamlined environmental impact assessment for the future X-Press Pearl disasters.

The X-Press Pearl disaster illustrates the urgent needs for streamlined environmental impact assessment to inform decision making. The environmental contamination caused by the disaster is complex, and the biological impact of different environmental stressors, and at different biological scales, needs to be determined. Traditional methods for analyzing complex environmental stressors are often inefficient and do not reflect the biological impact of pollution. The combination of chemical stressors and biological impacts is the key to environmental impact assessment based on integrated monitoring. Whole-cell bioreporters are tools for rapid, efficient and quantitative detection of the bioavailability, stressor effects, and toxicity of pollutants, i.e., spanning a wide range of applications. Here we propose the view that using whole-cell bioreporter technology to streamline short-term environmental impact assessment for maritime disasters such as the X-Press Pearl is more fit-for-purpose/practical than other approaches in use.

The X-Press Pearl disaster underscores gross neglect in the environmental management of shipping: Review of future data needs.

The X-Press Pearl disaster has the unfortunate hallmarks of other global container ship disasters that are becoming increasingly common and will continue to cause future environmental damage. Port container throughput is ~830 million twenty-foot equivalents (TEUs), projected to grow for the next four decades at minimum, and industry experts predict that ship sizes will continue to grow to 50,000 TEU. Misdeclared content, poor packing, inadequate stowage/lashing, ship-board fires, climate-change-driven intensity of storms, and increasing ship size all ensure increasing environmental damage from container ship maritime disasters. Industry consensus is that the number of containers lost at sea is conspicuously undercounted, and the impact of lost containers is unstudied. This paper reviews specific aspects of the container ship industry that contribute to environmental damage, and then addresses needs for risk, impact assessment and environmental management as pertains to industry operations and container ship disasters such as the X-Press Pearl.

Toxins from harmful algal blooms: How copper and iron render chalkophore a predictor of microcystin production.

Research on harmful algal blooms has focused on macronutrients, yet recent research increasingly indicates that understanding micronutrient roles is also important in the development of effective environmental management interventions. Here, we report results on metallophore production from mesocosms amended with copper and iron (enzymatic co-factors in photosynthetic electron transport) to probe questions of how cyanobacteria navigate the divide between copper nutrition, copper toxicity, and issues with iron bioavailability. These experiments utilized Microcystis, Chlorella and Desmodesmus spp., in mono- and mixed-cultures in lake water from a large, hypereutrophic lake (Taihu, China). To initiate experiments, copper and iron amendments were added to mesocosms containing algae that had been acclimated to achieve a state of copper and iron limitation. Mesocosms were analyzed over time for a range of analytes including algal growth parameters, algal assemblage progression, copper/iron concentrations and biomolecule production of chalkophore, siderophore and total microcystins. Community Trajectory Analysis and other multivariate methods were used for analysis resulting in our findings: 1) Microcystis spp. manage copper/iron requirements though a dynamically phased behavior of chalkophore/siderophore production according to their copper and iron limitation status (chalkophore correlates with Cu concentration, R2 = 0.99, and siderophore correlates with the sum of Cu and Fe concentrations, R2 = 0.98). 2) A strong correlation was observed between the production of chalkophore and the cyanobacterial toxin microcystin (R2 = 0.76)-Chalkophore is a predictor of microcystin production. 3) Based on our results and literature, we posit that Microcystis spp. produces microcystin in response to copper/iron availability to manage photosystem productivity and effect an energy-saving status. Results from this work underscore the importance of micronutrients in influencing harmful algal bloom progression and represents a major advance in understanding the ecological function for the cyanobacterial toxin microcystin as a hallmark of micronutrient limitation stress.

From speciation to toxicity: Using a "Two-in-One" whole-cell bioreporter approach to assess harmful effects of Cd and Pb.

Due to the sheer number of contaminated sites, bioavailability-based measurement and modeling of toxicity is used to triage response; despite advances, both remain relatively cumbersome. Cadmium (Cd) and lead (Pb) are two of the most toxic and globally prevalent pollutants, disproportionately impacting disadvantaged communities. Here we demonstrate the use of high throughput lights-on bioreporter technology to measure both speciation and toxicity. The organism's response is fit-for-purpose to parameterize the Biotic Ligand Model used in risk assessment of aquatic ecotoxicity and setting environmental Water Quality Criteria. Toxicity endpoints for analogous Cd and Pb models reported in literature average 71st and 44th rank-percentile sensitivity of Genus Mean Acute Values for acute toxicity (i.e., insensitive) in comparison to the bioreporter, the unique dual-mode measurement ability of which can predict toxicity endpoints from below the 5th percentile up to the 50th rank-percentile. These results are extensible to other reporters, paving the way to cost-efficient environmental risk assessment of aquatic ecotoxicity for a wide range of priority toxic pollutants.

Whole-cell biosensors for determination of bioavailable pollutants in soils and sediments: Theory and practice.

The bioavailability of pollutants is a key factor affecting environmental risk. Whole-cell bioreporters are a demonstratedly effective tool for the investigation of pollutant bioavailability in water and soil/sediment. Unlike aqueous samples, transmittance of bioreporter optical signal is reduced in direct-contact assays with soil/sediment, which affects the accuracy of bioreporter-detected pollutant bioavailability. No studies have measured the magnitude and variability of soil/sediment effects on signal in direct-contact assays or how associated uncertainties influence results. In this study, we investigate the optical effects of soil/sediment particles in suspensions on bioreporter signal transmittance and quantify how variable these optical effects are from sample-to-sample. We find that neglecting bioreporter signal diminution by soil/sediment, as many studies do, can lead to order-of-magnitude errors in results, underestimating risk. Correction based on methods in ad hoc use (e.g. comparison to signal from non-inducible reporter or use of reference soil/sediment) are also problematic for some types of experiment, and could lead to errors in excess of 30%. Our findings have a sound basis in theory, and we provide recommendations concerning the most suitable type of approach to use for different experimental settings. Generally, if best accuracy is not needed to quantify bioavailability, for samples that have been ground, sieved, and are of reasonably uniform color, it may be possible to use a single or average correction factor, particularly for experiments performed at a single slurry concentration. For investigations studying bioavailability under varying solid-phase:water ratios (e.g., sorption/desorption), detailed compensation measurements are needed for independent variables, including each specific soil/sediment sample, slurry concentration, and in some cases bioreporter signal intensity. Our measurements and calculations indicate that best results are obtained when working in the region of ballistic photon transmittance. Findings herein will be useful in areas that require information on bioavailability, such as ecotoxicology and environmental risk assessment.

Nanobiochar-rhizosphere interactions: Implications for the remediation of heavy-metal contaminated soils.

Soil heavy metal contamination has increasingly become a serious environmental issue globally, nearing crisis proportions. There is an urgent need to find environmentally friendly materials to remediate heavy-metal contaminated soils. With the continuing maturation of research on using biochar (BC) for the remediation of contaminated soil, nano-biochar (nano-BC), which is an important fraction of BC, has gradually attracted increasing attention. Compared with BC, nano-BC has unique and useful properties for soil remediation, including a high specific surface area and hydrodynamic dispersivity. The efficacy of nano-BC for immobilization of non-degradable heavy-metal contaminants in soil systems, however, is strongly affected by plant rhizosphere processes, and there is very little known about the role that nano-BC play in these processes. The rhizosphere represents a dynamically complex soil environment, which, although having a small thickness, drives potentially large materials fluxes into and out of plants, notably agricultural foodstuffs, via large diffusive gradients. This article provides a critical review of over 140 peer-reviewed papers regarding nano-BC-rhizosphere interactions and the implications for the remediation of heavy-metal contaminated soils. We conclude that, when using nano-BC to remediate heavy metal-contaminated soil, the relationship between nano-BC and rhizosphere needs to be considered. Moreover, the challenges to extending our knowledge regarding the environmental risk of using nano-BC for remediation, as well as further research needs, are identified.

Numerical approach to speciation and estimation of parameters used in modeling trace metal bioavailability.

Speciation affects trace metal bioavailability. One model used to describe the importance of speciation is the biotic ligand model (BLM), wherein the competition of inorganic and organic ligands with a biotic ligand for free-ion trace metal determines the ultimate metal availability to biota. This and similar models require natural ligand concentrations and conditional stability constants as input parameters. In concept, the BLM is itself an analogue of some analytical approaches to the determination of trace metal speciation. A notable example is competitive ligand equilibration/cathodic stripping voltammetry, which employs an artificial ligand for comparative assessment of natural ligand concentrations and discrete conditional stability constants (i.e., BLM parameters) in a natural sample. Here, we report a new numerical approach to voltammetric speciation and parameter estimation that employs multiple analytical windows and a two-step optimization process, simultaneously generating both parameters and a complete suite of corresponding species concentrations. This approach is more powerful, systematic, and flexible than those previously reported.

A new perspective of copper-iron effects on bloom-forming algae in a highly impacted environment.

Relatively little work has been done on the role of micronutrients in influencing development and progression of harmful algal blooms, yet micronutrients are ineluctably required for growth. Relatively small changes in micronutrient status have wide-ranging consequences. Here, we report results from mesocosm experiments with Microcystis and Desmodesmus spp., in mono- and mixed-cultures, to probe questions of how copper, iron, and copper-iron amendments affect growth, short-term assemblage progression, and production of siderophore, chalkophore, and microcystin in lake water from a large, hypereutrophic lake (Taihu, China). Our approach offers an entirely new perspective to understanding micronutrient dynamics in aqueous environments, as this is the first work to systematically screen for chalkophores and siderophores separately, as a function of copper/iron amendment, and using community trajectory analysis. Singular findings are summarized as follows: 1) unlike lab-based studies, in our work we observe neither dramatic copper-modulation of iron demand, nor evidence of an iron-protective effect from copper toxicity. 2) The interplay between chalkophore/siderophore production supports a concept model wherein Microcystis spp. varies behavior to individually and uniquely manage copper/iron requirements in a phased manner. In being able to specifically screen for chalkophores, we observe a previously unreported link between chalkophore and microcystin production that may relate to iron-limitation. 3) Regarding harmful algal bloom (HAB) persistance, the lake water itself influences mesocosm changes; differentiated effects for iron regarding growth indicators and/or reduction of Fe-limitation stress were found at an HAB-free field station (Xukou Bay), likely a consequence of low bioavailability of iron in this station as compared to HAB-impacted stations (half the initial dissolved iron concentration, persisting throughout experiments). The low dissolved iron accompanies more intense chalkophore/siderophore community trajectories.

Quantitative high-throughput approach to chalkophore screening in freshwaters.

There is an increasing need to study the effects of trace metal micronutrients on microorganisms in natural waters. For Fe, small Fe-binding ligands called siderophores, which are secreted from cells and bind Fe with high affinity, have been demonstrated to modulate bioavailability of this critical nutrient. Relatively little is known about secretion of strong Cu-binding ligands (chalkophores) that may help organisms navigate the divide between Cu nutrition and toxicity. A barrier to environmental chalkophore research is a lack of literature on chalkophore analysis. Here we report the development of a quantitative, high-throughput approach to chalkophore screening based on a popular competitive-ligand binding assay for siderophores wherein ligands compete for metal in a chromogenic ternary complex of chrome azurol sulfonate-metal-surfactant. We developed the assay for high-throughput analysis using a microplate reader. The method performance is slightly better than that of comparable screening approaches for siderophores. We find that levels of other metals in natural samples may be capable of causing matrix interferences (a neglected source of analytical uncertainty in siderophore screening) and that for our method this can be overcome by standard additions. In this respect the high-throughput nature of the technique is a distinct advantage. To demonstrate practical use, we tested samples from field mesocosm studies that were set up with and without Cu and Fe amendments; we find trends in results that are logical in the environmental context of our application. This approach will be useful in areas such as risk assessment for a rapid survey of metal speciation and bioavailability; investigators who perform structural studies might also benefit from this approach to rapidly screen and select samples with high Fe/Cu binding capacity for further study.

Regional-scale investigation of dissolved organic matter and lead binding in a large impacted lake with a focus on environmental risk assessment.

Environmental risk assessment (ERA) increasingly relies on speciation modeling of bioavailability. Heavy metals are the most prevalent pollutants globally, and dissolved organic matter (DOM) plays an important role in speciation and bioavailability of heavy metals. Due to the variation of DOM properties in natural aquatic systems, improvements to the standard one-size-fits-all approach to modeling metal-DOM interactions are needed for ERA. In this study, we investigate variations in DOM and lead (Pb)-DOM binding in Lake Tai (Taihu), a large, impacted lake in eastern China that is characterized by a complex drainage network and is an important water resource at a regional level, and we assess implications of our findings within the context of ERA needs. In our study, DOM in water samples collected from across the 2,400 km2 area of Taihu was characterized using three-dimensional excitation-emission matrix and synchronous fluorescence spectroscopy spectra, the latter being used to calculate conditional stability constants for metal binding. Parallel factor analysis and peak picking were used to assess contributions of protein- and humic-like components of DOM, and fluorescence indices indicative of diagenetic processes were calculated. These quantities calculated from spectroscopic studies, in addition to water quality parameters, were analyzed by bivariate and multivariate analysis. Results show that different DOM components are highly variable across different regions of Taihu, and bivariate and multivariate analyses confirm that water quality and DOM characterization parameters are strongly interrelated. This reflects the different inputs, diagenetic and transport processes across the large expanse of Taihu. We find that the conditional stability constant of Pb-DOM binding is strongly affected by the water chemical properties and composition of DOM, though the conditional stability constant is not itself a parameter that differentiates lake water properties in different regions of the lake. The variability of DOM composition and Pb-DOM binding strength across Taihu is consistent with prior findings that a one-size-fits-all approach to metal-DOM binding may lead to inaccuracies in commonly used speciation models, and therefore such generalized approaches need improvement for regional-level ERA in complex watersheds. The approach taken here to obtain site-specific metal-DOM conditional stability constants for use in increasing the accuracy of speciation modeling is fit-for-purpose for ERA applications at regional levels because the approach is relatively simple, inexpensive, and amenable to high throughput analysis.

Data on response of in situ algal phytoplankton assemblages to micronutrient treatment in small-scale mesocosms for a large hypereutrophic lake.

This article presents small-scale mesocosm data from "Effect of micronutrients on algae in different regions of Taihu, a large, spatially diverse, hypertrophic lake" [1]. The data is for limitation of the micronutrients boron (B), cobalt (Co), copper (Cu), iron (Fe), and molybdenum (Mo). Data is provided in raw form and includes data from analysis for chlorophyll-a, microscopic counts, and flow cytometry measurement from each sample collected for a total of 255 samples.

Effect of micronutrients on algae in different regions of Taihu, a large, spatially diverse, hypereutrophic lake.

Eutrophication or excessive nutrient richness is an impairment of many freshwater ecosystems and a prominent cause of harmful algal blooms. It is generally accepted that nitrogen and phosphorus nutrients are the primary causative factor, however, for systems subject to large anthropogenic perturbation, this may no longer be true, and the role of micronutrients is often overlooked. Here we report a study on Lake Tai (Taihu), a large, spatially diverse and hypereutrophic lake in China. We performed small-scale mesocosm nutrient limitation bioassays using boron, iron, cobalt, copper, molybdenum, nitrogen and phosphorus on phytoplankton communities sampled from different locations in Taihu to test the relative effects of micronutrients on in situ algal assemblages. In addition to commonly-used methods of chemical and biological analysis (including algal phytoplankton counting), we used flow cytometry coupled with data-driven analysis to monitor changes to algal assemblages. We found statistically significant effects of limitation or co-limitation for boron, cobalt, copper and iron. For copper at one location chlorophyll-a was over four times higher for amendment with copper, nitrogen and phosphorous than for the latter two alone. Since copper is often proposed as amendment for the environmental management of harmful algal blooms, this result is significant. We have three primary conclusions: first, the strong effects for Cu that we report here are mutually consistent across chlorophyll-a results, count data, and results determined from a data-driven approach to flow cytometry. Given that we cannot rule out a role for a Fe-Cu homeostatic link in causing these effects, future research into MNs and how they interact with N, P, and other MNs should be pursued to explore new interventions for effective management of HABs. Second, in view of the stimulatory effect that Cu exhibited, management of HABs with Cu as an algal biocide may not always be advisable. Third, our approach to flow cytometry offers data confirming our results from chemical and biological analysis, however also holds promise for future development as a high-throughput tool for use in understanding changes in algal assemblages. The results from this study concur with a small and emerging body of literature suggesting that the potential role of micronutrients in eutrophication requires further consideration in environmental management.

Arsenic mitigation in paddy soils by using microbial fuel cells.

Arsenic (As) behavior in paddy soils couples with the redox process of iron (Fe) minerals. When soil is flooded, Fe oxides are transformed to soluble ferrous ions by accepting the electrons from Fe reducers. This process can significantly affect the fate of As in paddy fields. In this study, we show a novel technique to manipulate the Fe redox processes in paddy soils by deploying soil microbial fuel cells (sMFC). The results showed that the sMFC bioanode can significantly decrease the release of Fe and As into soil porewater. Iron and As contents around sMFC anode were 65.0% and 47.0% of the control respectively at day 50. The observed phenomenon would be explained by a competition for organic substrate between sMFC bioanode and the iron- and arsenic-reducing bacteria in the soils. In the vicinity of bioanode, organic matter removal efficiencies were 10.3% and 14.0% higher than the control for lost on ignition carbon and total organic carbon respectively. Sequencing of the 16S rRNA genes suggested that the influence of bioanodes on bulk soil bacterial community structure was minimal. Moreover, during the experiment a maximum current and power density of 0.31 mA and 12.0 mWm-2 were obtained, respectively. This study shows a novel way to limit the release of Fe and As in soils porewater and simultaneously generate electricity.

Bioreporters: gfp versus lux revisited and single-cell response.

Genetically engineered organisms expressing spectroscopically active reporter molecules in response to chemical effectors display great potential as living transducers in sensing applications. Green fluorescent protein (gfp gene) bioreporters have distinct advantages over luminescent couterparts (lux gene), including applicability at the single-cell level, but are typically less sensitive. Here we describe a gfp-bearing bioreporter that is sensitive to naphthalene (a poorly water soluble pollutant behaving like a large class of hydrophobic compounds), is suitable for use in chemical assays and bioavailability studies, and has detection limits comparable to lux-bearing bioreporters for higher efficiency detection strategies. Simultaneously, we find that the exploitation of population response data from single-cell analysis is not an algorithmic conduit to enhanced signal detection and hence lower effector detection limits, as normally assumed. The assay reported functions to equal effect with or without biocide.

Advances in optical detection strategies for reporter signal measurements.

Many recent advances in bioreporter technology focus on challenges related to bioengineering, yet in many applications implementation of optical signal measurement is equally susceptible to improvement. For bioluminescent bioreporters, one area of effort lies in the development of semiconductor chip-based detector modules; this holds great promise for ultra-compact and field-deployable instrumentation, but has not yet had a palpable impact on improved detection limits. Regarding lower detection limits, single-molecule detection techniques have seen their first application to bioreporters, and preliminary results serve as an indication of future promise. Another technique applicable to fluorescent bioreporters is fluorescence flow cytometry, which is rapid, suitable for high-throughput screening, and lends itself to increased analytical specificity through simple algorithmic approaches to data treatment.

Improvement in CH4/CO2 ratio and CH4 yield as related to biomass mix composition during anaerobic co-digestion.

Sixteen data sets (two of which were measured in this study) with a combined total of 145 measurements of ultimate methane yield (UMY) during mono- and co-digestion of ternary biomass mixtures were used to assess impact of co-digestion on the relative change in UMY (ΔUMY) as a function of biomass mix composition. The data involved 9 biomass materials (brewery spent grains, chicken manure, cow manure, fresh grass clippings, pig manure, primary sewage sludge, vegetable food waste, wheat straw, and rice straw). Results of the assessment shows that co-digestion in 85% of yields positive values of ΔUMY regardless of the biomass materials used, however, a smaller fraction (15%) resulted in negative ΔUMY during co-digestion. The data further indicate that for each set of ternary biomass material mixtures there exists an optimal biomass mix composition at which ΔUMY is at a maximum. Statistical analyses based on the data used here indicate that the maximum value of ΔUMY (ΔUMYmax) is always positive regardless of biomass materials being co-digested.