Understanding the Chemical Cocktail Effect: Definition and Implications
Xenobiotics never appear as single, isolated substances in the environment but instead as multi-component mixtures. These mixtures, often referred to as "chemical cocktails," can have complex and unpredictable effects on ecosystems and human health. Understanding the ecotoxicology of these mixtures is crucial, yet our knowledge remains limited.
The Anthropocene has typically been characterized by an acceleration of climatic, biological, and geochemical signatures of human activity preserved in the geologic record beginning in the mid 20th century. In the Anthropocene, watershed chemical transport is increasingly dominated by novel combinations elements, which are hydrologically linked together as ‘chemical cocktails.’ Chemical cocktails are novel because human activities greatly enhance elemental concentrations and their probability for biogeochemical interactions and shared transport along hydrologic flowpaths.
What is a Chemical Cocktail?
Chemical cocktails are novel combinations of elements linked together due to human activities, these activities enhance elemental concentrations and their probability for biogeochemical interactions and shared transport along hydrologic flowpaths.
Urbanization contributes to the formation of novel elemental combinations and signatures in terrestrial and aquatic watersheds, also known as ‘chemical cocktails.’ The composition of chemical cocktails evolves across space and time due to:
- Elevated concentrations from anthropogenic sources
- Accelerated weathering and corrosion of the built environment
- Increased drainage density and intensification of urban water conveyance systems
- Enhanced rates of geochemical transformations due to changes in temperature, ionic strength, pH, and redox potentials
A new chemical cocktail approach advances our ability to: trace contaminant mixtures in watersheds, develop chemical proxies with high-resolution sensor data, and manage multiple water quality problems.
Conceptual model illustrating how groups of elements can be hydrologically linked as ‘chemical cocktails’ and transported along fluvial networks of the Anthropocene.
Emerging Micropollutants: Pesticides and Pharmaceuticals
Among the emerging micropollutants (EMPs), pesticides and pharmaceutical residues stand out for their widespread occurrence and highly diverse biological effects. The annual global use of pesticides is estimated to be around 3 million tons. Concurrently, projections indicate that spending on medicines will reach USD 1.6 trillion and 3335 billion doses in 2024. This highlights the substantial impact of these chemicals on the environment and underscores the need for comprehensive understanding and management of their presence and effects.
Pesticides are predominantly released into the environment through agricultural and horticultural applications. They can leach into deeper soil layers due to precipitation and ultimately enter groundwater or surface waters through agricultural run-off. There is an extensive body of literature, including thousands of studies, documenting the environmental occurrence of pesticide substances. These substances have been detected in every environmental compartment, from Antarctica to the Arctic, and even in rainwater.
Continuously used and released from wastewater treatment plants, pharmaceuticals and pesticides are considered to be pseudo-persistent contaminants. Once they enter the environment, they can persist in their original form or in structurally similar transformation products. The number and concentration of pesticides and their concentrations peak during and after the excessive agricultural application of said compounds in late spring and summer in water bodies.
The main pathways for active pharmaceutical ingredients (APIs) entering surface waters are through domestic wastewater, mainly due to inadequate removal of micropollutants during wastewater treatment processes. In recent years, increasing attention has been given to the monitoring of biologically active chemical residues in our environment. On a global scale and within the EU, 771 and 596 APIs have been detected in environmental matrices between 2010 and 2016, respectively.
Case Study: Cytotoxicity of Common Micropollutants
In a recent study, three active pharmaceutical ingredients (carbamazepine, diclofenac, and ibuprofen) and three pesticides (S-metolachlor, terbuthylazine, and tebuconazole) from the most frequently detected emerging micropollutants were examined for their acute cytotoxicity.
The objective of this study was to assess the acute cytotoxicity of the most frequently detected pesticides and pharmaceuticals, namely, metolachlor, tebuconazole, terbuthylazine, carbamazepine, diclofenac, and ibuprofen. We sought to examine their individual impact as well as the mixture effects of their binary, ternary, quaternary, quinary, and senary mixtures using the acute Aliivibrio fischeri assay. The synergistic, additive, and antagonistic effects between the chemicals in different mixtures at various effective concentrations were determined using the combination index (CI) method. Furthermore, we aimed to define the role of each compound in the cocktail effects using statistical analytical methods.
Materials and Methods
For toxicity experiments, 20 mg/mL carbamazepine, diclofenac-sodium, ibuprofen, S-metolachlor, tebuconazole, and terbuthylazine stock solutions were prepared in dimethyl sulfoxide (DMSO). For the mixtures, stock solutions containing the active ingredients were mixed in the same proportion (1:1 ratio) (from binary to senary).
To determine the acute cytotoxicity of pesticides, APIs, and their mixtures, a standard Microtox® acute assay was performed using the bioluminescence Aliivibrio fischeri (AVF) test organism. A decrease in light emission due to any negative changes in the metabolic status of the cells is easily detectable, and the results obtained are highly reproducible.
Key Findings
- Statistical analysis revealed a synergistic effect of diclofenac and carbamazepine, both individually and in combination within the mixtures.
- Diclofenac also exhibited synergy with S-metolachlor and when mixed with ibuprofen and S-metolachlor.
- S-metolachlor, whether alone or paired with ibuprofen or diclofenac, increased the toxicity at lower effective concentrations in the mixtures.
- Non-toxic terbuthylazine showed great toxicity-enhancing ability, especially at low concentrations.
- Several combinations displayed synergistic effects at environmentally relevant concentrations.
Combination Index (CI) Method
Synergistic, additive, and antagonistic effects for the combinations were characterized by combination index (CI) values at inhibition rates in the bioluminescence of 10%, 20%, 50%, 80%, and 95% (EC10, EC20, EC50, EC80, EC95, respectively). The CI values were calculated using the following equation, as described by Chou and Yang et al. The effects of mixtures were classified according to Chou and Talalay as synergistic if CI < 1, additive (concentration addition) if CI = 1, and antagonistic if CI > 1. To determine synergism, additive effect, or antagonism, 6 concentration-response data points (EC10, EC20, EC50, EC80, EC90, and EC95) were used for the combinations, consisting of 2, 3, 4, 5, and 6 compounds.
The type and intensity of interaction between chemical components are frequently expressed by combination indices (CIs) ranging from zero (extremely strong synergy) to positive infinity (extremely strong antagonism), where values close to one denote additivity.
Concentrations are expressed in mg/L resulting in 10, 20, 50, 80, 90, and 95% bioluminescence inhibition in Aliivibrio fischeri after 30 min of exposure. The effective concentrations resulting in bioluminescence inhibition in Aliivibrio fischeri varied over an extremely large range. Among the APIs, the NSAID ibuprofen and diclofenac had similar cytotoxic effects at lower concentrations; however, diclofenac showed higher toxicity with an increase in concentrations. Carbamazepine had significantly lower toxic effects at 50% effective concentration and above. Among pesticides, tebuconazole induced the highest inhibitions, while terbuthylazine, as described in our previous work , was non-toxic at any applied concentrations (up to its solubility limit).
Implications and Future Directions
Characterizing chemical cocktails and underlying geochemical processes is necessary for:
- Tracking pollution sources using complex chemical mixtures instead of individual elements or compounds.
- Developing new strategies for co-managing groups of contaminants.
- Identifying proxies for predicting transport of chemical mixtures using continuous sensor data.
- Determining whether interactive effects of chemical cocktails produce ecosystem-scale impacts greater than the sum of individual chemical stressors.
A watershed chemical cocktail approach significantly expands evaluations of water-quality signatures and impacts beyond single elements to mixtures.
Conceptual model illustrating how reactive chemical cocktails vary in formation and transport along the drying-rewetting cycle with water table, pre vs. post precipitation conditions, and soil redox conditions.
Overall, the currently available workflow for the analysis of mixture toxicity with QSAR is insufficient and limited.