Understanding the Cocktail Effect of Pesticides: Definition and Environmental Impact
Xenobiotics never appear as single, isolated substances in the environment but instead as multi-component mixtures. However, our understanding of the ecotoxicology of mixtures is far from sufficient.
Each year, a significant proportion of the approximately 2.3 billion tons of synthetic chemicals used globally, and 300 million tons used in the European Union, ultimately enters natural waters, which form the basis of our drinking water. Approximately one-fifth of the world’s population lacks access to safe drinking water of sufficient quality.
Although the chemicals released into the environment, in the majority of cases, are present at very low concentrations (µg/L or lower), their persistent presence represents long-term exposure to the elements of the ecosystem.
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 and will reach USD 1.6 trillion and 3335 billion doses (DDD-defined daily dose, the dose defined by the WHO for a given active substance) 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.
In the European Union, terbuthylazine (TRB) has emerged as one of the most frequently detected herbicides, replacing the banned atrazine. Terbuthylazine is commonly used in combination with metolachlor (MTC), which is also frequently identified as a residue. Tebuconazole (TBZ), a triazole fungicide, has gained increasing importance, mainly applied to cereals and grapes. In 2022, it was ranked as the fifth most widely marketed pesticide in Hungary, following metolachlor in the third position and terbuthylazine in the fourth position.
These compounds frequently occur together in environmental matrices, mainly in surface and ground waters. Terbuthylazine is most commonly used in combination with S-metolachlor in the EU to control broad-leaved weeds and annual grasses on both agricultural and non-agricultural soils.
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. Čelić et al. (2021) reported the co-occurrence of carbamazepine, metolachlor, tebuconazole, and terbuthylazine in all of the samples taken from the Ebro River. Xu et al. (2019) detected carbamazepine (0.02-4.34 ng/L), ibuprofen (0.70-22.91 ng/L), and tebuconazole (0.58-50.04 ng/L) in surface watersheds. Ibuprofen, diclofenac, metolachlor, and tebuconazole have been detected in Lake Guaíba. S-metolachlor, tebuconazole, and terbuthylazine have been detected in Lake Balaton and its sub-catchment area.
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.
Sampling locations in Lake Balaton
The Ecotoxicological Effects of Mixtures
The investigation of the ecotoxicological effects of mixtures is an arduous task, even though numerous studies on mixture toxicity have been published in recent years. In 2007, Belden et al. reviewed the results of 303 experiments from 45 publications on the cocktail effects of pesticides. In 2014, Cedergreen reviewed 194 two-component pesticide mixtures, 21 metal ion mixtures, and 136 antifoulant mixtures. Martin reported in his 2023 review that there were 761 different publications on mixture toxicology between 2007 and 2017.
The vast number of possible mixtures makes it nearly impossible to assess every mixture experimentally. Therefore, modeling can be a key approach to assessing the toxic properties of mixtures. However, current modeling methods also have their limitations in practical application.
Nowadays, there are two widely used and prominent reference models, concentration addition (CA) and independent action (IA), for predicting the combined effect of chemical mixtures; however, they are only suitable for the additive effect of mixtures. CA assumes that mixture components have the same or similar mode of action (MOA), whereas IA assumes they have a different or dissimilar MOA. According to Cedergreen et al. [39], the use of IA and CA to predict the cocktail effect of binary mixtures resulted in approximately 20% (of 158 mixtures), and 10% accuracy.
The Chou-Talalay method (combination index method) is one of the most widely used methods for detecting and quantifying synergistic interactions between two or more chemicals, having been cited over 7000 times over the past few decades. Considering that the chemicals can form a practically infinite number of combinations, quantitative structure-activity relationship (QSAR) models have been extensively used in forecasting not only the activity of single chemicals but the combined effects of components in mixtures.
Still, most of the QSAR models are feasible only for binary combinations and additive toxicities of mixtures. The QSAR models are limited by the accuracy of the dataset used to train them. The data available are typically composed of single or few experimental values, which may capture complex biological systems poorly.
Overall, the currently available workflow for the analysis of mixture toxicity with QSAR is insufficient and limited.
Study on Acute Cytotoxicity of Pesticides and Pharmaceuticals
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.
In this 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, both individually and in combination, by bioluminescence inhibition in Aliivibrio fischeri (NRRL B-11177). Synergy, additive effects, and antagonism on cytotoxicity were determined using the combination index (CI) method. Additionally, PERMANOVA was performed to reveal the roles of these chemicals in binary, ternary, quaternary, quinary, and senary mixtures influencing the joint effects.
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.
Materials and Methods
For toxicity experiments, 20 mg/mL carbamazepine (Supelco®, Budapest, Hungary, CAS 298-46-4, purity ≥ 99%), diclofenac-sodium (Supelco®, Budapest, Hungary, CAS 15307-79-6, purity ≥ 98.5%), ibuprofen (Sigma-Aldrich®, Budapest, Hungary, CAS 15687-27-1, purity ≥ 98%), S-metolachlor (Pestanal®, CAS 87392-12-9, purity 98.4%), tebuconazole (Supelco®, Budapest, Hungary, CAS 107534-96-3, neat), and terbuthylazine (Pestanal®,Budapest, Hungary, CAS 5915-41-3, purity 99.4%) stock solutions were prepared in dimethyl sulfoxide (DMSO, CAS 67-68-5, purity ≥ 99.99%, Fisher Scientific, Budapest, Hungary). 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) (DSM-7151, NRRL B-11177) 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. A total of 20 mg/mL stock solutions of carbamazepine (CBZ), diclofenac (DCF), ibuprofen (IBU), S-metolachlor (MTC), tebuconazole (TBZ), and terbuthylazine (TRB) were used in the acute assay, diluted from 100 mg/L to 6.25 mg/L in a 2 w/w% NaCl solution containing 1 v/v% DMSO. From binary to senary mixtures, chemicals were combined in equal concentrations and diluted from 200 mg/L to 12.5 mg/L.
The final concentration of DMSO was 0.5 v/v% in the assay, which is non-toxic to the test organism as described in Tóth et al. (2019) [53], and also did not result in any aberrance in bioluminescence in the negative control after 30 min of exposure, according to the ISO standard. Tests were performed in two parallels with a control and nine different concentrations of the chemicals at 15 ± 0.2 °C. The relative bioluminescence was detected by the Microtox® Model 500 Analyzer (SDI, Carlsbad, California) after 30 min of incubation, and bioluminescence inhibition was determined. For each compound and mixture, the effective concentration values resulting in 10, 20, 50, 80, and 95% inhibition in the bioluminescence (EC10, 20, 50, 80, 95) were calculated from the concentration-response curves using the MicrotoxOmni® software (version 1.1, AZUR Environmental Corp., Carlsbad, California, USA).
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 equation described by Chou and Yang et al. The effects of mixtures were classified according to Chou and Talalay [56] 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. Euclidean distances were calculated between the 26 samples using the transformed CI values at effect sizes of 10%, 20%, 50%, 80%, 90%, and 95%. In any case, where the cytotoxic effect was not detectable, meaning one-sided simple enhancement or potentiation [62], the enhancement of the non-toxic chemical in a mixture was expressed as a percent of the required dose (mg/L) change of the other compounds in the mixture not containing the non-toxic chemical to result in the same effect size as the mixture containing it.
Acute Cytotoxicity Results
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. 1: carbamazepine, 2: diclofenac, 3: ibuprofen, 4: S-metolachlor, 5: tebuconazole, 6: terbuthylazine. n.t.-non-toxic at the applied concentration. 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 [53], was non-toxic at any applied concentrations (up to its solubility limit).
At 50% effective concentrations, the binary DCF + IBU, IBU + TRB, and ternary CBZ + DCF + IBU mixtures were the most toxic cocktails, with 12, 18, and 12 mg/L EC50 values, respectively. The effective concentration values eliciting 10% inhibition in bioluminescence altered between 2 and 10 mg/L in most cases, but there were cocktails that could cause 10% inhibition only at high...
Visual representation of Synergistic and Antagonistic Effects