Risk Assessment Of Mixtures Research Articles

Neurotoxic Effects of Mixtures of Perfluoroalkyl Substances (PFAS) at Environmental and Human Blood Concentrations.

Per- and polyfluoroalkyl substances (PFAS) may cause various deleterious health effects. Epidemiological studies have demonstrated associations between PFAS exposure and adverse neurodevelopmental outcomes. The cytotoxicity, neurotoxicity, and mitochondrial toxicity of up to 12 PFAS including perfluoroalkyl carboxylates, perfluoroalkyl sulfonates, 6:2 fluorotelomer sulfonic acid (6:2 FTSA), and hexafluoropropylene oxide-dimer acid (HPFO-DA) were tested at concentrations typically observed in the environment (e.g., wastewater, biosolids) and in human blood using high-throughput in vitro assays. The cytotoxicity of all individual PFAS was classified as baseline toxicity, for which prediction models based on partition constants of PFAS between biomembrane lipids and water exist. No inhibition of the mitochondrial membrane potential and activation of oxidative stress response were observed below the cytotoxic concentrations of any PFAS tested. All mixture components and the designed mixtures inhibited the neurite outgrowth in differentiated neuronal cells derived from the SH-SY5Y cell line at concentrations around or below cytotoxicity. All designed mixtures acted according to concentration addition at low effect and concentration levels for cytotoxicity and neurotoxicity. The mixture effects were predictable from the experimental single compounds' concentration-response curves. These findings have important implications for the mixture risk assessment of PFAS.

S01-01 Risk assessment of complex chemical mixtures – an overview of approaches and recent developments

S01-01 Risk assessment of complex chemical mixtures – an overview of approaches and recent developments

Mixture effect of parental exposure to triazophos and fenvalerate on the early development of zebrafish offspring

Triazophos (TRI) and fenvalerate (FEN) have been extensively used in the world and frequently coexist in the water environments, might pose health risk to aquatic species. However, investigations of their mixture toxic effects on offspring after parental exposure have been neglected, especially for aquatic vertebrates such fish. To address this knowledge gap, parental zebrafish (F0 generation) were exposed to TRI, FEN and their mixture for 60 days, as well as the embryos (F1 generation) were hatched without or with continued corresponding exposures at the same concentrations until 7 days post fertilization. The results exhibited that exposure to TRI and FEN altered the expression levels of biomarkers associated with several biological processes, such as apoptosis and inflammatory response. Compared to individual exposure in the F1 generation, the co-exposure to TRI and FEN resulted in increased the expression of T4 and cc-chem mRNA and decreased the expression of ROS, trα, il-8, and gpx mRNA when the F0 generation was similarly exposed. These results revealed that the co-exposure to TRI and FEN has detrimental effects on fish progeny following parental exposure, even if the progeny are not directly exposed to the pesticides, and such negative effects may be intensified if the offspring continue to be exposed. This study enhances the understanding of the harmful impacts of parental exposure to the pesticide mixture on descendants and holds implications for the ecological risk assessment of pesticide mixtures in aquatic vertebrates. Further mechanistic studies are necessary to gain a deeper insight into the mixture effects of pesticides and other kinds of pollutants on subsequent offspring following parental exposure.

S13-03 Case studies showing the applicability of the mixture risk assessment strategy using HBM data

S13-03 Case studies showing the applicability of the mixture risk assessment strategy using HBM data

S13-01 Strategy for real-life mixture risk assessment using HBM data

S13-01 Strategy for real-life mixture risk assessment using HBM data

S01-04 Advancing mixture risk assessment from a regulatory point of view

S01-04 Advancing mixture risk assessment from a regulatory point of view

Mixture toxic mechanism of phoxim and prochloraz in the hook snout carp Opsariichthysbidens

Pesticides are usually found as mixtures in surface water bodies, even though their regulation in aquatic ecosystems is usually approached individually. In this context, this work aimed to investigate the enzymatic- and transcriptional-level responses after the mixture exposure of phoxim (PHX) and prochloraz (PRC) in the livers of hook snout carp Opsariichthys bidens. These data exhibited that co-exposure to PHX and PRC induced an acute synergistic impact on O. bidens. The activities of catalase (CAT), superoxide dismutase (SOD), carboxylesterase (CarE), and caspase3 varied significantly in most of the individual and combined challenges relative to basal values, indicating the activation of oxidative stress, detoxification dysfunction, as well as cell apoptosis. Besides, the transcriptional levels of five genes (gst, erα, mn-sod, cxcl-c1c, and il-8) exhibited more pronounced changes when subjected to combined pesticide exposure in contrast to the corresponding individual compounds. The findings revealed the manifestation of endocrine dysfunction and immune disruption. These results underscored the potential biochemical and molecular toxicity posed by the combination of PHX and PRC to O. bidens, thereby contributing to a deeper comprehension of the ecological toxicity of pesticide mixtures on aquatic organisms. Importantly, the concurrent presence of PHX and PRC might exacerbate hepatocellular damage in hook snout carps, potentially attributable to their synergistic toxic interactions. This study underscored the toxicological potency inherent in the co-occurrence of PHX and PRC in influencing fish development, thereby offering valuable insights for the risk assessment of pesticide mixtures and the safeguarding of aquatic organisms.

An innovative mixture sampling strategy with uniform design: Application to global sensitivity analysis of mixture toxicity

Global sensitivity analysis combined with quantitative high-throughput screening (GSA-qHTS) uses random starting points of the trajectories in mixture design, which may lead to potential contingency and a lack of representativeness. Moreover, a scenario in which all factor levels were at stimulatory effects was not considered, thereby hindering a comprehensive understanding of GSA-qHTS. Accordingly, this study innovatively introduced an optimised experimental design, uniform design (UD), to generate non-random and representative sample points with smaller uniformity deviation as starting points of multiple trajectories. By combining UD with the previously optimised one-factor-at-a-time (OAT) method, a novel mixture design method was developed (UD-OAT). The single toxicity tests showed that three pyridinium and five imidazolium ionic liquids (ILs) exerted stimulatory effects on Vibrio qinghaiensis sp.-Q67; thus, four stimulatory effective concentrations of each IL were selected as factor levels. The UD-OAT generated 108 mixture samples with equal frequency and without repetition. High-throughput microplate toxicity analysis revealed that all 108 mixtures exhibited inhibitory effects. Among these, type B mixtures exhibited increasing toxicities that subsequently decreased, unlike type C mixtures, which consistently increased over time. GSA successfully identified three of the eight ILs as important factors influencing the toxicities of the mixtures. When individual ILs produced stimulatory effects, mixtures containing two to three ILs exhibited either stimulatory effects or none. In contrast, mixtures containing five to eight ILs exhibited inhibitory effects, while those containing four ILs showed a transition from stimulatory to inhibitory effects. This study provides a novel mixture design method for studying mixture toxicity and fills the application gap of GSA-qHTS. The phenomenon of individuals being beneficial while mixtures can be harmful challenges traditional mixture risk assessments.

Classification and regression machine learning models for predicting the combined toxicity and interactions of antibiotics and fungicides mixtures

Antibiotics and triazole fungicides coexist in varying concentrations in natural aquatic environments, resulting in complex mixtures. These mixtures can potentially affect aquatic ecosystems. Accurately distinguishing synergistic and antagonistic mixtures and predicting mixture toxicity are crucial for effective mixture risk assessment. We tested the toxicities of 75 binary mixtures of antibiotics and fungicides against Auxenochlorella pyrenoidosa. Both regression and classification models for these mixtures were developed using machine learning models: random forest (RF), k-nearest neighbors (KNN), and kernel k-nearest neighbors (KKNN). The KKNN model emerged as the best regression model with high values of determination coefficient (R2 = 0.977), explained variance in prediction leave-one-out (Q2LOO = 0.894), and explained variance in external prediction (Q2F1 = 0.929, Q2F2 = 0.929, and Q2F3 = 0.923). The RF model, the leading classifier, exhibited high accuracy (accuracy = 1 for the training set and 0.905 for the test set) in distinguishing the synergistic and antagonistic mixtures. These results provide crucial value for the risk assessment of mixtures.

Synergistic risk in the gut and liver: Insights into the toxic mechanisms and molecular interactions of combined exposure to triazophos and fenvalerate in zebrafish

The simultaneous or sequential application of pesticides such as triazophos (TRI) and fenvalerate (FEN) in agriculture results in their residues co-existing in the environments. However, the impact of co-exposure to TRI and FEN on the gut-liver axis, along with the underlying mechanisms, remains unclear. Our results showed that exposure to FEN (96 h-LC50 value of 0.096 mg a.i. L−1) was more toxic to adult zebrafish compared to TRI (96 h-LC50 value of 6.75 mg a.i. L−1). Furthermore, the study aimed to reveal the toxic potencies of individual and combined exposure to TRI and FEN on the liver-gut axis in zebrafish (Danio rerio). Our results also indicated that pesticide exposure decreased tight junction molecule expression and increased intestinal inflammatory molecule expression in D. rerio, with co-exposure demonstrating enhanced toxicity. Co-exposure altered gut flora structure and species abundance. RNA-Seq sequencing revealed changes in liver gene expressions, particularly enrichment of P53 signaling. Molecular docking demonstrated FEN's stronger binding to P53 and Caspase3, correlating with its higher toxicity. Liver pathology confirmed exacerbated liver damage by individual and co-exposures, with co-exposure inducing more severe liver injury. qPCR results showed increased pro-apoptotic gene expression and decreased anti-apoptotic gene expression, with co-exposure exhibiting an interactive effect. Overall, this study identifies specific targets and pathways influenced by these pesticides, revealing toxicity mechanisms involving the gut-liver axis, which is crucial for environmental risk assessment of pesticide mixtures.

A review of Health Canada’s progress on human biomonitoring-based risk assessments and the path forward

Since the launch of the Chemicals Management Plan (CMP) in 2006, Health Canada has initiated screening-level risk assessments (RAs) of approximately 4300 priority substances under the Canadian Environmental Protection Act, 1999 (CEPA). With the availability of nationally representative human biomonitoring (HBM) data, over 300 of these substances were assessed using HBM-based RA approaches. Qualitative and quantitative HBM-based RA approaches for the regulatory risk assessment of the general population of Canada were developed to increase the efficiency of screening the potential health risk of CMP priority substances. To support HBM-based RAs, several biomonitoring equivalents (BE) were derived to interpret HBM data. For some CMP substances, Health Canada conducted cumulative risk assessments of chemical mixtures using HBM data as measures of exposure. In 2023, CEPA was amended to include the assessment of populations who may be disproportionately impacted (vulnerable populations) and the cumulative effects of multiple chemicals. Going forward, Health Canada is exploring modern approaches in HBM-based RAs, including biomarkers of effect and non-traditional biomarkers (e.g., hair, nails) to address CEPA amendments. This manuscript will discuss Health Canada’s progress in HBM-based RAs, and the possible path forward in using HBM data to strengthen human health risk assessments.

Co-exposure to cadmium and triazophos induces variations at enzymatic and transcriptional levels in Opsariichthys bidens

Heavy metals and pesticides are significant pollutants in aquatic environments, often leading to combined pollution and exerting toxic effects on aquatic organisms. With the rapid growth of modern industry and agriculture, heavy metal cadmium (Cd) and pesticide triazophos (TRI) are frequently detected together in various water bodies, particularly in agricultural watersheds. However, the combined toxic mechanisms of these pollutants on fish remain poorly understood. This experiment involved a 21-day co-exposure of Cd and TRI to the hook snout carp Opsariichthys bidens to investigate the toxic effects on liver tissues at both enzymatic and transcriptional levels. Biochemical analysis revealed that both individual and combined exposures significantly increased the content or activity of caspase-3 (CASP-3) and malondialdehyde (MDA). Moreover, the impact on these parameters was greater in the combined exposure groups compared to the corresponding individual exposure groups. These findings suggested that both individual and combined exposures could induce mitochondrial dysfunction and lipid peroxidation damage, with combined exposure exacerbating the toxicological effects of each individual pollutant. Furthermore, at the molecular level, both individual and combined exposures upregulated the expression levels of cu-sod, cat, and erβ, while downregulating the expression of il-1. Similar to the patterns observed in the biochemical parameters, the combined exposure group exhibited a greater impact on the expression of these genes compared to the individual exposure groups. These results indicated that exposure to Cd, TRI, and their combination induced oxidative stress, endocrine disruption, and immunosuppression in fish livers, with more severe effects observed in the combined exposure group. Overall, the interaction between Cd and TRI appeared to be synergistic, shedding light on the toxic mechanisms by which fish livers responded to these pollutants. These findings contributed to the understanding of mixture risk assessment of pollutants and were valuable for the conservation of aquatic resources.

Deep Learning Bridged Bioactivity, Structure, and GC-HRMS-Readable Evidence to Decipher Nontarget Toxicants in Sediments.

Identifying causative toxicants in mixtures is critical, but this task is challenging when mixtures contain multiple chemical classes. Effect-based methods are used to complement chemical analyses to identify toxicants, yet conventional bioassays typically rely on an apical and/or single endpoint, providing limited diagnostic potential to guide chemical prioritization. We proposed an event-driven taxonomy framework for mixture risk assessment that relied on high-throughput screening bioassays and toxicant identification integrated by deep learning. In this work, the framework was evaluated using chemical mixtures in sediments eliciting aryl-hydrocarbon receptor activation and oxidative stress response. Mixture prediction using target analysis explained <10% of observed sediment bioactivity. To identify additional contaminants, two deep learning models were developed to predict fingerprints of a pool of bioactive substances (event driver fingerprint, EDFP) and convert these candidates to MS-readable information (event driver ion, EDION) for nontarget analysis. Two libraries with 121 and 118 fingerprints were established, and 247 bioactive compounds were identified at confidence level 2 or 3 in sediment extract using GC-qToF-MS. Among them, 12 toxicants were analytically confirmed using reference standards. Collectively, we present a "bioactivity-signature-toxicant" strategy to deconvolute mixtures and to connect patchy data sets and guide nontarget analysis for diverse chemicals that elicit the same bioactivity.

Developmental toxicity and potential mechanisms exposed to polystyrene microplastics and polybrominated diphenyl ethers during early life stages of fat greenling (Hexagrammos otakii)

The occurrence of microplastics (MPs) in aquatic ecosystems and their ability to absorb hydrophobic pollutants, such as persistent organic pollutants (POPs), is currently a significant concern. MPs, which are the main breakdown product of plastics, have been frequently detected in the environment, posing serious threats to organisms’ health. One particular pollutant, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), is a dominant congener of PBDEs and is highly toxic to organisms. However, there is limited knowledge regarding the exposure of marine fishes to PBDEs through MPs and their combined toxic effects. In this study, the embryo toxicity of Hexagrammos otakii was conducted to investigate the combined effects of MPs and BDE-47. The results showed that MPs and BDE-47 co-exposure had detrimental effects on embryonic development, such as reduced hatchability, increased mortality, decreased heart rate, and body malformation. Moreover, the combined toxicity of these substances appeared more pronounced harmful effects compared to exposure to BDE-47 alone. Histopathological examination revealed that co-exposure can cause greater damage to hatching glands and yolk. The enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included phagosome, metabolism of xenobiotics by cytochrome P450, TCA cycle, and Wnt signaling pathway, which are closely related to embryonic growth. BDE-47 and MPs may activate the Wnt signaling pathway to affect the normal development of embryos. Our results suggest that MPs and BDE-47 exposure may cause growth disorders in the early life stages of H.otakii, leading to abnormal embryonic development. All these results will contribute to the further study of the ecological risk assessment and toxicity of MPs and organic pollutant mixtures in marine fish.

The use of effect biomarkers in chemical mixtures risk assessment – Are they still important?

Human epidemiological studies with biomarkers of effect play an invaluable role in identifying health effects with chemical exposures and in disease prevention. Effect biomarkers that measure genetic damage are potent tools to address the carcinogenic and/or mutagenic potential of chemical exposures, increasing confidence in regulatory risk assessment decision-making processes. The micronucleus (MN) test is recognized as one of the most successful and reliable assays to assess genotoxic events, which are associated with exposures that may cause cancer. To move towards the next generation risk assessment is crucial to establish bridges between standard approaches, new approach methodologies (NAMs) and tools for increase the mechanistically-based biological plausibility in human studies, such as the adverse outcome pathways (AOPs) framework. This paper aims to highlight the still active role of MN as biomarker of effect in the evolution and applicability of new methods and approaches in human risk assessment, with the positive consequence, that the new methods provide a deeper knowledge of the mechanistically-based biology of these endpoints.

The ConSoil project: An integrated framework for monitoring plant protection product residues in agricultural soil

AbstractPlant Protection Products (PPPs) are widely used to maintain high productivity and protect crops, but can have unintended toxic effects on beneficial non‐target soil organisms. To avoid unacceptable adverse effects of PPPs on soil organisms, a prospective risk assessment is carried out, which focuses on individual substances and their effects on a few individual species or groups. However, the reality of agricultural soils consists of complex networks of organisms exposed to mixtures of several PPP active substances. It is therefore essential to monitor PPP residues in soils. This paper describes the ConSoil project and its proposed framework for monitoring PPP residues in Swiss Agricultural soils which includes and integrates (1) risk‐based reference values for PPP residues in soil and (2) indicators of their effects on long‐term soil fertility. For risk‐based reference values, a proposal has been developed to derive Soil Guideline Values (SGVs) for PPP residues and a mixture risk assessment concept is being developed. Regarding indicators, a toolbox of ecological and ecotoxicological indicators will be proposed to reflect the protection goal of long‐term soil fertility in agricultural soils. For this objective, standardised and/or well‐established bioindicator methods will be selected for the key soil organisms that support soil fertility. To integrate SGVs and the biomonitoring toolbox, an adapted TRIAD approach is proposed, where generic SGVs are used as a screening tool to identify monitoring sites potentially at risk and to trigger more detailed monitoring. Detailed monitoring will refine the SGVs based on site‐specific characteristics and implement the bioindicator toolbox to measure the effects of PPP residues and their risk to long‐term soil fertility. As a novel integrated framework, it is essential to use the data generated in detailed assessments to calibrate and refine the SGVs and bioindicator tools and improve the monitoring over time.

Evaluation of a Proportional Response Addition Approach to Mixture Risk Assessment and Predictive Toxicology Using Data on Four Trihalomethanes from the U.S. EPA's Multiple-Purpose Design Study.

In this study, proportional response addition (Prop-RA), a model for predicting response from chemical mixture exposure, is demonstrated and evaluated by statistically analyzing data on all possible binary combinations of the four regulated trihalomethanes (THMs). These THMs were the subject of a multipurpose toxicology study specifically designed to evaluate Prop-RA. The experimental design used a set of doses common to all components and mixtures, providing hepatotoxicity data on the four single THMs and the binary combinations. In Prop-RA, the contribution of each component to mixture toxicity is proportional to its fraction in the mixture based on its response at the total mixture dose. The primary analysis consisted of 160 evaluations. Statistically significant departures from the Prop-RA prediction were found for seven evaluations, with three predications that were greater than and four that were less than the predicted response; interaction magnitudes (n-fold difference in response vs. prediction) ranged from 1.3 to 1.4 for the former and 2.6 to 3.8 for the latter. These predictions support the idea that Prop-RA works best with chemicals where the effective dose ranges overlap. Prop-RA does not assume the similarity of toxic action or independence, but it can be applied to a mixture of components that affect the same organ/system, with perhaps unknown toxic modes of action.

Potential health risk assessment of mixtures of heavy metals in drinking water

Despite the potential to cause chronic adverse effects, heavy metals screenings are not typically as frequent or exhaustive as those for bacterial contamination. Additionally, there is a pressing need for collectively assessing heavy metals as a mixture rather than individual allowed limits. We tested a hypothesis that comprehensive methodical wide screening for heavy metals can inform the magnitude of potential exposures and aid in classifying heavy metals collectively as hazardous rather than individual level comparisons. A total of 297 samples were taken by proportional cluster sampling from different households in Irbid governorate, depending on population density in each district. We analyzed the samples using Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS). Our analysis looked at individual levels of heavy metal clustering in different water sources and regions and calculated a heavy metals pollution index (HPI) in five different types of drinking water: harvested rainwater, bottled water, spring water, water shop, and tap water. Pb and Fe were the main contaminants in drinking water, especially tap and harvested rainwater. High amounts of essential metals were found in tap, harvested rain, and spring waters compared to water shop and bottled water. HPI was above critical value in 29 samples, all of which were harvested rainwater samples, and most of which came from Wasateya and Taiba regions. Spring and tap water had similar heavy metal patterns. This experimental approach provides a guide for clustering water sources in their heavy metal patterns and for classification of heavy metals as mixtures rather than individual limits; it also fills crucial gaps in prioritization of heavy metal testing according to water type and metal correlations. Furthermore, this study presents the first data on drinking water quality in Jordan to be published in over a decade.

Embryonic exposures to cadmium and PAHs cause long-term and interacting neurobehavioral effects in zebrafish

Developmental exposure to either polycyclic aromatic hydrocarbons (PAHs) or heavy metals has been shown to cause persisting and overlapping neurobehavioral effects in animal models. However, interactions between these compounds have not been well characterized, despite their co-occurrence in a variety of environmental media. In two companion studies, we examined the effects of developmental exposure to cadmium (Cd) with or without co-exposure to prototypic PAHs benzo[a]pyrene (BaP, Exp. 1) or fluoranthene (FA, Exp. 2) using a developing zebrafish model. Zebrafish embryos were exposed to Cd (0–0.3 μM), BaP (0–3 μM), FA (0–1.0 μM), or binary Cd-PAH mixtures from 5 to 122 h post fertilization (hpf). In Exp. 1, Cd and BaP produced independent effects on an array of outcomes and interacting effects on specific outcomes. Notably, Cd-induced deficits in dark-induced locomotor stimulation were attenuated by BaP co-exposure in the larval motility test and BaP-induced hyperactivity was attenuated by Cd co-exposure in the adolescent novel tank test. Likewise, in Exp. 2, Cd and FA produced both independent and interacting effects. FA-induced increases on adult post-tap activity in the tap startle test were attenuated by co-exposure with Cd. On the predator avoidance test, FA- and 0.3 μM Cd-induced hyperactivity effects were attenuated by their co-exposure. Taken together, these data indicate that while the effects of Cd and these representative PAHs on zebrafish behavior were largely independent of one another, binary mixtures can produce sub-additive effects for some neurobehavioral outcomes and at certain ages. This research emphasizes the need for detailed risk assessments of mixtures containing contaminants of differing classes, and for clarity on the mechanisms which allow cross-class toxicant interactions to occur.

Baseline Toxicity Model to Identify the Specific and Nonspecific Effects of Per- and Polyfluoroalkyl Substances in Cell-Based Bioassays.

High-throughput screening is a strategy to identify potential adverse outcome pathways (AOP) for thousands of per- and polyfluoroalkyl substances (PFAS) if the specific effects can be distinguished from nonspecific effects. We hypothesize that baseline toxicity may serve as a reference to determine the specificity of the cell responses. Baseline toxicity is the minimum (cyto)toxicity caused by the accumulation of chemicals in cell membranes, which disturbs their structure and function. A mass balance model linking the critical membrane concentration for baseline toxicity to nominal (i.e., dosed) concentrations of PFAS in cell-based bioassays yielded separate baseline toxicity prediction models for anionic and neutral PFAS, which were based on liposome-water distribution ratios as the sole model descriptors. The specificity of cell responses to 30 PFAS on six target effects (activation of peroxisome proliferator-activated receptor (PPAR) gamma, aryl hydrocarbon receptor, oxidative stress response, and neurotoxicity in own experiments, and literature data for activation of several PPARs and the estrogen receptor) were assessed by comparing effective concentrations to predicted baseline toxic concentrations. HFPO-DA, HFPO-DA-AS, and PFMOAA showed high specificity on PPARs, which provides information on key events in AOPs relevant to PFAS. However, PFAS were of low specificity in the other experimentally evaluated assays and others from the literature. Even if PFAS are not highly specific for certain defined targets but disturb many toxicity pathways with low potency, such effects are toxicologically relevant, especially for hydrophobic PFAS and because PFAS are highly persistent and cause chronic effects. This implicates a heightened need for the risk assessment of PFAS mixtures because nonspecific effects behave concentration-additive in mixtures.