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Chemometers for in situ risk assessment of mixtures of pollutants

Periodic Reporting for period 2 - CHEMO-RISK (Chemometers for in situ risk assessment of mixtures of pollutants)

Reporting period: 2018-11-01 to 2020-04-30

In the current reporting period, the CHEMO-RISK team addressed developing ”chemometer” passive samplers for the transfer of mixtures of environmental pollutants from different environmental media (sediment, water, biota of various trophic levels) and body compartments (blubber, brain, liver, kidney and blood of mammals) to the laboratory for their characterization. A special focus of the ”chemometer” concept is on comparability across media which is achieved by using silicone as the common sampling phase for all types of samples. The pollutants partition into the silicone according to their activity in the sample until both phases reach thermodynamic equilibrium. Then, the silicone is retrieved, its surface is thoroughly cleaned and the chemicals are solvent-extracted for chemical analysis or bioanalytical screening. The data (equilibrium partitioning concentrations in silicone, Csilicone⇌medium) can then be compared directly across media, e.g. to assess biota/sediment accumulation of various pollutants.

The issue of mixture risk characterization is of huge importance for society which on the one hand largely relies on multiple chemicals to provide or secure certain service functions (e.g. food, clean water, transport, medication), while on the other hand striving for a healthy, non-toxic environment for humans and ecosystems. Fulfilling both requirements calls for sound assessment of the hazards and risks arising from the presence of complex mixtures of chemicals in the multimedia environment covering a multitude of different ecosystems and environmental conditions. However, current risk assessment relies on a small number of individual, so-called ”indicator” chemicals being monitored and their effect testing in few indicator species, which implies uncertainty in terms of potential cocktail effects of multiple chemicals and does not cover diverse ecosystems where these chemicals may, e.g. be bioavailable to different degrees.

CHEMO-RISK aims for a novel scientifically sound chemical risk assessment paradigm that integrates exposure and effect assessment of a broad range of chemicals into a single procedure and provides information relevant to ecosystem and human health. The overall objectives are to answer the following questions: Which processes drive the enrichment of pollutants in aquatic biota on a thermodynamic basis (subproject A)? How do pollutants distribute within an organism, and which effects do they elicit at the key target sites (subproject B)? Can we apply everyday-life items such as silicone plasters to replace invasive sampling of blood in human health risk assessment (subproject C)? To which degree can pattern analysis of chemometer extracts help identify emerging chemicals and explain the observed toxicity profiles across media (subproject D)? The focus of these subprojects and their specific progress is described in detail in the following sections.
In the first 30 months of CHEMO-RISK, all four subprojects have been tackled, but are in different stages of completion as described below.

Subproject (A) Thermodynamic assessment of bioaccumulation in the aquatic environment: We developed methods for passive equilibrium sampling in lean biota tissues. A comprehensive sampling campaign was carried out in a Swedish lake that is characterized by lack of sources of pollutants other than the atmosphere and being shallow, which means there is no stratification throughout the year, ensuring mixed conditions across the water column. We sampled several individuals of a diverse set of fish species from Lake Angen close to Nyköping (pike, pikeperch, perch, eel, roach, bream), two species of mussels and crayfish, with silicone sheets. Furthermore, we collected sediment across the lake and sampled pollutants in water using large-volume solid-phase extraction (LV-SPE) and two different silicone-based passive sampler formats. The extraction and analysis is ongoing, and new insights are expected regarding bioconcentration (enrichment from the abiotic environment) and biomagnification (enrichment via the food chain). The „chemometer“ approach allows direct comparison of Csilicone⇌medium, a novel approach to assess bioaccumulation.

(B) For the assessment of internal exposure and effects in marine mammals, initial experiments tackling silicone-based passive equilibrium sampling in different tissues of a pig have been carried out. These experiments followed an automated approach, with stirring of slurries of the homogenized tissue, spiked with a test set of chemicals with widely varying physicochemical properties, and with small amounts of water added as needed to allow stirring. The tissues and body fluids that were investigated included blubber, liver, kidney, brain and blood. Even after up to 9 days of stirring, the system proved not to be at equilibrium yet, requiring further optimization of the experimental setup, in particular for marine mammal tissues. In addition, a comprehensive set of experiments was carried out to characterize the influence of co-dosed lipids on the response of cell-based reporter gene bioassays. These data served to set up a model that helps correct data for the reduced bioavailability in the bioassay system. For translation of the concentrations in the silicone ”chemometer” (Csilicone) into lipid-based concentrations common in monitoring and bioaccumulation assessment, lipid/silicone partition constants (Klipid/silicone) are being determined, covering a multitude of diverse chemicals. Upcoming experiments with automated in tissue sampling will further refine the ”chemometer” approach for future studies.

(C) The subproject ”Non-invasive assessment of human exposure” started only recently, in April, 2019. Progress has been made regarding the development of a novel silicone ”chemometer” passive sampling device, a plaster, to be applied on human skin to non-invasively determine human exposure to mixtures of pollutants. An initial experiment tested the transfer of a diverse set of chemicals from a pollutant-loaded ”donor” silicone sheet to a clean ”acceptor” silicone sheet, either in direct contact or separated by different wound dressing materials. Another focus of these initial experiments was to assess whether the uptake into the silicone occurs mainly from the skin or the overlying air. To tackle this issue, we applied activated carbon as an isolating layer, shielding the silicone from the one or the other side, respectively. A pilot study investigated different times of exposure of the silicone plasters on the thigh between 1 and 5 days for comparison with the rather well established silicone wristbands and partly blood. For chemical analysis, a separate method using thermodesorption and gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS) was established for a set of target compounds known to be relevant from literature studies. Future work aims to collect 500 extensive data sets of the participants a large cohort study.

(D) In terms of widening the pollutant domain of chemical analysis, this subproject has to date largely focused on (i) establishing a multi-target method for 160 chemicals and (ii) development of a clean-up method for silicone-based extracts of the ”chemometer” samples since direct injection of samples has been shown to severely contaminate the gas chromatography high-resolution mass spectrometry (GC/HRMS) system, impeding analysis of multiple extracts. The multi-target method has been applied to ”chemometer” extracts of sediment samples collected from four areas worldwide, which had been characterized toxicologically in a battery of cell-based reporter gene in vitro bioassays. The manuscript describing the coupling of using the chemical analytical data to explain the observed effects is under development. Pattern analysis in ”chemometer” extracts of sediment and diverse fish species covering different trophic levels from a contaminated site is scheduled to explore pattern analysis across compartments. Upcoming work includes the development of a method that allows for the challenging analysis of polychlorinated and polybrominated dibenzodioxins and furans in liver samples, and pattern analysis in multimedia „chemometer“ extracts generated in CHEMO-RISK subprojects A and B.
The first reporting period focused on establishing new methods that allow for:

(i) multimedia sampling using „chemometers“ based on silicone, and for the water phase also including other polymeric materials as well as large-volume solid-phase extraction of a broad range of compounds present in the dissolved phase;

(ii) undisturbed chemical analysis of multiple pollutants with largely varying physico-chemical properties in „chemometer“ extracts in a targeted way, with pattern analysis being optimized;

(iii) method and model development to improve understanding of the role of co-dosed lipids from in tissue „chemometer“ samplers in cell-based reporter gene bioassays.

Multimedia sampling covers aquatic ecosystems (sediment and biota irrespective of their lipid content, subproject A, with the particularly challenging water phase still under optimization) and currently addresses optimization of „chemometers“ to be used in different tissues and body fluids of mammals (subproject B) and in a non-invasive way on human skin (subproject C). The methods for different compartments of marine mammals are expected to be available soon and hence ready for application in a range of studies outlined above.

One of the first achievements was to establish a multi-chemical method to accurately determine 160 organic pollutants with a wide range of physico-chemical properties, covering legacy pollutants such as PCBs and polyaromatic hydrocarbons (PAHs), but also emerging compounds such as musk fragrances and UV stabilizers. Clean-up has shown to be a vital step to allow for chemical analysis using the GC/HRMS multi-target method without severely contaminating the analytical system after few injections, and the related manuscript is under development. The multi-target method is already being applied in a large range of studies, and will be complemented by a targeted dioxin/furan method for ultra-trace chemicals that are of high relevance due to their potencies to elicit toxicological effects.

Multimedia „chemometer“ extracts have in the meantime been generated and are ready for the extension of the pattern analysis approach from using the multi-target method to screening for unknown peaks in the GC/HRMS chromatograms. We will attempt to identify selected unknown peaks with interesting patterns, e.g. peaks that increase with trophic level (which is an indication of biomagnifying compounds) or peaks that decrease with trophic level (chemicals that might have been metabolized). This approach will be optimized using silicone-based „chemometer“ extracts of sediment and fish from a contaminated site, where it can be established for those pollutants known to be present that either are very persistent or metabolizable, and it can then be extended to unknown peaks to identify if they show relevant patterns.

When CHEMO-RISK nears completion, we expect to have achieved the following results:

(A) Thermodynamic bioaccumulation studies completed in two diverse aquatic ecosystems: (i) in a Swedish background lake and (ii) in a contaminated river. Ongoing work in the German Mulde river evaluated other media than silicone which may be suitable for „chemometer“ sampling of a more diverse set of pollutants.

(B) Assessment of internal exposure in the most relevant tissues and body fluids of marine mammals completed, covering either a temporal trend study, a spatial trend study, an interspecies trend study or, if sufficient sample material can be obtained, a mother/calf study.
C) Non-invasive human exposure study completed, comparing the newly developed „chemometer“ samplers with established formats (silicone wristbands) and assessing their prediction power of the blood levels of a range of participants of a cohort study.

(D) Chemical analytical tools for comprehensive „chemometer“ studies optimized and validated for multimedia und multi-target chemical application, so-called „iceberg“ studies completed that link the chemical analytical data in „chemometer“ extracts with observed effects, and pattern analysis in diverse „chemometer“ extracts completed, identifying, e.g. biomagnifying or metabolizable new compounds.