26 Jul 2021

MixTox I and II: Field Evaluation of Real-World Mixtures and Regulatory Approaches to Real-World Mixtures

Arnd Weyers, Bayer AG Crop Science; Beate Escher, Helmholtz Centre for Environmental Research; Marion Junghans, Swiss Centre for Applied Ecotoxicology Eawag; Thomas Backhaus, University of Gothenburg; and Leo Posthuma, RIVM Centre for Sustainability, Environment and Health

Mixtures have been recognized as relevant for environmental risk of chemicals, last but not least, by the current European Green Deal, which aims at improving mixture protection and management, such that protective policies protect against mixture exposures and impacts and curative ones help priority setting of programs of measures to mitigate existing pollution.

In the session “MixTox (I): Field Evaluation of Real-World Mixtures” at SETAC Europe 2021, the diversity of environmental mixtures was explored from different angles by ten oral presentations and three posters. Milo de Baat, KWR, opened the session with an overview presentation where he asked if we systematically underestimate the risk of micropollutants in the environment. His answer is “yes,” as he illustrated conceptually and with some case studies. The limitations of component-based mixture assessment can only be overcome by a combination of bioassays with non-target analysis. Preliminary associations indicated that the number of features in non-target analysis appears to be a predictor of the overall mixture effects. Several field studies supported a similar view: Sarah Stinson, UC Davis, demonstrated that effect on mortality in invertebrates and on behavior of fish and daphnids occurred for sites with high exposure to several substances.

Mixtures were explicitly addressed in monitoring programs that combined passive sampling with bioassays and chemical analysis as shown on a large scale for the Danube river by Zuzana Tousova, RECETOX, and on the small scale in agricultural ditches by Harry Boonstra, Wetterskip Fryslân. Passive sampler extracts were also investigated by Valentin Dupraz, EPOC, with non-target analysis to identify mixture risk drivers. It came as an initial surprise – which could be later rationalized – that it was not herbicides that were the risk drivers for algal toxicity, as in many previous studies, but non-herbicides, namely spiroxamine and clarithromycin that were the main risk drivers. Ron van der Oost, Waternet, expanded on his previous work in the SIMONI (smart integrated monitoring) strategy and showed how a smart combination of chemical analysis, bioassays and caged fish experiments even allows him to map effects onto key events within fish adverse outcome pathways with a single common key event, which is increased metabolic demands, identified. Mixtures are different wherever we look: two interesting presentations by Silvain Slaby and Francois le Cor, University de Lorraine, looked at the effects of the mixtures of pesticides and transformation products in fish ponds and how the ponds could actually improve water quality by natural attenuation.

One thing we need to keep in mind when we are monitoring mixtures in the environment are that the toxicokinetics in the organisms living in these aquatic environments might be highly dependent on system properties and chemical properties as demonstrated on uptake experiments with caged gammarids as shown by Benedikt Lauper, Eawag.

While the focus on the session was on field studies, there was also one relevant contribution by Silvain Bart, University of York, who presented methods for including synergistic and antagonistic effects in toxicokinetic-toxicodynamic modeling frameworks. They included variables that described dose-dependent degrees of over-additive effects, which were lower at low concentrations. Defined mixture experiments using passive dosing addressed the cytotoxicity of polycyclic aromatic hydrocarbons (PAHs) using the chemical activity concept (Bianca Jalvo, ACES) and the genotoxicity of PAH mixtures were tested in cell lines by Beatrix Matos, University of Lisboa.

The Zoom discussion session was well attended, and discussions elaborated on various aspects. Participants largely agreed that more integrative water bodies, such as larger rivers, have generally lower risk and positive trends in chemical status over the last decades, but more mixture responses driven by many chemicals. In contrast, smaller water bodies, such as ditches and small creeks, often encounter specific risk drivers and more temporal and spatial variability in exposure. Certain pesticides in a stream known for high exposure potentially may go up to very high levels for just a couple of hours in the study presented by Benedikt Lauper, Eawag. Temporal variability in exposure could be addressed through high-resolution monitoring and modeling taking into account known emission patterns and passive sampling techniques. Klara Hilscherova, RECETOX, also pointed out that the question of number of risk drivers also depends on specific versus nonspecific responses. Bioassays that respond to specific modes of action, such as inhibition of photosynthesis, often have a few highly potent mixture effect drivers, whereas for more general apical effects many more if not all chemicals contributed, albeit with different potencies. Whether we should focus on risk drivers or not was heatedly debated. Some argued that by focusing on risk drivers we incur the risk of losing unknown mixture effect drivers that have not been measured. Others felt the focus on known drivers was the only way forward to implementing mixtures into risk assessment and management. Ron van der Oost, Waternet, pointed out that the ecological status is often well predicted by effect-based method, but with respect to chemical status, you can have an overestimation of true risk (if just one chemical exceeds the EQS) or an underestimation (if there are many relevant mixture drivers that have no EQS). On the other hand, effect-based methods are not predictors of ecological effects but provide just a picture of the chemical contribution to ecological effects. A consensus was reached on the topic of synergy and antagonism, which is very interesting and relevant for some pesticide formulations, but plays a minor role for the combined effects of thousands of chemicals at environmentally low concentrations in complex environmental mixtures. That brought the discussion to parasites and diseases as confounding stressors that may lead to synergistic effects as put forward by Nina Cedergreen, University of Copenhagen. Sebastian Buchinger, BfG, pointed out that two-thirds of the ecological impact is caused by non-chemical stressors. Even when it comes to chemical stressors our focus has largely been on organics, but as Olukayode Jegede, Wageningen University, pointed out, metal mixtures also play a role. They pose a great challenge, though, because they might be essential at low concentrations, but become toxic only at high concentrations and because EQS are often close to natural background levels.

In the subsequent session, “MixTox (II): Regulatory Approaches to Real-World Mixtures,” the existing and novel insights in field evidence of real-world mixtures were evaluated in view of practice: if and how can those insights be used practicably such as to protect and/or restore environmental quality against chemical pollution? These questions were addressed by six pre-recorded oral and five e-poster presentations, together sketching an overview of the occurrence and diversity of mixture problems up to (inter)national approaches to effectively bridge the science-practice gap. Bridging that gap is key, as lectures in both sessions provided evidence for current exposures and effects, with high spatial and temporal variability in mixture compositions and associated pressure levels. A key question in view of the Chemical Strategy for Sustainability became evident: we apparently can characterize the “distance to target” to the toxic-free environment aspiration, but how can we minimize the distance in practice? The presentations concerned three cross-cutting themes:

  1. The knowns and the unknowns
  2. Effect-based methods for holistic assessments, and
  3. Needs for updated guidance

Under theme 1, Leo Posthuma, RIVM, presented a nationwide mixture study for the Netherlands, showing spatio-temporal variability in mixture toxic pressure for the Netherlands. Jessy Krier, University of Luxemburg, showed via suspect screening that the diversity of mixtures is vast, and that many compounds are present but not evaluated. As management priorities must be set in practice, Ismael Rodea Palomares, Bayer, presented that detected currently regulated chemicals do not contribute significantly to observed risk and legacy chemicals often act as ‘risk drivers,’ and – in the same vein – Emily Shaw, Michigan Technological University, showed that habitat characteristics influence ‘risk drivers’ of bioaccumulating compounds  to fish. Nele Markert, LANUV, expanded the mixture assessment with multiple non-chemical stressor insights, illustrating that those also count in determining impacts on ecological status. Although insights under Theme 1 thus expand vastly, Theme 2 adds information, by considering effect-based methods (EBMs). Presentations of Yves Marneff, ISSeP, and Beate Escher, UFZ, showed the power and relevance of EBMs, by showing clear evidence for improved insights in estrogenic activity patterns with EBMs as compared to chemical measurements, and by providing regulatory-relevant trigger values (effect-based trigger values), respectively. Moreover, Thomas Webster, Boston University, provided a further modeling tool to analyze data according to the concentration addition model, which is currently the operational method to address mixtures in practice. Thus, theme 2 shows a broad and meaningful contribution of science to move from the ‘known compounds’ of Theme 1 to a more holistic and meaningful assessment, also covering ‘unknowns.’ Theme 3 considered the final step to practice: useful guidance. Kunihiko Yamazaki, Ministry of the Environment, proposed an improvement to the World Health Organization’s International Classification for Patient Safety framework for mixture assessments. Anja Gladbach, Bayer, proposed a more efficient approach to assess plant protection products assessments, by using the risk quotients already calculated in the risk assessment, rather than apply complex additional calculations. And finally, Anne Gosselin, ECCC, described a large-scale plan to address mixtures in Canadian policies and practices. Together, the presentations showed that the diversity of mixture situations in the field had not hampered scholars to provide practically useful methods, as well as outlooks and plans to improve in the future.

The Zoom discussion of this session was well attended, with discussion focusing on five overarching questions, and trialing to conclude practicable summary conclusions. Regarding the handling of the spatio-temporal variability in the field (differences between bigger and smaller rivers) and how to use this insight remained open. Regarding the finding that there are frequently few chemicals acting as key ‘risk drivers’ this needs to be considered in chemicals assessment and management. Another major question was whether EBM-approaches are ready for regulatory implementation and how they can be used to calibrate (verify) component-based approaches. The question whether we need different approaches for prospective and retrospective risk assessment (the latter for management) was addressed. A last question was considering monitoring: do we monitor the right chemicals, to capture field mixture diversity?

In summary, vast progress towards practicable mixture approaches has been made, which is key to support the EU-Green Deal aspirations. There is a need to move forward, given the evidence for exposure and effects of real-life mixtures, there is ample room to expand on the practicable approaches, and there a policy need to adopt those. Given the Green Deal aspirations, there is a need to increase efforts in understanding real-life mixtures, impacts of non-chemical stressors and to develop knowledge-based protection and restoration approaches.

Authors’ contact information: arnd.weyers@bayer.com, beate.escher@ufz.de, marion.junghans@oekotoxzentrum.ch, thomas.backhaus@bioenv.gu.se and Leo.posthuma@rivm.nl

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