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Pyrethroids are a major insecticide class, suitable for biomonitoring in humans. Due to similarities in structure and metabolic pathways, urinary metabolites are common to various active substances. A tiered approach is proposed for risk assessment. Tier I was a conservative screening for overall pyrethroid exposure, based on phenoxybenzoic acid metabolites. Subsequently, probabilistic approaches and more specific metabolites were used for refining the risk estimates. Exposure was based on 95th percentiles from HBM4EU aligned studies (2014–2021) covering children in Belgium, Cyprus, France, Israel, Slovenia, and The Netherlands and adults in France, Germany, Israel, and Switzerland. In all children populations, the 95th percentiles for 3-phenoxybenzoic acid (3-PBA) exceeded the screening value. The probabilistic refinement quantified the risk level of the most exposed population (Belgium) at 2% or between 1–0.1% depending on the assumptions. In the substance specific assessments, the 95th percentiles of urinary concentrations in the aligned studies were well below the respective human biomonitoring guidance values (HBM-GVs). Both information sets were combined for refining the combined risk. Overall, the HBM data suggest a low health concern, at population level, related to pyrethroid exposure for the populations covered by the studies, even though a potential risk for highly exposed children cannot be completely excluded. The proposed tiered approach, including a screening step and several refinement options, seems to be a promising tool of scientific and regulatory value in future.

a b s t r a c tBioassays constitute a tool for pollution analysis providing a holistic approach and high-quality indication of the toxicity. Microbioassays allow evaluating the toxicity of many samples, implying lower costs and enabling routine monitoring and pollution control. But tests conducted so far are limited to the use of a small number of taxa. Lichens are excellent bioindicators of pollution with great ecological significance. Studies show that the phycobiont is more sensitive to pollutants than the mycobiont. Phycobiont have features such as adaptation to anhydrobiosis and relatively rapid growth in vitro, making them suitable for microbioassays. Our aim is to determine the sensitivity of phycobionts to the pharmaceutical micropollutants carbamazepine and diclofenac as a preliminary step for the development of a toxicity microbioassay based on phycobionts. Optical dispersion and chlorophyll autofluorescence were used as endpoints of toxicity on two algal species showing that suspensions present cyclic and taxon specific patterns of aggregation. Trebouxia TR9 suspensions present a very high grade of aggregation while Asterochloris erici cells do not. Both micropollutants alter optical properties of the suspensions of both species. No significant alteration of chlorophyll autofluorescence by carbamazepine is observed. A. erici chlorophyll autofluorescence is extremely sensitive to diclofenac but the effect is not dependent on the drug concentration or on the time of exposure. Differently, TR9 only shows punctual chlorophyll alterations. Fluctuations in optical dispersion may indicate changes in the population structure of the species, including reproductive strategy. A. erici seems more sensitive to micropollutants, is better characterized and is available from commercial collections.