Key messages: Human biomonitoring surveys provide information on European residents’ exposure to mixtures of chemicals. Human biomonitoring data indicate a potential health risk from exposure to a combination of 15 substances (or groups of substances). Seven of these substances drive the mixture risk: bisphenol A, the perfluorinated chemicals PFOS and PFOA, arsenic, cadmium and two phthalates (monobutyl phthalate and monoisobutyl phthalate). Monitoring results from two periods indicate a decrease in the overall human health risks in the EU.

Risk of a mixture of 15 substances prioritised for human biomonitoring in the European population in the periods 2007–2014 and 2014–2021

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Unintended mixtures of chemicals do not currently need to be assessed under EU legislation. However, the Chemicals Strategy for Sustainability does emphasise that the effects of chemical mixtures need to be considered across policy domains. Monitoring chemicals in humans unlocks broad insights on the cumulative effect of exposure from all sources. Such insights are independent of exposure pathways (whether via e.g. clothing and other consumer articles, diet, the environment) and policy specific domains.

Several studies investigated the presence of certain chemicals in the general European population between 2007 and 2021. The 15 chemicals under investigation were selected based on their EU policy relevance and human health concerns linked to exposure (based on e.g. information on hazardous properties, exposure characteristics and public concern). The results were then used to calculate the risk of chemical mixtures posed to humans in Europe with a comparison of two time periods: 2007-2014 and 2014-2021.

The risk is calculated as the sum of risk quotients i.e., ratios of the internal body exposure levels measured in the European population and established health-based guidance values (HBGVs).

Comparing the results from the two time periods helps inform whether EU chemical policy is effective in reducing the overall risks to human health from the combination of 15 prioritised substances. However, concluding on the likelihood of specific adverse effects from exposure to the mixture requires higher tier mixture risk assessments.

Results from the two periods indicate that health risks associated with the combined exposure cannot be ruled out. Seven of the 15 substances are the main drivers of the risk: bisphenol A, the perfluorinated chemicals PFOS (perfluorooctane sulfonic acid) and PFOA (perfluorooctanoic acid), arsenic, cadmium and two phthalates (monobutyl phthalate and monoisobutyl phthalate). Bisphenol A, in particular, is responsible for 97.8% to 98.5% of the risk from the studied mixture.

The results also indicate a decreasing trend over time, suggesting that policy interventions designed to reduce the exposure and risks associated with chemicals prioritised for human biomonitoring in the EU might have been successful. For example, the use of PFOS has been restricted since 2010, while PFOA has been restricted since 2020.

However, care should be taken when interpreting the results since there may have been differences in sampling designs (e.g. in terms of representativeness of sample populations across EU countries). Further analysis revealed that differences in the age categories of participating subjects have a minimal effect on the overall result. A clearer interpretation of trends will become apparent in the future if European human biomonitoring data are further harmonised.

To meet policy targets, people’s exposure to identified risk drivers needs to be further decreased. At the same time, it is essential that people are not exposed to emerging chemicals of concern which would add to the existing toxic pressure. Future human biomonitoring will be essential in tracking long-term exposure trends for previously and currently used chemicals like per- and polyfluoroalkyl substances (PFAS). At the same time, it will allow emerging chemicals of concern to be detected in e.g. cases of regrettable substitutions (i.e. where harmful chemicals are substituted with others which have hazardous properties similar to those they replace).

The European Human Biomonitoring Initiative (HBM4EU) collected and harmonised previous human biomonitoring studies into a dataset called HBM4EU. It aggregated and generated new studies collected in the HBM4EU-aligned dataset, both available on IPCHEM. The datasets were used to extract data to populate the two reference periods 2007–2014 and 2014–2021. Data from unrepresentative populations, for example contamination hotspots, clinical and occupational studies, were excluded.

A total of 15 substances (or groups of substances) were identified to calculate the risk of chemical mixture. This includes biomarkers that have been measured in both reference periods and for which internal reference values exist.

The risk is calculated as the sum of risk quotients, which correspond to the ratios between the country population-weighted mean exposure levels at the 50th percentile, measured in the general European population (teenagers, adults and the elderly), and established internal HBGVs (see Table below). The sum of risk quotients is based on the conservative assumption of concentration addition, i.e. where substances in the same mixture are assumed to have a joint adverse effect. The calculation of the risk is done independently of the modes of action underpinning HBGVs. This means that the used HBGVs may originate from studies of different types of adverse effects. Data with values reported as below the limit of detection or limit of quantification were not considered in the calculations.

HBGVs were derived from epidemiological or toxicological studies as described in Apel et al. (2020). A complete list of values is provided in the Table below.

The reported values should be interpreted cautiously due to differences in the sampling designs and analytical methods within the different studies, uncertainties around HBGVs, and the limited temporal range and resolution of human biomonitoring data.

The approach builds on the study by Socianu et al. (2022). Detailed documentation of the methodology and the R code is in preparation.

Health-based guidance values (HBGVs) used in the calculation of the risk of chemical mixture



Metabolite biomarker

Substance abbreviation




Arsenic, inorganic

Inorganic arsenic (As(iii) + As(v)), monomethylated arsenic (MMA), dymethylated arsenic (DMA)


6.4 ug/L


Hays et al. 2010

Perfluorooctanoic acid


2 ug/L


Hölzer et al. 2021

Perfluorooctanesulfonic acid


5 ug/L


Hölzer et al. 2021



1 ug/L


Apel et al. 2017

Di-iso-butyl phthalate

mono-isobutyl phthalate (MiBP)


230 ug/L


Lange et al. 2021

Di-n-butyl phthalate

mono-n-butyl phthalate (MnBP)


190 ug/L


Lange et al. 2021

Di(2-ethylhexyl) phthalate

sum of mono(2-ethylhexyl) phthalate, mono(2-ethyl-5-hydroxy- hexyl) phthalate, mono(2-ethyl-5-oxo-hexyl) phthalate and mono(2-ethyl-5-carboxy-pentyl) phthalate

DEHP ∑(4 met)

1000 ug/L


Aylward et al. 2009

Di-isononyl phthalate

sum of 7-carboxy-(mono-methyl- heptyl) phthalate (cx-MiNP) and 7-OH-(mono-methyl-octyl) phthalate (OH-MiNP)

DiNP ∑(2 met)

1400 ug/L


Hays et al. 2011

Di(2-propylheptyl) phthalate

6-oh-mono-propyl-heptyl phthalate (OH-MiDP)


220 ug/L


Lange et al. 2021

Diethyl phthalate

mono-ethyl phthalate (MEP)


18000 ug/L


Aylward et al. 2009

Butylbenzyl phthalate

mono-benzyl phthalate (MBzP)


3000 ug/L


Lange et al. 2021

Bisphenol A


0.0115 ug/L


Ougier et al. 2021

Pyrethroid insecticides

3-phenoxybenzoic acid (3-PBA)


87 ug/L


Aylward et al. 2018


3,5,6-trichloro-2-pyridinol (TCPγ)


2100 ug/L


Arnold et al. 2015

Benzophenone 3


340 ug/L crt


Rousselle et al. 2022

References and footnotes

  1. Luijten, M., et al., 2023, ‘Mixture risk assessment and human biomonitoring: Lessons learnt from HBM4EU’, International Journal of Hygiene and Environmental Health 249, p. 114135 (DOI: 10.1016/j.ijheh.2023.114135).
  2. Apel, P., et al., 2020, ‘Human biomonitoring initiative (HBM4EU) - Strategy to derive human biomonitoring guidance values (HBM-GVs) for health risk assessment’, International Journal of Hygiene and Environmental Health 230, p. 113622 (DOI: 10.1016/j.ijheh.2020.113622).
  3. Socianu, S., et al., 2022, ‘Chemical Mixtures in the EU Population: Composition and Potential Risks’, International Journal of Environmental Research and Public Health 19(10), p. 6121
  4. Hays, S. M. et al., 2010, ‘Biomonitoring equivalents for inorganic arsenic’, Regulatory Toxicology and Pharmacology 58(1), pp. 1–9.
  5. Hölzer, J. et al., 2021, ‘Human Biomonitoring (HBM)-I values for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) – Description, derivation and discussion’, Regulatory Toxicology and Pharmacology 121, p. 104862.
    a b
  6. Apel, P. et al., 2017, ‘New HBM values for emerging substances, inventory of reference and HBM values in force, and working principles of the German Human Biomonitoring Commission’, International Journal of Hygiene and Environmental Health 220(2 Pt A), pp. 52–166.
  7. Lange R, Apel P, Rousselle C, Charles S, Sissoko F, Kolossa-Gehring M, Ougier E. The European Human Biomonitoring Initiative (HBM4EU): Human biomonitoring guidance values for selected phthalates and a substitute plasticizer. Int J Hyg Environ Health. 2021 May;234:113722. doi: 10.1016/j.ijheh.2021.113722. Epub 2021 Mar 9. PMID: 33711757.
    a b c d
  8. Aylward LL, Hays SM, Gagné M, Krishnan K. Derivation of Biomonitoring Equivalents for di(2-ethylhexyl)phthalate (CAS No. 117-81-7). Regul Toxicol Pharmacol. 2009 Dec;55(3):249-58. doi: 10.1016/j.yrtph.2009.09.001. Epub 2009 Sep 12. PMID: 19751789.
  9. Hays SM, Aylward LL, Kirman CR, Krishnan K, Nong A. Biomonitoring equivalents for di-isononyl phthalate (DINP). Regul Toxicol Pharmacol. 2011 Jul;60(2):181-8. doi: 10.1016/j.yrtph.2011.03.013. Epub 2011 Apr 3. PMID: 21466828.
  10. Aylward LL, Hays SM, Gagné M, Krishnan K. Derivation of Biomonitoring Equivalents for di-n-butyl phthalate (DBP), benzylbutyl phthalate (BzBP), and diethyl phthalate (DEP). Regul Toxicol Pharmacol. 2009 Dec;55(3):259-67. doi: 10.1016/j.yrtph.2009.09.003. Epub 2009 Sep 12. PMID: 19751787.
  11. EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP), et al., 2023, ‘Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs’, EFSA Journal 21(4), p. e06857 (DOI: 10.2903/j.efsa.2023.6857).
  12. Original HBGV from Ougier et al. 2021 was adapted by dividing proportionally to the decrease from the 2015 provisional TDI of 4 μg kg/bw/day to the most recent TDI of 0.0002 μg kg/bw/day (EFSA, 2023).
  13. Ougier E, Zeman F, Antignac JP, Rousselle C, Lange R, Kolossa-Gehring M, Apel P. Human biomonitoring initiative (HBM4EU): Human biomonitoring guidance values (HBM-GVs) derived for bisphenol A. Environ Int. 2021 Sep;154:106563. doi: 10.1016/j.envint.2021.106563. Epub 2021 Apr 23. PMID: 33894553.
  14. Aylward LL, Irwin K, St-Amand A, Nong A, Hays SM. Screening-level Biomonitoring Equivalents for tiered interpretation of urinary 3-phenoxybenzoic acid (3-PBA) in a risk assessment context. Regul Toxicol Pharmacol. 2018 Feb;92:29-38. doi: 10.1016/j.yrtph.2017.11.002. Epub 2017 Nov 4. PMID: 29113940.
  15. Arnold SM, Morriss A, Velovitch J, Juberg D, Burns CJ, Bartels M, Aggarwal M, Poet T, Hays S, Price P. Derivation of human Biomonitoring Guidance Values for chlorpyrifos using a physiologically based pharmacokinetic and pharmacodynamic model of cholinesterase inhibition. Regul Toxicol Pharmacol. 2015 Mar;71(2):235-43. doi: 10.1016/j.yrtph.2014.12.013. Epub 2014 Dec 25. PMID: 25543108.
  16. Rousselle C, Meslin M, Berman T, Woutersen M, Bil W, Wildeman J, Chaudhry Q. Using Human Biomonitoring Data to Support Risk Assessment of Cosmetic Ingredients-A Case Study of Benzophenone-3. Toxics. 2022 Feb 19;10(2):96. doi: 10.3390/toxics10020096. PMID: 35202282; PMCID: PMC8877280.