Plastic Products Leach Chemicals That Induce In Vitro Toxicity under Realistic Use Conditions

Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies.


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(Electrolytes for batteries). We downloaded the lists from https://echa.europa.eu/information-onchemicals/registered-substances on 13.12.2020, combined all available CAS numbers and removed duplicates, 3) the chemicals (pre)registered under the REACH regulation in 2017 (NORMANdb) as provided by the NORMAN Suspect List Exchange. 4 We combined all available SMILES codes and removed duplicates.
To generate molecular structures for in silico fragmentation, we annotated the available CAS numbers of the chemicals in the ECHAdb with SMILES codes using the CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard/dsstoxdb/batch_search). For the CPPdb and the NORMANdb, we used the SMILES codes available in the respective list. We converted the SMILES to CID numbers using PubChem's Identifier Exchange Service (https://pubchem.ncbi.nlm.nih.gov/idexchange) and downloaded the structural information as individual sdf file per database using PubChem Entrez (https://pubchem.ncbi.nlm.nih.gov). Each annotation step resulted in the loss of compounds that either had no SMILES codes, CID or structural information (Table S2). The resulting structural databases contained 2680 (CPPdb), 7092 (ECHAdb), and 65,738 chemicals (NORMANdb) for in silico fragmentation. effective concentration leading to a luciferase induction ratio of 2.0 over the control (IR 2); -, no effect at measured concentrations; n.a., not assessed (e.g., due to cytotoxicity).
a Luminescence inhibition induced by migrates from 600 mg plastic well -1 .
b Number of independent experiments performed with two technical replicates, each. Note: -, no oxidative stress response observed (induction rate < 2) at the analyzed concentrations.
a Maximal luciferase induction ratio induced by the highest measured non-cytotoxic concentration.
b Highest analyzed concentration at which no cytotoxicity was observed. Concentrations between 1.56 and 200 mg plastic well -1 were tested.
c Number of independent experiments performed in two technical replicates, each. d IR of two replicates exceeded 2 and thus, the ECIR2 was still calculated using all 3n.
e Only of n = 2 since 1n was cytotoxic at 200 mg plastic well -1 and thus, excluded.
S8 Table S6. Estrogenic and antiandrogenic activities (mean ± SEM) of procedural blanks (PB), SPE blanks (SPE) and samples in YES and YAAS as well f All measured concentrations (0.20-100 mg plastic well -1 ) were cytotoxic, such that antiandrogenicity could not be assessed S10 Note: -, no effect at concentrations analyzed or value not calculable.  Note: Note: -, no effect at concentrations analyzed or value not calculable.
Bioassay results of migrates and extracts at concentration ranges or points assessed for both leaching conditions: Baseline toxicity: EC20 at 0-5.0 mg plastic well -1 and luminescence inhibition (LI) at 3 mg plastic well -1 , antiandrogenic activities: 3.00 mg plastic well -1 (exception: PVC 2: 0.78 mg plastic well -1 ). Relative antiandrogenic activities < LOD are given in brackets and correspond to 27.3 % (migrates) and 29.2 % (extracts).. Note: a listed in the CPPdb as associated with plastic packaging. S15 Figure S1. D S18 Figure S4. Baseline toxicity of plastic migrates in the Microtox assay presented as means (lines) EC20

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(A) and EC50 (B) baseline toxicity as well as luminescence inhibition (C). >600 indicates that the migrate of 600 mg plastic well -1 did not inhibit the bioluminescence by >20 or 50 %, respectively. S19 Figure S5. Oxidative stress response induced by migrates in the AREc32 assay as mean (lines) ECIR2 (A) and induction ratios of the highest measured non-cytotoxic concentrations (B, see Table S5) >200 indicates that migrates of 200 mg plastic well -1 (highest analyzed concentration) did not exceed an induction ratio of 2. Activity data of migrates which were antiandrogenic (mean effects > LOD) at 100 mg plastic well -1 presented with their cytotoxic (c) concentrations (grey) and non-cytotoxicity concentrations (black).
S23 Figure S8. Baseline toxicity of aqueous plastic migrates in the Microtox assay presented as means (lines) EC20 (A) and EC50 (B) baseline toxicity as well as luminescence inhibition (C) from two to three independent experiments (dots). >5 indicates that migrates of 5.00 mg plastic well -1 did not inhibit the bioluminescence by > 20% or 50 %, respectively. S24 S25 S26 Figure S9. Concentration-response relationship for baseline toxicity of migrates (with SPE) and aqueous migrates (without SPE). Only samples for which both, migrates and aqueous migrates, induced luminescence inhibition ≥ 20 % at measured concentrations are shown. Data is presented as mean ± standard error of the mean (SEM). EC20, effect concentration inducing 20 % baseline toxicity.
S27 Figure S10. Comparison of the abundance of chemical features detected in the extracts and migrates of each plastic product. Red lines highlight the band of an abundance ratio of 0.1-10.