2 4 dichlorophenoxyacetic acid

Juglone (Mw: 174.15 g/mol, CAS Number: 481-39-0), polyvinyl alcohol (PVA, Mw 31–50 kDa, 87–89 % hydrolyzed, CAS Number: 9002-89-5), PLGA (50:50 lactide:glicolide; inherent viscosity 0.45−0.60 dL/g, Mw ~38−54 kDa, CAS Number: 26780-50-7), and dichloromethane (DCM, CAS Number: 75-09-2) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Plant suspension cultures were established from alfalfa (Medicago sativa cv Verco) seeds kindly provided by Dr Satı Uzun (University of Erciyes, Turkey ) . 2,4-dichlorophenoxyacetic acid, kinetin, sucrose, and agar used for preparing the Murashige and Skoog (MS) medium were purchased from Sigma-Aldrich.
The mouse fibroblast cell line L929 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) / Ham’s Nutrient Mixture F-12 medium, foetal bovine serum (FBS), penicillin, and L-glutamine used in the preparation of fibroblast cell cultures were purchased from Sigma-Aldrich. The Ames Microplate Format Mutagenicity Assay™ 98/100 kit and strains were provided from Anaria-Xenometrix (Mason, OH, USA). All the chemicals and solvents used for mutagenicity study were of analytical grade. Ultra-pure water was obtained from the Millipore MilliQ Gradient system (Merck Millipore, Burlington, MA, USA)

Chemicals from the ToxCast Phase II library (ph 2 subset) were obtained from the National Center for Computational Toxicology (NCCT), U.S. EPA. The test library contained 777 samples, representing 768 unique chemicals; 9 chemicals were in duplicate to serve as internal quality controls. Chemicals were solubilized in dimethyl sulfoxide (DMSO) (≤2 × 10⁻²M, Table S1) and supplied in thirteen 96-well plates (62 samples per plate) (Evotec Inc., Branford, CT). Stock chemical plates were visually inspected under an inverted microscope to confirm solubility in each well (Table S1). All control chemicals, including sodium perchlorate (NaClO₄), sodium nitrate (NaNO₃), sodium thiocyanate (NaSCN), 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,3-dichloro-1,4-napthoquinone (DCNQ) (Sigma Aldrich, St. Louis, MO), were initially solubilized in DMSO (EMD Millipore Corp., Darmstadt, Germany) at 2 × 10⁻²M. All chemicals in the ToxCast inventory supplied for testing undergo manual quality curation review prior to registration in EPA's DSSTox structure database (Richard et al., 2016 (link)), and are assigned DSSTox chemical identifiers (DTXSIDs, chemical names, CAS numbers) and mapped to chemical structures (DTXCIDs), wherever possible. These identifiers and maximum concentrations used in this study are provided in Table S1.

Some of the chemicals, including 2,4-dichlorophenoxyacetic acid (the chemical formula of C₈H₆Cl₂O₃ and the purity of 99%) with a molecular weight of 221.04 g mol⁻¹, lead nitrate (Pb(NO₃)₂, >99% purity) and nitric acid (HNO₃, 95% purity) and sodium sulfate (Na₂SO₄, 99% purity) were provided by Sigma Aldrich (St. Louis, MO, USA). Other chemicals, i.e., CaCl₂·2H₂O, MgSO₄·7H₂O, KI, MnCl₂·4H₂O, FeCl₃·6H₂O, CuSO₄·5H₂O, H₃BO₃, CoCl₂·6H₂O, ZnSO₄·7H₂O, Na₂MnO₄·2H₂O, EDTA, CaCl₂·H₂O and MgSO₄·7H₂O were bought from Merck Co, Germany. Sodium hydroxide (NaOH) and sulfuric acid (H₂SO₄) were also supplied by Merck Co (Darmstadt, Germany). The regulation of solution pH was carried out using 0.01 N H₂SO₄ and 0.01 N NaOH.

Metsulfuron methyl, MM (CAS: 74223-64-6, 99.3% purity, pK_a: 3.3), 2,4-dichlorophenoxyacetic acid, 2,4-D (CAS: 94-75-7, 99.3% purity, pK_a: 2.98) and paraquat, PQ (CAS: 75365-73-0, 98.0% purity) were purchased from Riedel-de Haën, Sigma-Aldrich. The 10 mM stock solutions at pH 6.0 ± 0.2 were prepared in 0.01 M NaCl. pH 6.0 ± 0.2 was adjust for all solutions using small volumes of HCl and/or NaOH solutions (1 M). pH measurements were performed by using a Cole Parmer Model 59003-25 pH meter with an Alpha® model PY-41 electrode.
The molecular structures of all contaminants are shown in Table S1 of the ESI†. Considering the pK_a values of 2,4-D and MM their anionic species are predominant at working pH; on the other hand, BAC-12, BAC-14 and PQ are quaternary ammonium salts, therefore they only form cations in aqueous solution.⁴⁴ Calcium chloride CaCl₂·2H₂O (CAS: 10035-04-8, 99.0% purity) was supplied by Sigma-Aldrich Company. All other chemicals were analytical grade.