3 4 dichloroaniline

A binary mixture of insecticide was tested for 5 (d-tetramethrin: 0.01 – 1.20 μmolL⁻¹ and cyphenothrin: 0.02 – 3.20 μmolL⁻¹) concentrations prepared as dilutions with standard dilution water. Thirty fertilized eggs were randomly selected and transferred into 60 mm petri dish containing different concentrations of binary mixture of d-tetramethrin and cyphenothrin that are used for the assay, the positive control (4 mgL⁻¹ (24.69 μmolL⁻¹) 3,4-dichloroaniline) [27 ] (Sigma, USA), and negative control (only dilution water) [27 ], respectively, to minimize the dilution of concentration from the transfer straight from dilution water and to minimize the delay in exposure.
Twenty embryos were individually exposed in 24-well microtiter plates (Corning, USA), each well containing 2 ml of the respective concentrations of a mixture of insecticide. Plates were pretreated with respective solutions to minimize the absorption of the plastic material [27 ]. A single 24-well plate was used for each concentration of test, negative control, and positive control. Twenty wells of each plate were used for test solution and the remaining 4 wells served as internal control (containing dilution water). The plates were covered with non-adhesive foil and kept at RT (26°C) away from direct sunlight for 24, 48, and 72 hrs [27 ]. Assays were initiated within 2 hrs of post-fertilization.

Benzo[a]pyrene (BaP; CAS no. CAS No. 50–32–8), 5-fluorouracil (CAS no. 51–21-8) and 3,4-dichloroaniline (CAS no. 95–76-1) were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). All stocks and dilutions of test compounds were prepared in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany).
DMSO extracts of the sixteen complex petroleum substances (Fig. 1) were tested, including a distillate (sample SN100-DIS, CAS No. 64741–50-0), an aromatic extract (sample SN100-AE, CAS No. 64742–05-8), waxy raffinates (samples SN100-WR and SN400-WR; CAS No. 64741–89-5), base oils (sample SN100-LBO, CAS. No 64742–65-0; sample SN500-LBO, CAS. No 64742–65-0; sample SN100-BO, CAS No. 64742–56-9), a technical white oil (sample SN100-TWO, CAS No. 8042–47-5), a medicinal white oil (sample SN100-MWO, CAS No. 8042–47-5), a slackwax (sample SN400-SW, CAS No.64742–61-6), a hard paraffin wax (sample SN400-HPW, CAS No. 8002–74-2), a highly refined hard paraffin wax (sample SN400-HPHPW, CAS No.64742–51-4), a waxy brightstock (sample WBS, CAS No. not available), a brightstock slackwax (sample BSSW, CAS No. not available), a microcrystalline wax (sample MCW, CAS No. 63231–60-7), and a hydrogenated microcrystalline wax (sample HGMCW, CAS No. 64742–60-5).

Analytical grade dimethyl phthalate, monomethyl phthalate, dibutyl phthalate, dioctyl phthalate, propanil, monalide, chlorpropham, carfentrazone-ethyl, methomyl, 3-chloroaniline, 4-chloroaniline, and 3,4-dichloroaniline were purchased from Sigma-Aldrich (Castle Hill, Australia). Sulfosulfuron, metsulfuron-methyl, pyrazosulfuron-ethyl, imazethpyr, and pendimethalin were gifted by Dow Agrosciences (India). All other chemicals were used at the highest purity available commercially.

All chemicals were obtained from Sigma-Aldrich (Buchs, Switzerland) unless indicated otherwise. The OTA (Sigma Cat. No. 01877; produced by Petromyces albertensi, lot No. 067M4011V) and 3,4-Dichloroaniline (Sigma Cat. No. 437778; lot No. 13509KQV) were solubilized in pure ethanol before use.

Bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), 3,4‐dichloroaniline, atrazine desethyl (6‐Chloro‐N,N‐dimethyl‐1,3,5‐triazine‐2,4‐diamine), N‐(1‐naphthyl)‐ethylene‐diamine, 2‐benzimidazole propionic acid, aflatoxin‐BSA conjugate (hapten density of 8‐12), N‐hydroxysuccinimide (NHS) ester, O‐phenylenediamine dihydrochloride (OPD), 2‐(N‐morpholino) ethanesulfonic acid (MES), and dimethylformamide were purchased from Sigma‐Aldrich (St. Louis, MO). Goat anti‐human IgG+IgM+IgA‐HRP labeled Abs were purchased from Abcam (Cambridge, MA). Vacutainer tubes were obtained from BD Diagnostics (Franklin Lakes, NJ). EDC [1‐ethyl‐3‐(3‐dimethyl aminopropyl) carbodiimide] and Nunc maxiSorp flat‐bottom plates were purchased from Thermo Fisher Scientific (Waltham, MA). All the reagents and solvents were of analytical grade.

Thiram (98.4% purity) and 3,4-dichloroaniline (98% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thiram stock solutions were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C for further use. Test solutions (40, 20, 10, 5, 2.5, 1.25, and 0.625 μg/L) were prepared by dissolving the required volume of stock solutions in E3 buffer (290 µg NaCl; 8.3 µg KCl; 48 µg CaCl₂; 81.5 µg MgCl₂ to each mL of deionized water and 0.1% methylene blue; pH 7.2). All the chemicals used in this investigation were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.