This
study reports the distribution coefficient between phospholipid bilayer
membranes and phosphate buffered saline (PBS) medium (DMW,PBS) for 19 cationic surfactants. The method used a
sorbent dilution series with solid supported lipid membranes (SSLMs).
The existing SSLM protocol, applying a 96 well plate setup, was adapted
to use 1.5 mL glass autosampler vials instead, which facilitated sampling
and circumvented several confounding loss processes for some of the
cationic surfactants. About 1% of the phospholipids were found to
be detached from the SSLM beads, resulting in nonlinear sorption isotherms
for compounds with log DMW values above
4. Renewal of the medium resulted in linear sorption isotherms. DMW values determined at pH 5.4 demonstrated
that cationic surfactant species account for the observed DMW,PBS. Log DMW,PBS values above 5.5 are only experimentally feasible with lower LC-MS/MS
detection limits and/or concentrated extracts of the aqueous samples.
Based on the number of carbon atoms, dialkylamines showed a considerably
lower sorption affinity than linear alkylamine analogues. These SSLM
results closely overlapped with measurements on a chromatographic
tool based on immobilized artificial membranes (IAM-HPLC) and with
quantum-chemistry based calculations with COSMOmic. The SSLM data
suggest that IAM-HPLC underestimates the DMW of ionized primary and secondary alkylamines by 0.8 and 0.5 log
units, respectively.
The nominal concentration
is generally used to express concentration–effect
relationships in in vitro toxicity assays. However, the nominal concentration
does not necessarily represent the exposure concentration responsible
for the observed effect. Surfactants accumulate at interphases and
likely sorb to in vitro system components such as serum protein and
well plate plastic. The extent of sorption and the consequences of
this sorption on in vitro readouts is largely unknown for these chemicals.
The aim of this study was to demonstrate the effect of sorption to
in vitro components on the observed cytotoxic potency of benzalkonium
chlorides (BAC) varying in alkyl chain length (6–18 carbon
atoms, C6–18) in a basal cytotoxicity assay with
the rainbow trout gill cell line (RTgill-W1). Cells were exposed for
48 h in 96-well plates to increasing concentration of BACs in exposure
medium containing 0, 60 μM bovine serum albumin (BSA) or 10%
fetal bovine serum (FBS). Before and after exposure, BAC concentrations
in exposure medium were analytically determined. Based on freely dissolved
concentrations at the end of the exposure, median effect concentrations
(EC50) decreased with increasing alkyl chain length up
to 14 carbons. For BAC with alkyl chains of 12 or more carbons, EC50’s based on measured concentrations after exposure
in supplement-free medium were up to 25-times lower than EC50’s calculated using nominal concentrations. When BSA or FBS
was added to the medium, a decrease in cytotoxic potency of up to
22 times was observed for BAC with alkyl chains of eight or more carbons.
The results of this study emphasize the importance of expressing the
in vitro readouts as a function of a dose metric that is least influenced
by assay setup to compare assay sensitivities and chemical potencies.
Working with and analysis of cationic surfactants can be problematic since aqueous concentrations are difficult to control, both when taking environmental aqueous samples as well as performing laboratory work with spiked concentrations. For a selection of 32 amine based cationic surfactants (including C8- to C18-alkylamines, C14-dialkyldimethylammonium, C8-tetraalkylammonium, benzalkonium and pyridinium compounds), the extraction from aqueous samples was studied in detail. Aqueous concentrations were determined using solid phase extraction (SPE; 3 mL/60 mg Oasis WCX-SPE cartridges) with recoveries of ≥80% for 30 compounds, and ≥90% for 16 compounds. Sorption to glassware was evaluated in 120 mL flasks, 40 mL vials and 1.5 mL autosampler vials, using 15 mM NaCl, where the glass binding of simple primary amines and quaternary ammonium compounds increased with alkyl chain length. Sorption to the outside of pipette tips (≤20% of total amount in solution) when sampling aqueous solutions may interfere with accurate measurements. Polyacrylate solid phase microextraction (PA-SPME) fibers with two coating thicknesses (7 and 35 μm) were tested as potential extraction devices. The uptake kinetics, pH-dependence and influence of ionic strength on sorption to PA fibers were studied. Changing medium from 100 mM Na to 10 mM Ca decreases K with one order of magnitude. Results indicate that for PA-SPME neutral amines are absorbed rather than adsorbed, although the exact sorption mechanism remains to be elucidated. Further research remains necessary to establish a definitive applicability domain for PA-SPME. However, results indicate that alkyl chain lengths ≥14 carbon atoms and multiple alkyl chains become problematic. A calibration curve should always be measured together with the samples. In conclusion, it seems that for amine based surfactants PA-SPME does not provide the reliability and reproducibility necessary for precise sorption experiments, specifically for alkyl chain lengths beyond 12 carbon atoms.
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