Influence of surfactants and humic acids on Artemia Franciscana's embryonic phospho-metabolite profile as measured by 31P NMR

Abstract

Surfactants, such as triton X-100 (Tx-100), cetylpyridinium chloride (CPC), and sodium dodecyl sulfate (SDS) are known to be toxic to Artemia Franciscana (Artemia) - an organism, frequently used to monitor the health of the aquatic environment. The phospho-metabolite profile of a living organism is often indicative of imbalances that may have been caused by environmental stressors, such as surfactants. This study utilizes in vivo³¹P NMR to monitor temporal changes in the phospho-metabolite profile of Artemia caused by Tx-100, CPC, and SDS and the ability of humic acid (HA) to mitigate the toxicity of these surfactants. It was found that, while Tx-100 does not have any effect on the phospho-metabolite profile, both CPC and SDS cause a complete retardation in growth of the phosphodiester (PDE) peak in the ³¹P NMR spectrum, which is indicative of the inhibited cell replication. This growth inhibition was independently verified by the decreased guanosine triphosphate (GTP) concentration in the CPC and SDS-exposed Artemia. In addition, upon introduction of HA to the CPC and SDS-exposed Artemia, an increase of PDE peak over time is indicative of HA mitigating toxicity.

Keywords: Artemia; Environmental; NMR; Surfactants; Toxicity.

Figure 1

A) Representative ³¹P NMR spectrum of live Artemia Franciscana. Peak identities are 1) phosphomonoesters (PME; 3.7-4.2 ppm), 2) inorganic phosphate (P_i; 2.5-3.5 ppm), 3) phosphodiesters (PDE; 1.2-1.8 ppm), 4) phosphocreatine (PCR; 0-1 ppm), 5, 6, 7, and 8) α-adenosine triphosphate (ATP)/α-adenosine diphosphate (ADP), β-ATP (–5.5 - –4.5 ppm; –11.8 - –9.8 ppm) 9) γ-ATP/β-ATP (–19.0 - –18.6 ppm) and B)³¹P NMR example spectrum of dead Artemia embryos (embryo's death was induced on purpose for illustrative purposes by leaving the embryos in the hypochlorite solution for too long).

Figure 2

Stacked representative ³¹P NMR spectra for Artemia franciscana embryos in A) 35 ppt NaCl, B)100 ppm Tx-100, C) 5 ppm CPC and D) 35 ppm SDS

Figure 3

³¹P NMR intensities of PDE/P_i of Artemia Franciscana embryos with 35 ppt NaCl, 5 ppm CPC in 35 ppt NaCl, 35 ppm SDS in 35 ppt NaCl, and 100 ppm Tx-100 in 35 ppt NaCl.

Figure 4

The changes in micromoles of guanosine triphosphate (GTP) per milligram of dry Artemia Franciscana tissue extracts for a 5h exposure to as measured by HPLC (*p < 0.05 versus the control)

Figure 5

³¹P NMR intensities of PDE/P_i of Artemia Franciscana embryos under 35 ppt NaCl, 35 ppm LAHA in 35 ppt NaCl, 5 ppm CPC in 35 ppt NaCl, and 5 ppm LAHA and 5 ppm CPC in 35 ppt NaCl

Figure 6

³¹P NMR intensities of PDE/P_i of Artemia Franciscana embryos under 35 ppt NaCl, 35 ppm LAHA in 35 ppt NaCl, 35 ppm SDS in 35 ppt NaCl, and 35 ppm LAHA and 35 ppm SDS in 35 ppt NaCl (*last three time points only repeated in duplicate).