A cost-effective strategy combining chemical analysis and bioassays
for the identification of polar toxic compounds in sewage sludge is reported.
ToxAlert®100 bioluminescence inhibition assay was used in combination
with chemical analysis involving extraction, clean-up, chromatographic separation
and mass spectrometry detection. This methodology was applied to real samples
of sludge from three wastewater treatment plants (WWTP) located
in Catalonia (Spain) during a 3 month period. In the first step,
sewage sludge was lyophilized, treated by sonication with a mixture of methanol
and chloroform and finally cleaned up using a sequential solid phase extraction (SSPE)
with an octadecylsilica cartridge (C18) in series with
a polymeric Lichrolut EN cartridge (Lic EN). In the second step,
the toxicity of each fraction of the sludge sample was investigated using
the ToxAlert®100. The unequivocal identification and quantification
of polar organic cytotoxic substances present in the fractionated extracts
were determined by liquid chromatography-mass spectrometry (LC-MS).
Major toxic compounds identified were: non-ionic polyethoxylated surfactants (nonylphenol
polyethoxylates, alcohol polyethoxylates), their intermediates (polyethylene
glycol polyethoxylated, nonylphenol carboxylates and polyethoxylated alcohol
carboxylates), linear alkylbenzenesulfonates and heavy metals. The toxic
response (in terms of bioluminescence inhibition using ToxAlert®100),
defined by the 50% effective concentration (EC50),
and the toxicity units (TU) for every standard non-ionic surfactant
were calculated. The results provided the identification of polar cytotoxic
compounds as well as the evaluation of their contribution to the total toxicity
observed in sewage sludge.
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