proposed a physical extraction method to separate the diffusible from the cell-bound
EPS and microorganism fractions to further measure pesticide concentrations accumulated in each of these two fractions. Solvent extraction of organic contaminants
from dried biofilm is then performed by pressurized liquid extraction (Writer et al.
2011; Huerta et al. 2016), ultrasonic extraction (Headley et al. 2001), shaking
extraction (Wang et al. 1999; Du et al. 2015), or Soxhlet extraction (Schorer and
Eisele 1997). Organic extracts are sometimes purified by solid-phase extraction
(Coat et al. 2011; Writer et al. 2011; Du et al. 2012, 2015) or directly analyzed by
chromatographic techniques (Headley et al. 2001). Liquid or gas chromatography
coupled to mass spectrometry (LC-MS or GC-MS, depending on the compounds) is
preferred to achieve the requisite selectivity and sensitivity (Coat et al. 2011; Dobor
et al. 2012; Du et al. 2012; Huerta et al. 2016; Ruhí et al. 2016). As an example,
liquid chromatography with UV detection was not sufficiently sensitive for analysis
of N-methyl pyrrolidinone in biofilm extracts with a limit of quantification (LOQ) at
100 ng g
À1 , whereas LC-MS was able to reach a LOQ of 2 ng g
À1 (Headley et al.
2001; Huerta et al. 2016). While current identification and quantification methods
generally include extraction and purification steps, Headley et al. (1995) proposed in
the 1990s a method based on the direct injection of a small biofilm sample using an
insertion probe. Subsequent detection and identification of contaminants and metabolites were performed by tandem mass spectrometry (MS-MS) (Headley et al. 1995).
Unfortunately, the absence of separation of biofilm components prior to sample
introduction in the ion source induced interferences that limit the detection of a wide
range of contaminants at low-level concentration.
As for metal detection (Sect. 2.1), imaging techniques, such as CLSM and STXM
(Neu et al. 2010), have been successfully used to investigate organic contaminant
bioaccumulation in biofilms (Lawrence et al. 2001, 2016; Dynes et al. 2006b). These
nondestructive imaging techniques were applied on hydrated biofilm samples and
allowed direct observation of contaminants localization in the complex structure
of preserved biofilms. Fluorescence was usually used to detect contaminant with
CLSM, either by investigating fluorescent contaminants (Wolfaardt et al. 1994) or
by using specific probes targeting the contaminant investigated (Lawrence et al.
2001). Contaminant identification by STXM was probe-independent and based on
comparison with suitable reference spectra (Neu et al. 2010). Thus, coupling CLSM
with monoclonal antibodies specific to atrazine, Lawrence et al. (2001) showed that
bioaccumulation of this pesticide in river biofilms resulted from atrazine sorption to
specific microcolonies. Using STXM, Dynes et al. (2006b) revealed differences in
bioaccumulation patterns of the antimicrobial chlorhexidine between pennate and
centric diatoms within a complex microbial biofilm. To our knowledge, STXM has
not been used for quantification of organic contaminant accumulation in biofilms;
however the optical density obtained by STXM reflected the amount of contaminant
and could therefore be used for relative comparison between samples (Dynes et al.
2006b). Up to now, these techniques have only been applied on complex microbial
biofilms exposed in laboratory at relatively higher concentrations of organic contaminants than those found in the aquatic environments.
Role of Biofilms in Contaminant Bioaccumulation and Trophic Transfer in Aquatic. . .
125
EPS and microorganism fractions to further measure pesticide concentrations accumulated in each of these two fractions. Solvent extraction of organic contaminants
from dried biofilm is then performed by pressurized liquid extraction (Writer et al.
2011; Huerta et al. 2016), ultrasonic extraction (Headley et al. 2001), shaking
extraction (Wang et al. 1999; Du et al. 2015), or Soxhlet extraction (Schorer and
Eisele 1997). Organic extracts are sometimes purified by solid-phase extraction
(Coat et al. 2011; Writer et al. 2011; Du et al. 2012, 2015) or directly analyzed by
chromatographic techniques (Headley et al. 2001). Liquid or gas chromatography
coupled to mass spectrometry (LC-MS or GC-MS, depending on the compounds) is
preferred to achieve the requisite selectivity and sensitivity (Coat et al. 2011; Dobor
et al. 2012; Du et al. 2012; Huerta et al. 2016; Ruhí et al. 2016). As an example,
liquid chromatography with UV detection was not sufficiently sensitive for analysis
of N-methyl pyrrolidinone in biofilm extracts with a limit of quantification (LOQ) at
100 ng g
À1 , whereas LC-MS was able to reach a LOQ of 2 ng g
À1 (Headley et al.
2001; Huerta et al. 2016). While current identification and quantification methods
generally include extraction and purification steps, Headley et al. (1995) proposed in
the 1990s a method based on the direct injection of a small biofilm sample using an
insertion probe. Subsequent detection and identification of contaminants and metabolites were performed by tandem mass spectrometry (MS-MS) (Headley et al. 1995).
Unfortunately, the absence of separation of biofilm components prior to sample
introduction in the ion source induced interferences that limit the detection of a wide
range of contaminants at low-level concentration.
As for metal detection (Sect. 2.1), imaging techniques, such as CLSM and STXM
(Neu et al. 2010), have been successfully used to investigate organic contaminant
bioaccumulation in biofilms (Lawrence et al. 2001, 2016; Dynes et al. 2006b). These
nondestructive imaging techniques were applied on hydrated biofilm samples and
allowed direct observation of contaminants localization in the complex structure
of preserved biofilms. Fluorescence was usually used to detect contaminant with
CLSM, either by investigating fluorescent contaminants (Wolfaardt et al. 1994) or
by using specific probes targeting the contaminant investigated (Lawrence et al.
2001). Contaminant identification by STXM was probe-independent and based on
comparison with suitable reference spectra (Neu et al. 2010). Thus, coupling CLSM
with monoclonal antibodies specific to atrazine, Lawrence et al. (2001) showed that
bioaccumulation of this pesticide in river biofilms resulted from atrazine sorption to
specific microcolonies. Using STXM, Dynes et al. (2006b) revealed differences in
bioaccumulation patterns of the antimicrobial chlorhexidine between pennate and
centric diatoms within a complex microbial biofilm. To our knowledge, STXM has
not been used for quantification of organic contaminant accumulation in biofilms;
however the optical density obtained by STXM reflected the amount of contaminant
and could therefore be used for relative comparison between samples (Dynes et al.
2006b). Up to now, these techniques have only been applied on complex microbial
biofilms exposed in laboratory at relatively higher concentrations of organic contaminants than those found in the aquatic environments.
Role of Biofilms in Contaminant Bioaccumulation and Trophic Transfer in Aquatic. . .
125