which consists of removing a soil circle of 40 cm diameter and 25 cm of depth using
a cleaned metal-blade spade, and then undamaged earthworms were extracted from
the collected soil. It is an older protocol used by Salagovic and coworkers in 1996
[194] in order to assess the genotoxicity of polluted soil on earthworm.
Usually, the very first step before earthworm’s tissues extraction is to let them
empty their guts. For this depuration step, earthworms are left on wet filter paper for
24 h, and then they are washed with deionized water, dried with a towel, and frozen
and/or lyophilized. Only one study reported the use of freeze-drying the earthworms
[181]. After lyophilization, earthworms are crushed, homogenized, and stored at
À20
C until analysis. Other studies used homogenized fresh earthworm tissue for
the analysis [20, 183].
The weights of the samples vary according to the extraction method followed,
since for QuEChERS weights of 0.25 and 0.5 g were employed, respectively
[20, 181]; when ASE was performed, weights between 3 and 5 g were needed [183].
We have been able to identify at least four different methods applied to the
analysis of drugs in earthworms (Table 7). Kinney et al. managed to detect 77 anthropogenic organic waste indicators means of the ASE methodology, although 20 were
detected in real samples. Approximately, 50 mL of a 70:30 ACN/water solvent
mixture were employed during five static cycles at a temperature of 130
C and
10,300 kPa. Recoveries from 27 to 117% were observed, but overall good recoveries
and accuracy results were reported [183].
One of the first studies used a previous extraction method used for the soil but
also applicable to earthworms. The method consists of an ultrasonic extraction for
the determination of four pharmaceutical drugs (carbamazepine, diclofenac, fluoxetine, and orlistat). Different extraction solvents have been used depending on the
compound. For carbamazepine, 20 mL of MeOH was used while for fluoxetine and
orlistat 20 mL of ACN/water (7:3, v/v). Finally, 20 mL of ethyl acetate was used for
diclofenac. After centrifugation, no cleaning process was performed to avoid reducing recovery results [182].
The use of the most versatile QuEChERS method has been reported by two
different studies. In the most recent work by Montemurro et al., an analytical method
was developed for the determination of over 50 pollutants present in earthworm
wastewater using a rapid extraction method based on QuEChERS with an innovative
cleaning step with SPE using Oasis PRiME HLB. The method shows good recovery
results from 70 to 99% for most of the studied compounds. By applying this method,
the authors managed to detect and quantify 19 PhACs in earthworms grown under
controlled conditions, whereas 8 analytes were detected in earthworm samples
collected from a cultivated field irrigated with treated wastewater [181].
Bergé and colleagues also used the QuEChERS method as an extraction method
to detect 11 steroids, 14 veterinary antibiotics, and 6 human pharmaceutical products. In this case, the AOAC-buffered salt kit was used. For the hydration phase, an
emulsion of 9 mL of water/hexane (67:33, v/v) was used, while acetonitrile was the
extraction solvent. The cleaning procedure was performed using PSA and C18
(950 mg of MgSO 4 , 150 mg of PSA, and 150 mg of C18). Recoveries between
45.2 and 105% have been observed with good results of linearity and precision [20].
Development of Methods for the Determination of PhACs in Soil/Earthworm/Crop. . .
475
a cleaned metal-blade spade, and then undamaged earthworms were extracted from
the collected soil. It is an older protocol used by Salagovic and coworkers in 1996
[194] in order to assess the genotoxicity of polluted soil on earthworm.
Usually, the very first step before earthworm’s tissues extraction is to let them
empty their guts. For this depuration step, earthworms are left on wet filter paper for
24 h, and then they are washed with deionized water, dried with a towel, and frozen
and/or lyophilized. Only one study reported the use of freeze-drying the earthworms
[181]. After lyophilization, earthworms are crushed, homogenized, and stored at
À20
C until analysis. Other studies used homogenized fresh earthworm tissue for
the analysis [20, 183].
The weights of the samples vary according to the extraction method followed,
since for QuEChERS weights of 0.25 and 0.5 g were employed, respectively
[20, 181]; when ASE was performed, weights between 3 and 5 g were needed [183].
We have been able to identify at least four different methods applied to the
analysis of drugs in earthworms (Table 7). Kinney et al. managed to detect 77 anthropogenic organic waste indicators means of the ASE methodology, although 20 were
detected in real samples. Approximately, 50 mL of a 70:30 ACN/water solvent
mixture were employed during five static cycles at a temperature of 130
C and
10,300 kPa. Recoveries from 27 to 117% were observed, but overall good recoveries
and accuracy results were reported [183].
One of the first studies used a previous extraction method used for the soil but
also applicable to earthworms. The method consists of an ultrasonic extraction for
the determination of four pharmaceutical drugs (carbamazepine, diclofenac, fluoxetine, and orlistat). Different extraction solvents have been used depending on the
compound. For carbamazepine, 20 mL of MeOH was used while for fluoxetine and
orlistat 20 mL of ACN/water (7:3, v/v). Finally, 20 mL of ethyl acetate was used for
diclofenac. After centrifugation, no cleaning process was performed to avoid reducing recovery results [182].
The use of the most versatile QuEChERS method has been reported by two
different studies. In the most recent work by Montemurro et al., an analytical method
was developed for the determination of over 50 pollutants present in earthworm
wastewater using a rapid extraction method based on QuEChERS with an innovative
cleaning step with SPE using Oasis PRiME HLB. The method shows good recovery
results from 70 to 99% for most of the studied compounds. By applying this method,
the authors managed to detect and quantify 19 PhACs in earthworms grown under
controlled conditions, whereas 8 analytes were detected in earthworm samples
collected from a cultivated field irrigated with treated wastewater [181].
Bergé and colleagues also used the QuEChERS method as an extraction method
to detect 11 steroids, 14 veterinary antibiotics, and 6 human pharmaceutical products. In this case, the AOAC-buffered salt kit was used. For the hydration phase, an
emulsion of 9 mL of water/hexane (67:33, v/v) was used, while acetonitrile was the
extraction solvent. The cleaning procedure was performed using PSA and C18
(950 mg of MgSO 4 , 150 mg of PSA, and 150 mg of C18). Recoveries between
45.2 and 105% have been observed with good results of linearity and precision [20].
Development of Methods for the Determination of PhACs in Soil/Earthworm/Crop. . .
475