Comparison of different extraction methods for the

determination of booster biocides in marine organisms

through liquid chromatography-mass spectrometry
Lia Gracy Rocha Diniz1, Eny Maria Vieira1, Teresa Cristina Rodrigues dos Santos Franco2,
Cristina Afonso-Olivares3, Rayco Guedes-Alonso3, Sarah Montesdeoca-Esponda3, M
Esther Torres-Padrn3, Zoraida Sosa-Ferrera3, Jos Juan Santana-Rodrguez3
1

Institute of Chemistry of So Carlos, Trabalhador So Carlense Ave, 400, So Carlos, So Paulo, Brazil
Laboratrio de Qumica Analtica e Ecotoxicologia, Universidade Federal do Maranho, Brazil
3
Departmento de Qumica, Universidad de Las Palmas de Gran Canaria, Spain.
2

E-mail contact: eny@iqsc.usp.br

1. Introduction
Since restrictive regulation of the antifouling agent tributyltin,the study of alternatives for the use antifouling
paints tin free is increasing due to high level of contamination in the marine and coastal ecosystems. around
eighteen compounds worldwide non-metallic organic are currently used or promoted as agents in antifouling
paints. Some of the most common booster biocides are are 4-chloro-3-methylphenol, chlorothalonil,
dichlofluanid, diuron, thiram, irgarol 1051 and the latters degradation product or a mixed together with up to
five these components.
Although they same these are less persistent in the environment, many of these compounds have been
associated with noxious effects such as metabolic disorders, infertility and inhibition of growth, reduction in
immunity and even death of some organisms.
Few studies related to methods for determination of booster biocides in biological samples Some studies
have reported diuron, irgarol 1051 and M1 (degradation product of irgarol) in tissues of marine algae and
molluscs level ng g-1. The use of soft tissue of clams may be a viable option for the chemical analysis of the
presence of anti-fouling in port areas, considering reports of some studies on the cause-and-effect
relationship between exposure to organotin antifouling and the development of imposex in Stramonita
haemastoma a mollusc often found in rocky shores, anatomically related species of the genus Thais,
commonly found in tropical and temperate waters.

2. Materials and methods

In laboratory the shells of organisms were removed for the analysis of soft tissues, the samples were dried
by lyophilization followed by homogenized. The samples were spiked with a mixture of the selected
compounds in methanol to obtain a final concentration of 500 ng g-1 to determine the optimal extraction
conditions. The samples were stirred until homogenized and air-dried overnight in the dark at room
temperature.
For multiwave microwave extraction a oven with a 6 EVAP rotor and 6 MF 100 PTFE vessels (Anton Paar,
Graz, Austria) was used.
Influential parameters of the MAE procedure (sample amount, extractant solvents, solvent volume,
microwave power, and extraction time) were studied through single factor experiments.
The first parameter, which was checked for the optimization of MAE, non-optimum conditions (200W; 6 min),
was amount of sample, between 50 and 500 mg of spiked sample were diluted with methanol was
transferred to PTFE vessels. The vessels symmetrically placed on the microwave, the temperature and
pressure were controlled automatically using the system's interface. After the processing, the samples were
allowed to cool followed by clean-up with Chromafil Xtra syringe filter 0.45m.
The similar procedure was followed so as to determine the, extractant solvent (CH3CN, Methanol and a
mixture 50% v/v of CH3CN, Methanol), solvent volume (5 and 10 mL), the duration of the microwave
extraction (1; 1.5 and 2 min) and the microwave power (500, 700, 1000 W). The eluates were dried under a
gentle stream of nitrogen gas and reconstituted with 1 mL of methanol.
Ultrasonic Extraction procedure.
In this set of experiments 100 mg of sample spiked were sonicated with extractant solvent (CH3CN,
Methanol and a mixture 50% v/v of CH3CN, Methanol). The extracts were filtered and reduced in volume to
1 mL with methanol after selection best extraction solvent, for the optimization, parameters were modeled

simultaneously by the use of an experimental design (23). An experimental design was chosen, because of
possible interaction effects between the different parameters, volume of extraction solvent (2.5, 5 and 10 ml)
and the time of sonication (10, 15, and 20 min).

Clean-up procedure.
For cleanup with SPE was used a Varian Vac Elut 20 SPE manifold coupled to a Sartorius Model 16692
vacuum, the extraction solutions were filtered through a 0.45 m syringe filter and diluted with 100 mL Milli-Q
water. The cartridges (EnvirElut, 500 mg) were conditioned with 2x5 mL methanol and 2x5 mL Milli-Q water.
The extracts were passed through the cartridges under vacuum at a flow rate of 1 mL.min-1 followed by
clean-up with 2x5 mL Milli-Q water and the elution step utilized 2x1 mL methanol. The eluates were dried
under a gentle stream of nitrogen gas (purity > 99.999%) and reconstituted with 1 mL of methanol.
Chromatographic analysis
The analysis of the extracted samples was carried out with HPLC-DAD analysis, a chromatography system
(Varian Inc., Madrid, Spain)equipped with a pump fitted with an auto-sampler and volume selector, a column
valve module with an internal oven and a DAD detector was employed. The system, acquisition and
processing of data were controlled using Star software 6.5 version (Varian Inc., Madrid, Spain). Analytical C18 column SunFire C18 (3.0x100 mm) 3.5m was obtained from Waters (USA). Twenty microliters of
sample was injected into the chromatography system. The mobile phase consisted of MilliQ water and
methanol, starting with (50:50 v/v) for 3 min up to a proportion of 80:20 v/v in 14 min at a flow-rate of 0.5 mL
min-1. The diode array detector allowed UV spectra in the range 190-300 nm. Based on absorbance signals
observed in the DAD spectrum of the standard solutions, diuron and irgarol were detected and quantified at
248 nm and 230 nm, respectively. To ensure the presence of the booster biocides in real samples, retention
times were used and comparisons made between the absorbance spectra in the sample and in the standard
solution. The retention times and wavelengths of the compounds used for their measurement

3. Results and discussion

In this study, three solvents; CH3CN, Methanol and a mixture 50% v/v of CH3CN and methanol were selected
considering some factors such as suitability to analytes polarity, suitability to standards solvent. The most
proper (which extracts better than the others) was methanol, chosen as best solvent and it was used in both
MAE and ultrasonic bath extractions. For the amount of sample was selected as preliminary conditions 100
mg of sample for extraction.
Results were compared to obtain the best analytical method to extract booster biocides in soft tissues of this
type of molluscs.

A 23 factorial design was performed with two variables (irradiation time and
power) and three levels (1, 1.5, and 2 min at 500, 750, and 1000 W, respectively) was
followed for optimization of MAE. All analyses were performed with 100mg of sample
(containing 500ng g-1 of each analytes), and were carried out in triplicate using the
polynomial fits of the results. The response surface for each analyte was created (Figure
1).

Figure 1: Response surface for the effect of time and power on the extraction of (a) diuron and (b) Irgarol
1051.

Higher analytical signals were obtained at low power and shorter times. It is
possible that high power and the longer irradiation time on the small peak areas were due
to volatilization or degradation of the analytes. Based on these results, the MAE optimized
procedure for these analytes utilized 100 mg of tissue sample in 5 mL of methanol solvent
with an irradiation time of 1 min at 1000 W of power.

4. Conclusions
Analytical methods are studied for the extraction of Diuron and Irgarol 1051 in biological tissue samples from
molluscs.
Based in this initial study, MAE is the extraction method suitable for diuron and irgarol in biological tissue
sample although it is necessary an preconcentration and clean-up.

5. References
[[1] Diniz, LGR (2013) First Appraisal of Water Contamination by Antifouling Booster Biocide of 3 Generation
at Itaqui Harbor (So Luiz - Maranho - Brazil). J. Brazil Chem Soc 25: 380-388.
[2] Snchez-Rodrguez A, Sosa-Ferrera Z, Santana-Rodrguez JJ (2012) Analytical methods for the
determination of common booster biocides in marine samples. Cent Eur J Chem 10: 521-533.

ACKNOWLEDGEMENT - The authors thank to FAPESP for the their financial support to this work.