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, Mª

Esther Torres-Padrón3, Zoraida Sosa-Ferrera3, José Juan Santana-Rodríguez3

1Institute of Chemistry of São Carlos, Trabalhador São Carlense Ave, 400, São Carlos, São Paulo, Brazil

2Laboratório de Química Analítica e Ecotoxicologia, Universidade Federal do Maranhão, Brazil

3Departmento de Química, Universidad de Las Palmas de Gran Canaria, Spain.

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 latter’s 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 0.45µm. 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 μm 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 C-18 column SunFire® C18 (3.0x100 mm) 3.5μm 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 (São Luiz - Maranhão - Brazil). J. Brazil Chem Soc 25: 380-388.

[2] Sánchez-Rodríguez A, Sosa-Ferrera Z, Santana-Rodríguez 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.

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