Pll: 80273-1223(99)00553-3

~ Pergamon Wol. Sci Tech Vol. 40, No.6, pp. 165-169,1999

IC1999Published by ElsevierScienceUd on behalfoflhe IAWQ

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COLORIMETRIC APPROACH TOCYANOBACTERIAL OFF-FLAVORDETECTION

Dwight Miller*, Eric D. Conte**, C. Y. Shen** andPeter W. Perschbacher***

• FDAlNationalCenterfor Toxicological Research. 3900 NCTRRoad. Jefferson.AR 70279, USA•• Western Kentucky University. DepartmentofChemistry, Bowling Green.KY 4101.USA••• Aquaculture/Fisheries CenterBox 4912. University ofArkansasat Pine Bluff,AR 71611. USA

ABSTRACT

A colorimetric method 10 quantify methylisobomeol and geosmin in water and fish flesh based on a spot testby Wood and Snoeyink was developed. To test water samples, one liter of filtered water is pumped throughI solid phase extraction device and the off-flavor eluted from the device with toluene, The toluene iscombined with I 1 percent vanillin in concentrated sulfuric acid and agitated for 30 min to produce the colorchange. The standard solution is yellow and al increasing concentrations of 2-mcthylisobomeol or geosminthe color changes from light orange 10 blood red. Extraction from I liter of water results in I sensitivity of Ippb, This is suitable for quality control of seafood. For use in measuring action levels (high and low pptlevels for aquaculture and drinking water, respectively), extraction from greater volumes will be required. C1999 Published by Elsevier Science Lid on behalfof the IAWQ. AU rights reserved

KEYWORDS

Colorimetric; geosmin; Mffi.

INTRODUCTION

Detection of the two major cyanobacterial off-flavors 2-methylisobomeol (Mffi) and geosmin (GEO) hasrelied solely on taste and odor (organoleptic) testing, until the ready availability of gas chromatography­mass spectrometry (GC-MS) procedures allowed measurement of these compounds at ppt levels. HoweverGC-MS determinations require expensive equipment and materials and are time-consuming. Organolepti~testing is the only practical method available to quality control managers, but it remains subject to the abilityof an individual to detect Mffi and GEO as well as variable or subjective rejection criteria. The limitationsofsensory analysis have been described by McGuire et al. (1981).

Methods for Mffi and GEO determinations by GC-MS in water and seafood samples have not beenstandardized (Karahadian and Lindsay, 1994). Typically, these tertiary alcohols are extracted by an organicsolvent such as methylene chloride. The solvent is sent to a laboratory for determination; however, thenumber of laboratories capable of this procedure is limited. Some larger water districts maintain GC-MScapability for Mffi and GEO. Because accuracy of the measurement varies depending on conditions of

165

166 D. MILLERet al.

extraction and detection methods, continued research to improve efficiency is needed. For example, use offunctionalized solid phase adsorbents have resulted in very high extraction efficiencies.

Enzyme-linked immunosorbent analysis (ELISA), using antibodies to MIB or GEO linked to colorimetricdevelopment, have been developed for water samples (Chung et al., 1990, 1991). Although ELISA providesa rapid field test, sensitivity is I ppm. This is substantially above the low ppt and low ppb levels at whichoff-flavor in water and fish occur, respectively (Brett and Dionigi, 1997). Water samples must therefore beconcentrated (Chung, 1992).

The most widely used colorimetric method for the determination of alcohols is that for ethanol in blood.This method involves the oxidation of alcohol with chromium (VI) to chromium (III), and is morecommonly known as the Jone's oxidation . Other potential classic tests include: 1) Lucas test, 2) Cericammonium nitrate, and 3) coordination of alcohols with bis(8-hydroxyquinoline) vanadium oxinate (Feigelet al., 1966). Except for the vanadium oxinate method, they do not ofTer application in the determination oftertiary alcohols such as MIB or GEO. The first colorimetric test for an off-flavor was a Tortelli and Jaffereaction for GEO (Hensarling and Waage, 1990). This reaction lacked sensitivity and used bromine, ahazardous material. A very obscure reagent used as a TLC stain is 1 percent vanillin in sulfuric acid,reported to produce a detectable red color in the presence of 1 ng of MIB on a TLC plate (Wood andSnoeyink, 1977).

Based on the results of Wood and Snoeyink (1977), we have developed a colorimetric method to quantifyMIB in water and fish flesh. GEO determination was also found to be applicable to the same technique.The test has the advantages of being simple, inexpensive, and relatively fast. At a rate of 12 samples perhour, the cost will be approximately SUS 4 per sample.

METHODS AND MATERIALS

The chemicals and equipment used in this method were commercially procured. The chemicals were allreagent grade and used as described.

Standards

Standards of MID and GEO were prepared in sulfuric acid concentrate, previously washed with reagentgrade toluene at 1000,500, 100,50, 10,5 and I ug per mllevels. Color was developed in one mL each ofthese solutions for 30 min. The color was measured with a Hach colorimeter. Linear regression analysis ofthe absorption data as a function of concentration at 540 IU11 and 560 IU11 for MID were slopes of 0.0063 and0.0049 with intercepts of 0.0052 and 0.001, respectively, and for GEO were slopes of 0.0004 and 0.0004with intercepts of 0.0052 and 0.0001, respectively, and for GEO were slopes of 0.0004 and 0.0004 withintercepts of0.0062 and 0.0199, respectively. All regressions had ~ values of0.999 or better.

OfT-flavor in water

A minimum of 1.5 I ofwater was collected in a sealable container. One hundred mg ofcalcium chloride wasadded to a water sample and mixed. The water-calcium mixture was allowed to stand approximately 2 min.then the water was filtered through a glass wool plug. (A rough filter is all that is required). One liter offiltered water was pumped through a preconditioned C-IS sep-pak at 40 ml per min into a I liter graduatedcylinder to measure the volume. Preconditioning was accomplished by passing a I ml of ethylacetate, 1 mlof toluene, I ml of methanol, and 2 ml of water through the sep-pak in sequence. After the ofT-flavor hasbeen extracted from I liter of water, 5 ml ofclean water and 5 ml of air were passed through the sep-pak toclean the residual pond water and water out of the sep-pak. The semi-dry sep-pak was then eluted with 2 mlof sulfuric acid washed with toluene through a drying tube containing anhydrous sodium sulphate, directlyinto a 13 by 100 rom test tube. To the toluene off-flavor solution, I ml of I percent vanillin in sulfuric acidwas added and rotated for 30 min. The absorbance was measured and compared to the calibration curve toestimate the level of ofT-flavor. The conformation and recovery determinations have been published (Conteet al., 1996).

Colormetric approachto cyanobacterial otT-flavor detection 167

RESULTS

Calibration curves were developed for Mill and GEO using a Hach colorimeter. This was accomplished byspiking toluene with 1,5, 10,50, and 500 llg ofMIB and developing the color with 1 percent vanillin insulfuric acid. The same procedure was followed for GEO. The visible spectra for the color complex ofMill and GEO exhibits Amax of540 run and 560 run, respectively (Figure 1). Calibration curves ofMB andGEO are shown for the 1 to 100 llg at 540 run and 560 run (Figure 2). The visible absorption data for the500 ug sample was well below the regression line. This was attributed to the oligermization of themolecules or chemical stacking. The time dependence of the color formation for MID at 100 ug is shown inFigure 3. As shown, the measurement of absorbance at 30 min into the color development demands thetiming must be precise, whereas, measurements made at 240 min would be less subject to precise timeconstraints because of the differences in the slope of the curve is closer to zero at 30 min than at 240 min.

700

.5

2.0

1.5

1.0

o.o~ ~~...........~"!"""",""~"",,,__

400

w(JZCma:oenInC

FREQ

700

GEOCOMPLEX

500 600FREQ

.5

1.0

0.01--o------..-~----400

w(JZCma:oenmc

Figure J.Thevisibleadsorpncnspectra (or Mill and OEO vanillincomplexes.

168 D. MILLER et al.

0.70 1.00

0.60 MI8at 540nm w GEOat540nmlU 0 0.80U 0.50Z Z41( 0.40 < 0.60m mex: 0.30 a:

0.400 0(/) 0.20 enm m 0.2041( 0.10 <

0.000 20 40 60 80 100 0.00

0 40 60 80 100

0.50 1.20

MIS at 560 nmw GEO at 560 nm

lU 0 1.00U 0.40 ZZ < 0.8041( 0.30 CDm a:ex: 0 0.600 0.20 UJ(/) m 0.40III41( 0.10 <

0.20••0.00

0 20 40 10 80 100 0.000 20 40 60 80 100

CONCENTRAnON, uglliter water CONCENTRATION, uglllterwater

Figure 2. Calibration curves forMIBandGEOat 540DID and560nm.

M18100 ug at 540nm •

800 800 1000 1200 1400 1GOO 1800 2000

nME(hra.)

FigureJ. Colordevelopment forMIBas a function of timeat 540nm.

The outline of the procedure to examine a water sample is shown in Figure 4. One liter of filtered water ispumped through a Waters C-IS solid-phase extraction sep-pak or Varian bond-elute Jr. The Mm or GEO iscaptured on the adsorbent and is eluted in place with 2-5 ml of toluene solvent. One ml of I percent vanillinin 36N sulfuric acid was added to the toluene eluent and agitated for 30 min to produce the color change.The standard solution is yellow and at increasing concentrations of Mm or GEO it changes from lightorange to blood red. The conformation is best accomplished by examining the An-l or by GC·MS (Conte et01.• 1996a). Recoveries for the extraction method is 86 to 93 percent for 0.1 and 200 ~g as determined byGC-MS. We have also examined a water sample containing Mm. The colorimetric method gave 50 ~gIl

and the GC-MS method gave 67 J.1g1l.

Colonnetric approachto cyanobacterial off-flavordetection 169

-.-

./ FIn... I FI.....through --. through

gl_wooll abeorbilnt42mIInIin

•, Waatl ~

WIthIS mI Tolueue

Colorimetric Method In Water

Rr=l~

~-==-l

§J,-:---____.-.....-"'i..:-I .....-----".Figure4. Flow diagramfor the analysisof a water sample.

Measurement of MID and GEO lev~ls in fish. fles~ involves purging in a microwave oven as described byConte et 01. (1996b). After collection, colorimetric development and measurement process is the same aswith water samples.

CONCLUSIONS

Extraction from I liter of water results in a sensitivity of 1 ppb. Interferences from other alcohols, organicacids and phenols in water samples may be possible, although to date interferences have not been a problem.Any colorimetrically measurable level of MID of GEO in water from a 1 liter sample would result in tasteproblems in fish flesh (Tucker and Martin, 1991). Clearly, the issue of a simple and timely test resides in adefined target of unacceptable level of MID or GEO in water and fish tissue and developing more creativemethods ofconcentrating the analyte.

REFERENCES

Bett, K. L. and Dionigi,C. B. (1997). Detectingseafood off-flavors: limitationsof sensoryevaluation. Food Tech.; !l1(8),70-79.Chung. S.-Y. (1992). Attemptsto improvethe sensitivityofan enzymeIinked-immunosorbent assay for 2-methylisobomeol. Waf.

Sci. Tech., 25(2), 89-95.Chung, S.-Y., Johnsen, P. B. and Klesius, P. H. (1990). Developmentof an ELISA using polyclonal antibodies specific for 2­

methylisbomeoJ. J. Agric. FoodChem., 38, 410-415.Chung, S.-Y., VerceUoti, 1. R., Johnson,P. B. and KJesius. P. H. (1991). Developmentof an enzyme-linked immunosorbentassay

for GEO. J. Agric. FoodChem., 39,764-769.Conte, E. D~ Conway, S. C~ Miller, D. W. and Perschbacher, P. W. (1996a). Determination of methylisobomeol in channel

catfish pond water by solid phase extraction followed by gas chromatography-mass spectrometry. Waf. Res., 30(9),2125-2127.

Conte, E. D., Shell,C. Y., Miller, D. W. and Perschbacher,.P. W. (1996b~ . Microwa~e distil1ati~n-solid phase absorbent trappingdevice for the detennination of off-flavors geosmm and methyhsobomeol, m catfish nssue below their rejection levels.Anal. Chem., 68(15), 2713-2716.

Fieg!,F. (1966). Spot Testin Organic Analysis. Elsevier PublishingCompany,Amsterdam.Hensarling, T. P. and Waage, S. K. (1990). A bromine-based color reaction for detection of geosmin.J. Agric. Food Chem., 38,

1236-1237.Karahadian,C. and Lindsay,R. C. (1994). Chemicalisolationofodorife~us. components. In: Analysuo/Contaminants InEdible

AquaticResources, J. W. Kiceniukand S. Ray (eds). VCH PubhshingInc., New York,NY, pp. 275-283.McGuire, M.l., Krasner, S. W., Hwang,C.l. and Izaguirre,G. (1981). Closed-loopstrippinganalysis as a tool for solVing taste

and odor problems. J. Amer. Water Works Assoc., 73, 530-S37 .,Tucker, C. S. and Martin, J. F. (1991). Environment-related off-flavors In fish. In: Aquaculture and WaterQuality, D. E. Brune

and 1. R. Tomasso (eds) , The WorldAquacultureSoci~ty, Baton Rouge,.pp. 113-179: .Wood, N. F. and Snoeyink,V. L. (l9n). 2_Methylisobomeol, unproved synthesIsand quantitative gas chromatographicmethod

for trace concentrationsproducingodor in water. J. Chromatogr. 132,405-420.