mercury ( hg ) is a ubiquitous heavy metal that is neurotoxic to humans and wildlife . , more than 50% of water bodies are under fish consumption advisories largely due to hg contamination . in addition , hg levels in the tissues of most fish - eating wildlife are within 10-fold of levels known to cause overt damage . in particular , organic hg ( generally found as methyl hg , mehg ) is of health concern since it penetrates lipid bilayers , biomagnifies through aquatic food chains , and about 95% of the ingested dose is absorbed into the blood stream . methyl hg can also cross the blood brain barrier by conjugating with l - cysteine and exploiting the methionine - uptake pathway . as hg has a high affinity for protein thiols , multiple neural components are vulnerable to its toxic action . in an effort to assess hg concentrations in biological samples , various analytical schemes mercury strongly absorbs light at 253.7 nm and total hg can be quantified via established spectroscopic methods . brooksrand , perkin elmer ) have provided direct mercury analyzers to the market which permit rapid and straightforward analysis of total hg . however , the detection of organic hg is still hindered by sample treatments steps that are time consuming , wet chemistry digestions that generate ample chemical waste , and/or need for complex instrumentation . one common method to assess organic hg involves pretreatment of sample with acid followed by alkaline digestion , and then extraction with an organic solvent ( benzene or toluene ) and finally back - extraction into an aqueous solution using cysteine or thiosulphate[4,10 - 12 ] . organic hg compounds may be quantified on the basis of separation techniques ( e.g. , gc , hplc ) with a sensitive detector ( e.g. , ecd , cv - aas , icp - ms ) . it is postulated that the presence of se within cells may mitigate hg toxicity by forming an inert crystalline hg - se complex . further , strict hg - se molar ratios of 1:1 have been observed in biological tissues from several organisms , though few hg monitoring studies co - report on se levels . the essential role of se in physiology has encouraged the development of analytical methods for its quantification at trace levels . among available techniques , fluorescence - based methods are considered to be easier and more cost - effective than conventional spectroscopic techniques ( e.g. , icp - ms , gfaas or hgaas ) though few have outlined in detail key methodological considerations for the assessment of total se in biological samples[17 - 19 ] . owing to the need to determine both organic hg and total se in biological samples , there is a need to develop and apply of fast and reliable analytical methods . here we detail a series of methods that may be used to simply and rapidly detect organic hg ( < 1.5 ml total sample volume , < 1.5 hour ) and total se ( < 3.0 ml total volume , < 3 hour ) in a range of biological samples . figure 1 provides a graphical schematic of the procedure to extract and detect organic hg . about 10 to 50 mg of standard reference materials ( dolt-3 : dogfish liver , tort-2 : lobster hepatopancreas ; both from the national research council of canada , nist 1566b : oyster tissue from national institute of standards and technology , usa ) was homogenized in 50 mm tris - hcl buffer ( ph 8.5 ) containing protease ( subtilisin a , 99% ; 100 g ) , and next incubated at 50c for 1 hr . following this digestion step , naoh ( 40% ; 125 l ) , cysteine ( 1% ; 50 l ) , cuso4 ( 0.5 m ; 50 l ) , acidic nabr ( 3.1% ; 500 l ) , and toluene ( 500 l ) were sequentially added to the digest and vortexed . following centrifugation at 13,000 g for 5 minutes at room temperature , the top toluene layer was transferred to a test tube and mixed twice ( 60 - 80% of toluene ) with na2s2o3 ( 5 mm ; 150 l ) to permit back - extraction of organic hg into an aqueous phase . the aqueous phase was re - centrifuged ( 13,000 g for 2 minutes ) and then placed into another test tube ( 1.5 ml tube ) for organic hg analysis . the final extracts , once obtained , can be analyzed for hg content within a few days . all samples ( srms for total hg , extracts for organic hg ) were directly analyzed by a dma-80 ( dma-80 milestone , inc . , briefly , the direct mercury analyzer liberates mercury by introducing samples into a controlled heated environment . nickel and quartz boats are used to introduce solid and liquid samples , respectively , into the machine 's autosampler . the autosampler delivers the sampling boats into a quartz catalytic tube , where it is dried and then thermally decomposed in a continuous flow of ultra pure oxygen . the remaining decomposition products are carried by oxygen to a gold amalgamator that selectively traps mercury vapour . after the system is flushed with oxygen to remove any remaining decomposition products , the amalgamator is rapidly heated to release the mercury vapour from the gold trap . then , flowing oxygen carries the mercury vapour through absorbance cells positioned in the light path of a single wavelength atomic absorption spectrophotometer . this method is endorsed by the u.s . environmental protection agency ( epa method 7473 ) . here , the detection limit for the direct hg analyzer was 0.020 ng and ranged from 0.010 to 0.029 ng . total and organic hg concentrations are expressed as g / g ( ppm ) dry weight . schematic procedure of organic mercury analysis ( direct mercury analyzer ; time estimates are based on the analysis of one sample , though several can be batch processed ) . a simplified microplate - based fluorometric assay ( total volume < 3 ml ) for biological samples has been developed , based on the tube - based method of sheehan and gao developed for urine and plasma . the method involves sample digestion ( hno3-hclo4 and hcl ) , conjugation as a piazselenol ( 2,3-diaminonaphthalene , dan ) , and cyclohexane extraction ( figure 2 ) . approximately 10 to 50 mg of srms ( dolt-3 , tort-2 , nist 1568a : rice flour , nist 1577b : bovine liver , nist 1515 : apple leaves , selenite and selenate stock standard solution ) and lemon shark ( negaprion brevirostris ) samples were digested in the presence of 400 l of hno3(70% ) and 100 l hclo4(40% ) for 80 min at 195c , followed by the addition of 500 l of hcl for 30 min at 150c in a borosilicate tube . these acid digestions facilitate the oxidation of all forms of se ( particularly selenide and selenate ) into selenite ( selenous acid ) . to the digest , 10 mm of edta with sequential additions of 6.3 mm of dan in 0.1 m hcl ( 500 l ) and 1 ml of cyclohexane was added to form a fluorescent complex as a 4,7-dichloro-5,6-benzopiazselenol ( cl2-se - dan complex ) in an organic phase . though endogenous metals present in working solutions may interfere with dan , the interference may be masked by the addition of 10 mm of edta , which we tested here ( 250 l ; tested ranged from 1 to 100 mm ) . the fluorophore extracts , which reflect total se , can be determined by fluorescence spectroscopy ( emission at 560 nm ; excitation at 360 nm ) using 96 well microplates . concentrations of total se in shark kidney samples obtained by the fluorometric assay were compared to values obtained using a conventional graphite furnace atomic absorption spectroscopy method as previously described . the detection limit of total se for the fluorometric assay ranged from 2 to 3 ng , with standard curve linearity for total se concentrations up to 600 ng ( figure 3 ) . schematic procedure of total selenium analysis ( fluorescence spectrometry ; time estimates are based on the analysis of one sample , though several can be batch processed ) . relationship between predicted and measured total se ( ng ) values in dolt-3 using the fluorescence method described here . average recovery rates from the certified values of dolt-3 , tort-2 , and nist 1566b for total hg were 98.9  4.3% ( n = 61 ) , 97.5  5.5% ( n = 23 ) , and 95.4 4.9% ( n = 3 ) , respectively ( table 1 ) . for organic hg , the average recovery rates from the certified values of dolt-3 , tort-2 , and nist 1566b were 98.6  5.7% ( n = 47 ) , 97.9  4.7% ( n = 19 ) , and 97.2  9.6% ( n = 4 ) , respectively ( table 1 ) . for total hg and organic hg , the relative standard deviation ( rsd ) was lower than 10% for all replicate measures . recovery rates of organic and total mercury concentrations in the certified reference materials  detection limit : 0.020 ng by dma-80 for total se ( table 2 ) , the recovery rates were within the certified values (  18% ) for dolt-3 ( n = 35 ; 103  18% ) , tort-2 ( n = 20 ; 99.2  15.7% ) , nist 1568a ( n = 8 ; 102  16% ) , and nist 1577b ( n = 8 ; 97.3  11.7% ) . total se was not detected from apple leaves ( nist 1515 ; 0.050 g / g ) which is likely due to the low concentration ( close to detection limits ) of se in this material . selenite ( which is converted from all forms of se ) reacts with dan to yield a fluorescent se - dan complex . the recovery rates of selenate standard stock solution ( even at higher levels such as 100 or 250 g / g ) were similar to those from the selenite solution ( table 2 ) . in addition , se concentrations in shark kidney samples ( n = 10 ) that were measured using the fluorometric method described here were comparable to those observed obtained using graphite furnace atomic absorption spectroscopy ( gfaas ) method ( r = 0.978 ; p < 0.001 ) ( figure 4 ) . results of the current assay may provide a rapid and reliable analytical approach for measuring total se , although the fluorophore stability results remained to be resolved . recovery rates of total selenium in the certified reference materials nd : not detected ,  detection limit : 2 - 3 ng by fluorescence spectroscopy pearson correlations of total se in lemon shark kidneys measured between fluorometric assay and atomic absorption spectroscopy ( gfaas ) . overall , the outlined approaches provide an easy , rapid ( < 1.5 hr for organic hg , < 5 min for total hg , < 3 hr for total se ) , reproducible , and cost - effective platform for measuring organic hg and total se . as the methods are down - scaled , they require very small quantities of sample ( 10 - 50 mg ) and can be performed on precious materials , and they generate less chemical waste than conventional approaches . dn and nb designed the study , analyzed data , and drafted the manuscript . both have read and approved the final manuscript . we thank doug adams ( florida fish & wildlife conservation commission ) for lemon shark tissues .
backgroundorganic mercury ( hg ) is a global pollutant of concern and selenium is believed to afford protection against mercury risk though few approaches exist to rapidly assess both chemicals in biological samples . here , micro - scale and rapid methods to detect organic mercury ( < 1.5 ml total sample volume , < 1.5 hour ) and total selenium ( se ; < 3.0 ml total volume , < 3 hour ) from a range of biological samples ( 10 - 50 mg ) are described.resultsfor organic hg , 
 samples are digested using tris - hcl buffer ( with sequential additions of protease , naoh , cysteine , cuso4 , acidic nabr ) followed by extraction with toluene and na2s2o3 . 
 the final product is analyzed via commercially available direct / total mercury analyzers . 
 for se , a fluorometric assay has been developed for microplate readers that involves digestion ( hno3-hclo4 and hcl ) , conjugation ( 2,3-diaminonaphthalene ) , and cyclohexane extraction . 
 recovery of organic hg ( 86 - 107% ) and se ( 85 - 121% ) were determined through use of standard reference materials and lemon shark kidney tissues.conclusionsthe approaches outlined provide an easy , rapid , reproducible , and cost - effective platform for monitoring organic hg and total se in biological samples . owing to the importance of organic hg and se in the pathophysiology of hg , integration of such methods into established research monitoring efforts ( that largely focus on screening total hg only ) will help increase understanding of hg 's true risks .