methylmercury is efficiently accumulated 
 through estuarine food 
 webs , such that concentrations in fish are controlled by initial ch3hg assimilation at the base of the food web .
sources and pathways of ch3hg and hg accumulation in lower trophic levels 
 are therefore fundamental to understanding hg cycling in estuaries 
 and controlling hg levels in top predator fish .
estuarine sediments 
 are a sink for hg and also a dominant 
 site for methylation of hg to the more bioavailable species , 
 ch3hg .
benthic organisms , which 
 dwell at the sediment  water interface , are exposed to hg from 
 both sediments and the water column and are also a food source to larger invertebrates 
 and fish . as such , they present an important 
 link between hg bioaccumulation in benthic and pelagic food webs , 
 yet dominant sources and pathways of hg uptake in benthic fauna are 
 not well understood .
benthic 
 species are exposed to dissolved metals from porewater and overlying 
 water and also take up metals from dietary sources , which can originate in both the sediment and water column .
identifying 
 the uptake and trophic transfer between sediment and water food sources 
 and primary consumers is important to linking water and sediment quality 
 data to bioaccumulation .
an important benthic species along 
 the east coast of north america 
 is the estuarine amphipod , leptocheirus plumulosus , which is abundant over a wide range of sediment types and salinity 
 and temperature conditions.l .
plumulosus is also used as a test organism to 
 assess both acute and chronic toxicity of estuarine sediments .
l. plumulosus are facultative feeders ; they live in u - shaped burrows through which 
 they filter water and ingest suspended particles and also roam the 
 surface of the sediment and deposit feed , thus deriving their nutrition and exposure to contaminants from 
 both the water column and sediments .
microcosm and modeling studies 
 have shown dietary ingestion of organic matter , rather than uptake 
 from the dissolved phase , to be the dominant process for ch3hg and hg accumulation in l. plumulosus(6,18 ) over a range of assimilation 
 efficiencies ( ae ) and ingestion rates ( ir ) , the relative contribution 
 of food sources to body burden was modeled to be > 80% for ch3hg and 1060% for hg , although 
 accumulation 
 was determined in water with no appreciable dissolved organic carbon 
 ( doc ) , and uptake of both species from 
 natural water is inversely correlated to doc concentrations .
ingestion pathways are ignored in toxicity testing , 
 which assumes metal exposure is predominantly from sediment porewater .
biokinetic models of metal accumulation currently lack estimates 
 of uptake from deposit vs suspension feeding . feeding behavior in l. plumulosus
is a critical piece of information to understanding hg accumulation 
 and trophic transfer . in this study , we used enriched isotopic 
 ch3hg and hg tracers in microcosm 
 experiments to directly 
 determine hg uptake pathways in l. plumulosus .
two experiments were conducted to study feeding pathways of ch3hg ( experiment 1 ) and hg ( experiment 
 2 ) .
this approach had several advantages : ( 1 ) using multiple enriched 
 isotopes of hg allowed us to simultaneously decipher uptake from different 
 pathways .
( 2 ) we could expose amphipods to ch3hg and hg at environmentally relevant levels .
( 3 ) unlike radiometric tracer studies , this 
 technique enabled hg species transformation to be traced throughout the experiment .
because ae of ch3hg is much higher than that of hg , methylation of hg will increase 
 availability to the biota .
methylation 
 occurs in anoxic zones in sediment and 
 has also been observed in algal cultures .
if hg speciation is not monitored 
 during incubation in uptake experiments , bioaccumulation of hg species 
 may be misinterpreted .
enriched stable isotopes of hg species have 
 been used as a powerful tool to study hg cycling and uptake into freshwater 
 foodwebs . in this study , we applied the technique to identify trophic transfer 
 of hg species from live phytoplankton in water and sediment compartments 
 in estuarine benthic infauna .
all solutions were made up 
 using ultrapure water ( > 18 m cm ) produced 
 by a purelabpluswater purifier ( us filter , ma , usa ) . enriched stable 
 isotopes of hg ( as hgo , hgo , and hgo )
single isotope spikes of hg and hg were prepared by dissolution of 
 hgo in hcl , followed by dilution to 400 mg / l in 5% hcl ( fisher optima 
 grade , pittsburgh , pa , usa ) .
solutions of ch3hgcl and ch3hgcl were synthesized by conversion 
 of hgo to hgcl2 followed by 
 reaction with methylcobalamin .
stock 
 solutions were diluted to 50 mg / l of ch3hg and 5 mg / l of ch3hg in 
 0.5% acetic acid and 0.2% hcl ( fisher optima ) .
isotopic abundances 
 of natural hg and the enriched isotope spikes are given in the supporting information .
the marine 
 phytoplankton species , i. galbana , 
 was used as the food source for the ch3hg and 
 hg exposures of l. plumulosus .
cells of i. galbana were grown in 
 a 19 l carboy of 20 ppt artificial seawater ( instant ocean prepared 
 in ultrapure water ) to a density of 1.3  10 cells 
 per milliliter and then reduced by centrifugation to 800 ml .
the concentrated 
 culture was then divided into four 200 ml solutions , and each algal 
 solution was spiked with one isotopically enriched hg species . in 
 experiment 1 ( ch3hg uptake ) , the suspensions 
 were tagged with 2.5 g ch3hg or ch3hg and in experiment 
 2 ( hg uptake ) , with 34 g of hg or hg .
the isotopically labeled 
 cultures were allowed to equilibrate for 24 h and then spun down into 
 pellets and the supernatant discarded .
aliquots ( 1 ml ) of each cell suspension 
 were removed and then centrifuged , and the resulting algal pellets 
 were freeze - dried and weighed .
the pellet and supernatant solutions 
 ( both n = 4 ) were analyzed for ch3hg and thg , and the fraction of algal - bound hg was determined 
 ( 94  1% for ch3hg , and 
 95  2% for ch3hg ; 98.1 
  0.1% for hg and hg ) .
concentrations of hg spikes in the algal slurries are 
 included in tables 1 and 2 .
leptocheirus 
 plumulosus was purchased 
 from aquatic research organisms ( hampton , nh ) and cultured according 
 to epa method 1994 .
similarly sized ( > 1 
 mm ) amphipods were retrieved from culturing tanks prior to the experiment 
 and depurated for 4 h in salt water .
microcosm 
 experiments were conducted in 
 100 ml ( 5 cm diameter , 10 cm height ) glass jars ( qorpak , bridgeville , 
 pa , usa ) .
the 
 organic carbon content was determined to be 3.2  0.1% by loi 
 ( dry sediment was heated at 550 c for 4 h ) . for each experiment
, 
 16 identical microcosms were assembled . in each microcosm , 1 ml of 
 algal suspension containing enriched isotope ch3hg ( experiment 1 ) or hg ( experiment 2 ) was mixed 
 with 16 g of wet sediment , and the other enriched isotope - tagged 
 algae was mixed with 60 ml of 20 ppt artificial seawater ( table 2 ; henceforth isotopically enriched hg species are 
 denoted by a subscript ( hgsed or hgwater ) indicating 
 the compartment to which the spiked algae was added ) .
the sediment 
 was added to a jar ( 1 cm depth ) , along with 5 amphipods , and 
 overlying water was added .
microcosms were kept in an environmental 
 chamber ( 20 c , 10:14 ( l / d ) h photoperiod ) , covered loosely with 
 plastic to minimize evaporation , and slowly agitated on an orbital 
 shaker ( 100 rpm ) to maintain aeration and suspension of algae .
a water 
 sample was taken from four microcosms immediately after assembly ( t = 0 ) ; then four microcosms were dismantled for analysis 
 after 12 , 24 , 36 , and 48 h. this two - day time series was chosen to 
 monitor relative accumulation of hg from different sources in amphipods 
 over time , and to minimize the effects of algal death and decay on 
 bioavailability of ch3hg and hg   associated with longer incubation 
 periods .
changes in hg concentration and speciation were monitored 
 in whole and filtered water and sediment throughout the time series . 

a second set of microcosms was assembled under the same conditions 
 but using algae suspensions not spiked with hg species , to monitor 
 dissolved oxygen ( do ) , ph , and oxidation reduction potential ( eh ) 
 throughout the experiment .
results are reported in the supporting information ( si ) . to dismantle 
 microcosms after each time point ,
a 15 ml aliquot was filtered 
 through a 0.45 m poresize 25 mm syringe tip filter ( fisherbrand ) , 
 and a second 15 ml sample was collected unfiltered . both filtered 
 and whole water samples were acidified to 0.5% hcl ( fisher optima ) . 

amphipods from each microcosm were removed from the sediment by pasteur 
 pipet and thoroughly rinsed in two sequential water baths and placed 
 in a preweighed 15 ml glass vial ( i - chem ) .
sediments were removed 
 from the microcosm jars and placed in 60 ml pfa vials ( sarstedt , nmbrecht , 
 germany ) .
amphipod and sediment samples were freeze - dried ; all concentrations 
 are reported on a dry weight basis .
all sample containers were double 
 bagged and stored refrigerated in the dark until analysis .
freeze - dried 
 amphipod samples were weighed and extracted in glass vials . to each 
 vial , 1.5 ml 4 m hno3 was weighed , and samples were heated 
 overnight at 60 c .
a 50 l 
 aliquot of each extract was transferred to a 40 ml amber glass vial 
 with teflon - lined septa ( brooks rand laboratories , seattle , usa ) . samples ( 8 ml ) of filtered and whole overlying 
 water were weighed into 40 ml vials .
portions ( 0.5 g ) of freeze - dried , 
 homogenized sediment samples were leached with kbr / h2so4/cuso4 , extracted into ch2cl2 , then back extraction into 10 ml water ; then 2 ml of the extract was weighed into 40 ml glass vials .
samples were buffered and derivatized , then analyzed using 
 a merx - m methylmercury analysis system ( brooks rand laboratories , 
 seattle , usa ) coupled with a 7700x agilent icp - ms ( agilent technologies , 
 santa clara , usa ) ; the details of this technique are given in taylor 
 et al .
isotope deconvolution was used 
 to separate hg species derived from three sources : the ambient hg 
 in the sample ( determined from the hg signal ) and hg 
 inputs from two enriched hg - tagged algal spikes added to the water 
 and sediment compartments .
external calibration was 
 performed using six standards ( 0.025 to 25 ng / l ) , and a secondary 
 source calibration check was analyzed every 10 samples .
method detection 
 limits ( mdl ) were 0.05 ng / g ( sediment ) , 5 ng / g ( amphipods ) , and 0.13 
 ng / l water .
recoveries for ch3hg were 85  13% ( n = 2 ) in bcr-580 estuarine sediment ( irmm , geel , belgium ) 
 and 106  10% ( n = 3 ) in nist 2976 mussel ; spike 
 recoveries in water were 99  5% ( n = 5 ) .
quantification of total 
 hg from biological tissues was determined from 4 m hno3 extracts .
aliquots ( 0.5 ml ) of amphipod 
 extracts were diluted 10 times by weight and analyzed directly by 
 icp - ms ( agilent 7700x ) , with a mdl of 20 ng / g .
portions of sediment 
 samples ( 0.25 g ) were weighed into 60 ml pfa tubes ( sarstedt ) and 
 5 ml of 45% hno3 : 5% hcl v / v was added to each tube .
samples 
 were digested in an open vessel microwave ( mars xpress , cem corps , 
 matthews , nc , usa ) at 95 c for 1 h. digested samples were diluted 
 to 50 ml with ultrapure ( 18 m ) water , and weighed , then analyzed 
 by icp - ms , with a mdl of 0.1 ng / g .
signal intensities were deconvoluted 
 ( si ) prior to conversion to concentration 
 by external calibration .
recovery of standard reference materials 
 nist 2711a ( n = 2 ) and nist 2976 ( n = 3 ) were 80  1% and 115%  11% , respectively .
filtered and whole overlying water and supernatant samples from algal 
 suspensions were analyzed by cold vapor - icp - ms , using an automated 
 merx 
t purge and trap system ( brooks rand ) coupled with icp - ms 
 based on epa method 1631 . samples ( 8 
 ml overlying water or 0.4 ml supernatant ) were weighed into 40 ml 
 clear glass vials with teflon - lined septa ( brooks rand ) and diluted 
 to 25 ml with 1% hcl .
samples were digested overnight in capped vials , 
 by the addition of 0.1 ml freshly prepared brcl ( 0.16 g kbr was dissolved 
 in 15 ml hcl , then 0.38 g kbro3 added and stirred for 1 
 h ) .
to each sample , 0.1 ml sncl2 and 0.1 ml hydroxylamine 
 ( brooks rand ) were added , then vials were recapped for analysis .
bioaccumulation 
 factors ( baf ) were calculated for each uptake pathway to normalize 
 body burden to different exposure concentrations .
body burdens of 
 isotopically enriched hg from 36 and 48 h time points were divided 
 by sediment concentrations in each microcosm ; e.g. , in experiment 
 1 , baf for ch3hgsed was calculated as the concentration of ch3hgsed ( ng / g ) in amphipods relative to its 
 concentration in sediment .
metal accumulation from ingestion has been described 
 by the model 
 in eq 1(40,41)1where the body burden of a metal species taken 
 up from food , css , is calculated from 
 the metal concentration in food ( cf ) , 
 the assimilation efficiency of the metal species ( ae , unitless ) , the 
 ingestion rate , ir ( mg food / g body wt / d ) , the metal efflux rate constant 
 following uptake from food , kef ( 1/d ) , 
 and the growth rate constant , g ( 1/d ) . the equation 
 was rearranged to calculate ir . reported values of ae ( ch3hg = 80% , hg = 6% ) , kef ( ch3hg = 0.052 d , hg= 0.089 d ) , and g ( 0.07 d ) for l. plumulosus were applied , and 
 concentrations of isotopic tracers in algae cf and amphipods css were used to 
 distinguish ingestion from benthic and pelagic sources .
nonparametric 
 analyses were chosen because distribution could not be ascertained 
 due to small sample sizes .
significant trends in spike concentrations 
 in sediment , water , and amphipods with time were assessed by kruskal 
wallis 
 rank tests ; specific differences in concentration between time points 
 were determined using the mann  whitney rank sum statistic . 

using the two enriched isotopes , 
 ch3hg concentrations 
 from spiked algae added simultaneously to the water column and sediment 
 were tracked in the sediment , water column , and in amphipods over 
 time ( table 1 ) . for sediment , differences in 
 concentrations of both enriched isotope hg species were not significant 
 between time points ( kruskal  wallis : d.f . 

= 3 , p > 0.05 ) , so data for all time points were 
 pooled for each experiment .
only 0.2% of the recovered ch3hgwater was suspended in the 
 water column after 12 h , whereas concentrations of ch3hgwater and ch3hgsed in the homogenized sediment samples 
 were similar .
variation in ch3hg tracer 
 levels occurred in the water column between time points ( table 1 ) .
levels of ch3hgwater in the initial whole water sample ( t = 0 ) were elevated and variable between microcosms .
concentrations 
 of ch3hgwater decreased 
 from 0 to 24 h and then reached a steady state between 24 and 48 h 
 ( mann  whitney u , p > 0.05 ) . in the filtered 
 water fraction ,
low levels of 
 the ch3hgsed tracer 
 were also present in the water compartment . enriched isotope 
 ch3hg tracers were present 
 in amphipods after 12 h ( figure 1 ) , and concentrations 
 of both tracers in amphipods did not vary significantly across time 
 series ( kruskal  wallis , d.f .
= 3 , p > 0.05 ) , although variance between replicates at each 
 time point was large ( 1646% ) .
body burdens of isotopically enriched ch3hg tracers in l. plumulosus over time 
 ( experiment 1 ) .
the two 
 enriched isotopes of hg were tracked over time in microcosms 
 ( table 2 ) . as with experiment 1 , both tracers 
 were present in the bulk sediment , including 99.9% hg - tagged 
 algae added to the water column .
concentrations of hg tracers in sediment did not vary with time ( kruskal  wallis d.f.= 3 , p > 0.05 ) , whereas tracer concentrations 
 in the whole water fraction were variable .
whole water samples were 
 elevated in hgwater at t = 0 ( mann  whitney u , z = 2.3 , p = 0.02 ) immediately following the addition of spiked algae 
 to the surface water but reached an apparent steady state from 24 
 to 48 h ( p > 0.05 )
. concentrations of hgwater in the filtered water were also highly 
 variable at t = 0 but did not change significantly 
 from 0 to 48 h ( kruskal  wallis , d.f .
methylation of the hg spikes was evident during incubation 
 of the i. galbana suspensions ( table 2 ; 0.70.8% ch3hg ) . 

the % ch3hg of tracers in sediment increased 
 slightly relative to the starting algal suspension ( mann  whitney 
 u , % ch3hgsed : z = 2.5 , p = 0.01 ; % ch3hgwater : z = 
 2.0 , p = 0.04 ) .
the % ch3hg in 
 the hgsed tracer , mixed with 
 the sediment , was slightly higher than in the hgwater tracer initially added to the water column ( mann  whitney 
 u , z = 2.2 p = 0.03 ) , although 
 variability in % ch3hg in tracers among microcosms 
 was high ( rsd = 5863% ) . in the whole and filtered water samples , 
 % ch3hgsed was higher 
 than in the algal suspensions ( table 2 ) prior 
 to addition to the sediment ( mann  whitney u , z = 2.5 , p = 0.01 ) . in the overlying water , 
 % ch3hgsed was not 
 significantly different than in the sediment ( p > 
 0.05 ) .
there was no difference between % ch3hgwater in the water column and in the algal 
 suspensions or sediments ( p > 0.05 ) .
neutral ph 
 ( 8) 
 and oxic conditions ( do > 6 mg / l ) in the sediment and water column 
 were maintained over the time course under these experimental conditions 
 ( si ) .
body burdens of isotopic tracers 
 in amphipods , as thg , ch3hg , and % ch3hg are shown throughout the time 
 series in figure 2 .
ambient thg in amphipods 
 ( from hg ) was 15 ng / g with 39  12% ch3hg ( all time points ; n = 16 ) . at t
= 12 h , body burdens of hgwater were higher 
 than in later time points , even when one high value ( 1812 ng / g ) was 
 removed ( mann  whitney u , z = 2.3 , p = 0.02 ) .
conversely , increases in hgsed body burden were significant with time ( figure 2a ) .
( a ) body burdens of isotopically enriched hg tracers , 
 as thg concentrations .
( b ) ch3hg concentrations 
 and % ch3hg in l. plumulosus over time ( experiment 2 ) . letters ( a , b , c for hgsed ; or y , z for hgwater ) signify statistical differences between time points 
 by mann 
amphipods accumulated methylated 
 forms of both tracers , comprising 
 up to 18% of thg ( figure 2b and c ) .
uptake 
 of ch3hgwater was 
 evident , but there was no relationship between accumulation and time 
 ( kruskal  wallis , d.f .
= 3 , p > 0.05 ) , whereas ch3hgsed body burden was significantly related with time .
the 
 trend 
 was also significant when normalized to total hgsed ( as % ch3hgsed ) . because 99.8% ch3hgwater and 99.9% hgwater added to the water column
were recovered 
 in the bulk sediment , bafs for both enriched isotopes were calculated 
 as body burdens normalized to sediment concentrations .
the baf for 
 ch3hgwater was 682 
  442 ( unitless ) , whereas for ch3hgsed , the baf was 30  19 , such that uptake 
 of ch3hg from organic material of pelagic origin 
 was 23 higher than from algae mixed with the sediment . 
 for experiment 2 ,
bafs for hg were 0.9  0.2 for hgsed and 5.8  2.0 for hgwater , and bioaccumulation from 
 settling algae was only 6 higher than for sediment sources .
for the ch3hg experiment , 
 ir was determined to be 65 mg / g / day from ingestion of 
 algae added to the water column and 3.1 mg / g / day for ingestion of 
 algae mixed in with the sediment . for the hg experiment , 

ir was 30 mg / g / day for algae originating in the water column and 5.2 
 mg / g / day for sediment feeding .
the ir is a measure of the milligrams 
 of food consumed ( per body wt / day ) and should not vary with metal 
 species , although its calculation does depend on the accuracy of the 
 applied constants ( ae , kef , g ) as well as the experimentally derived food concentration and body 
 burden .
microcosm studies using 
 enriched stable isotopes showed trophic 
 transfer from sedimenting algae was a major source of hg to benthic - dwelling 
 amphipods .
this suggests that newly deposited phytoplankton is an 
 important and potentially overlooked source of hg to benthic infauna 
 in ecosystem studies and toxicity testing .
enriched stable 
 isotopes are a powerful tool for studying accumulation 
 of hg in aquatic food webs ; we applied this technique to a microcosm study to compare trophic 
 transfer of hg by two ingestion pathways .
microcosms were scaled down 
 from recommended conditions for culturing l. plumulosus , to minimize the quantity of enriched isotopes needed , but maintaining 
 recommended amphipod densities ( < 1.5 cm ) , sediment 
 depth ( 1 cm ) , and water conditions ( 20 ppt , 20 c , do > 4.4 
 mg
concentrations of ch3hg and thg in sediments and whole surface waters 
 in microcosms 
 were within ranges found in new england and estuaries worldwide , although dissolved 
 water concentrations were slightly higher .
body burdens of hg from 
 tracers were up to 6 higher than those found in new england 
 estuaries ( chen , unpublished : 17105 ng / g ) . in estuaries , 

sediment characteristics 
 and food quality have been shown to affect ae and kef in amphipods , as well as survival 
 and reproduction , but specific effects 
 on amphipod feeding behavior have not been reported . because amphipods 
 feed selectively , behavior may change with habitat and food availability . 

accumulation of hg species from sediment mixed with algae was shown 
 to be higher than from sediment alone , suggesting the abundance of high quality food spiked with hg in 
 this experiment may lead to higher accumulation in amphipods than 
 in a natural environment .
homogenized sediment and standardized conditions 
 used in this experiment allow for direct comparison of metal accumulation 
 from different feeding modes under optimal conditions .
algae 
 added to the water column was rapidly deposited to the sediment 
 surface , such that > 99.8% of ch3hg and hg were recovered from the 
 bulk sediment 
 within 12 h. concentrations of hg species added to the water column 
 varied over time , due to settling of algae added to the water , and 
 diffusion / suspension of tracers added to the sediment . variability 
 between samples was high at initial time points due to the capture 
 of large suspended particles during sampling . while sediment concentrations 
 were similar for both tracers added to the microcosm , spiked algae 
 originating in the water column was deposited to the sediment surface , 
 whereas the spikes originating in the sediment were thoroughly mixed 
 in the bulk sediment .
tracking speciation throughout the experiment 
 proved important 
 as methylation of hg spikes was observed . in radiometric 
 methods
, hg species can not be distinguished , and species transformation 
 alters the bioavailability of hg , which 
 could lead to inaccuracies in uptake modeling .
methylation of tracers 
 was observed in spiked algal cultures ( 0.70.8% ) and has previously 
 been observed in cultures of periphyton .
further methylation was also 
 evident during sediment incubation ( 23% ch3hg ) , despite maintaining oxic conditions throughout the experiment . 

this may also occur during toxicity testing , where sediment incubation 
 conditions are similar to those used here , altering the bioavailability 
 of hg to test organisms relative to natural environments .
because 
 the experiment focused on uptake , a short exposure duration 
 was used to minimize artifacts from algal decay , water quality changes , 
 and amphipod death / reproduction .
the body 
 burden of ch3hg reaches an apparent steady 
 state after 12 h in this experiment , although variability at each 
 time point was high , possibly due to feeding behavior , and to variable 
 metal distribution in the algal food source .
lawrence and mason reported 
 an apparent steady state in amphipods sampled at three and six days , 
 but calculated much longer exposures ( 50 day ) are needed to 
 achieve equilibrium .
the apparent steady 
 state in body burden may be due to the long time intervals ( 12 h ) 
 used in this study , relative to reported gut passage times of 35228 
 min for l. plumulosus fed i. galbana .
longer exposure 
 to hg sources may lead to higher accumulation if a true steady state 
 was not reached in this experiment , but body burdens represent relative 
 accumulations from different feeding pathways . because tracers were 
 ingested simultaneously and prepared from the same algal source , ae 
 and kef will be the same for both ingestion 
 pathways .
amphipods 
 in this study were not depurated , although for ch3hg , this is likely to have little effect on the determined body 
 burden due to its high ae where rate 
 of loss from excretion is negligible relative to growth dilution .
variability in body burden of hg tracers was high , 
 likely due to spatial variability of tracers in both food sources . 

the body burden of hgsed increased 
 significantly between time points , but hgwater did not .
the pattern may be explained 
 by preferential uptake of hgwater from settling algae during the first time interval ( t = 0 to 12 h ) , then increased consumption of hgsed as amphipods stir up sediment while they burrow . 

this may be explained by the 
 higher ae of ch3hg ( 80% ) than hg ( 6% ) , causing hg to be 
 excreted while ch3hg is mostly retained .
this 
 difference in assimilation should be consistent for both isotopic 
 tracers , however , but was not significant for ch3hgwater .
uptake 
 of hg tracers initially added to the water column and deposited on 
 the sediment surface was significantly higher than for tracers mixed 
 in the sediment , suggesting ingestion from the sediment surface is 
 the major feeding mode in amphipods .
while bafs are useful to normalize 
 body burden to different exposure concentrations , they can be difficult 
 to evaluate between different food sources and habitats . in this study , 
 because the same algal food source was added to both compartments , 
 bafs were used to compare relative uptake from different sources . 
 for both spikes
, bafs were determined using concentrations in amphipods 
 relative to the sediment ; this assumes that ingestion from the sediment 
 surface or the bulk sediment were distinct uptake pathways and ignores 
 accumulation from the water column .
conversely , if both sediment sources 
 of ch3hg were assumed to have the same baf , 
 then bafch3201hg+ ( for bulk sediment ) could be applied 
 to the concentration of ch3hgwater in the sediment compartment , to predict an amphipod 
 body burden of 45 ng / g ch3hgwater .
to 
 accumulate another 625 ng / g of ch3hgwater from the fraction of algae suspended in the 
 water , a 5 mg amphipod would have to ingest 3.9 ng of ch3hgwater ( assuming ae = 80% ) . 

whole water ch3hgwater concentrations were 2.7 to 16.5 ng / l ( 12 to 48 h ) , meaning that 
 only 0.15 to 1 ng of ch3hgwater was available for uptake from the entire in 60 ml water 
 compartments .
similar calculations suggest amphipods would need to 
 acquire 40 ng of hgwater from 
 suspended algae to achieve determined body burdens , whereas only 1.54 
 ng of hgwater were present in 
 the microcosms .
only a small portion of hg spikes was present 
 in the dissolved water fraction .
lawrence and mason found an inverse 
 effect of doc - binding on hg uptake and derived a laboratory - based 
 relationship between doc concentration ( mg / l ) and water bioaccumulation 
 factor ( wbaf ) : log wbafch3hg = 1.740.173[doc ] , where wbafch3hg is the 
 concentration of ch3hg in amphipods ( ng / g wet 
 wt ) divided by the dissolved concentration of ch3hg in the water column ( ng / l ) . using this equation ,
wbafch3200hg was estimated to be 7.5 for a doc concentration 
 of 5 mg / l measured in this study . for a dissolved ch3hg concentration of 2 ng / l ( experiment 1 ; time 
 points 12 to 48 h ) , amphipod concentrations are calculated to increase 
 100 ng / g ( dry wt , assuming 15% dry / wet weight ratio ) , which is less than 15% of the determined ch3hgbody burden .
wbafhg2 + is reportedly 
 10 times lower than wbafch3hg , under the same 
 conditions .
ng / l ( experiment 2 ; time points 12 to 48 h ) , 
 amphipod concentrations are calculated to increase 80 ng / g ( dry wt ) , 
 which is also 15% of the determined body burden .
uptake from both 
 dissolved and suspended algae sources was therefore considered minor 
 in this study , and while newly deposited algae may be considered part 
 of the sediment , this study suggests accumulation was much higher 
 from this new surface layer than from the bulk sediment . as 
 expected , bafs for ch3hg were significantly 
 higher than for hg . in phytoplankton
, ch3hg accumulates predominantly in the cytoplasm of algal cells , 
 whereas hg adheres to the cell membrane ; the fraction of metal in the 
 cytoplasm is attributed with higher ae during trophic transfer . comparing the bioaccumulation of the two hg 
 species ,
bafch3hg was relatively higher than 
 bafhg for tracers originating in the water 
 column than for sediment sources .
uptake of ch3hg into the cytoplasm of phytoplankton only occurs in live cells , whereas 
 both hg species are bound to the cell membrane in dead cells , causing lower assimilation of ch3hg during trophic transfer .
accumulation of ch3hg in some invertebrates ( copepods ) was shown to decrease 
 with decay of the marine algae , thalassiosira weissflogii ; however , ch3hg assimilation in l. plumulosis was not affected , which is thought 
 to be due to this species being well adapted to detrital feeding .
release of trace metals from decaying algae 
 decreases exponentially with time and varies between elements . in this study , death and decay
may have occurred 
 more rapidly in the phytoplantkon mixed in the sediment , which would 
 explain this relative difference , but the microcosm incubation time 
 was relatively short ( 48 h ) , and algal decay has been shown not to 
 affect ch3hg assimilation in amphipods .
although amphipods were rinsed , residual particle 
 binding may lead to adsorption of hgsed to the amphipod exoskeleton elevating apparent body burdens 
 from the sediment . given that hg is strongly particle 
 reactive
, adsorption of hgsed to the exoskeleton may have been higher 
 than for ch3hgsed , 
 causing the relatively higher baf from sediment sources .
to 
 advance biokinetic modeling studies , ir for both feeding pathways 
 of l. plumulosus were calculated .
values 
 of ir for surface feeding determined here are in reasonable agreement 
 with values for suspension feeding determined from five different 
 metal species using radiometric techniques ( 50 to 151 mg / g / day ) . in this study and in williams et al . , ir was 
 calculated using values of ae determined by radiometric techniques , which may have led to overestimation of ae for 
 hg if methylation of hg spikes occurred 
 during incubation .
this would lead to underestimation of ir , which 
 was lower for hg than for ch3hg in both studies .
no other studies report ir from deposit feeding 
 in amphipods , although shlekat et al . reported a single measurement 
 of 3 g / g / day , which is much higher than was observed here .
this may reflect variation in feeding behaviors 
 and effects of food substrate on test organisms .
phytoplankton rapidly accumulate ch3hg , but rapid algal growth 
 has been shown to dilute hg in algal food sources and therefore decrease 
 uptake into higher trophic levels .
deposition 
 of phytoplankton blooms is a major source of organic carbon flux to 
 sediment surfaces , and this study suggests 
 deposited algae are a source of hg species to primary benthic infaunal 
 consumers . in estuaries , increases in growth and reproduction of l. plumulosis have been correlated to chl 
 a production associated with sedimenting algae . in a study of c - labeled 
 tracers , however , phytodeposition was found to be a less prominent 
 food source than microphytobenthos production for benthic fauna , including 
 amphipods .
however , the study also demonstrated that pelagic organic 
 carbon sources were consumed during pulses which mimicked deposition 
 of spring phytoplankton blooms .
. 
 turbulence and resuspension of sediment can increase transfer of sediment 
 ch3hg into organisms , and sedimenting algal blooms have also been shown to increase ch3hg production in surface sediments .
previously , bioavailability of hg species in 
 estuaries has been inversely correlated with sediment organic matter .
this study suggests hg in suspended and freshly deposited algal 
 cells are an important uptake source , and that newly deposited algal 
 particles should be distinguished from bulk sediment organic carbon 
 as compartments controlling hg accumulation . in field collection , 
 bulk sediments are typically collected from the top 4 to 15 cm .
field 
 collection of amphipods is reported within the top 57 cm of 
 the sediment , but imaging of amphipod 
 burrows in sediment incubations show the highest burrow volume within 
 the top 1 cm of sediment .
our findings 
 suggest that amphipods feed primarily from the sediment surface , suggesting 
 much smaller sampling depths ( < 1 cm ) are appropriate for assessing 
 hg sources to benthic infauna .
l. plumulosus are routinely used 
 in both acute and chronic toxicity tests for estuarine sediments . in acute ( 10 d ) tests , sediments are the only food source , whereas 
 during 28 day chronic toxicity tests , l. plumulosus are fed by an external uncontaminated food source 23 times 
 per week . if in natural systems , algae 
 originating in the water column are a preferred food source , these 
 tests do not accurately assess exposure to contamination , as both 
 tests overlook the influence of contamination originating in the water 
 column , and acute toxicity tests ignore ingestion pathways completely . 

bioaccumulation varies with feeding behavior , suggesting uptake pathways 
 in test organisms need careful investigation to relate to sediment 
 and water quality criteria .
 mercury 
 is a widespread contaminant in marine food webs , and identifying 
 uptake pathways of mercury species , ch3hg+ and 
 hg2 + , into low trophic level organisms is important to 
 understanding its entry into marine food webs . 
 enriched stable isotope 
 tracers were used to study benthic vs. pelagic pathways of ch3hg+ and hg2 + uptake via food to the 
 infaunal estuarine amphipod , leptocheirus plumulosus . 
 algal cells differentially labeled with isotopically enriched ch3hg+ or hg2 + were added simultaneously 
 to the sediment and water column of microcosms , and hg species were 
 monitored in amphipods and in sediment and water compartments . 
 methylation 
 of hg2 + occurred during the course of the experiment , enhancing 
 the uptake of hg2 + spikes . 
 trophic transfer of hg from 
 algae added to the water column was determined to be the major uptake 
 route for amphipods , suggesting inputs of contaminated organic matter 
 from the pelagic zone are important to mercury bioaccumulation even 
 in organisms living in sediments . 