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Organic matter in marine sediments can be sufficiently labelled for ingestion experiments with deposit-feeders by allowing 14C-formaldehyde to bond chemically to it. Microbes, which otherwise would account for much of the I4C-formaldehyde uptake, are reversibly inhibited with 30 % NaC1. When incubated in 30 % NaC1, at least 95 % of the I4C uptake appears to be due to chemical bonding with sedimentary organics. There is a positive relationship between '4~-formaldehyde uptake and organic content of sediment. INTRODUCTION use of radiotracers to study the ingestion of natural sedimentary organic matter by deposit-feeders. SimiThe goal of this study was to develop a method of lar methods have been described by Banks and Wolfinradiolabelling sedimentary organic matter for use in barger (1981). feeding studies on deposit-feeding animals. Two For ingestion experiments, 2 conditions must be met approaches have been used previously for such by the radiolabelling procedure: The label must be studies: (1) Chemical/gravimetric analyses of natural associated with sedimentary organic matter, rather material (Newell, 1965; Hargrave, 1970; Lopez et al., than be adsorbed to surfaces or taken up by microbes, 1977); (2) radiolabelled 'synthetic' detritus (Kofoed, and the label should not migrate during the time 1975a; Tenore, 1977; Cammen, 1980). One disadvancourse of the feeding experiment. It is important to tage of the first approach is its lack of precision and note that the conditions for ingestion experiments are sensitivity in measuring small changes, a typical probless rigorous than those to study assimilation, which lem in estimating assimilation of organic debris by requires the added condition of knowing the distribuanimals. For example, it is usually impossible to distintion of radioisotope within the labelled substrate (Lamguish 5 % assimilation from 0 % assimilation. Chemipert, 1977; Banks and Wolfinbarger, 1981). cal/gravimetric analyses are not easily applied to study ingestion selectivity of organic matter, a subject of considerable interest in deposit-feeding research (Self MATERIALS AND METHODS and Jumars, 1978; Taghon et al., 1978; Lopez and Kofoed, 1980; Lopez and Cheng. 1982). The use of 14C-formaldehyde was chosen as labelling agent, radiolabelled detritus prepared in the laboratory because formaldehyde reacts with a wide variety of allows greater sensitivity for measuring small changes functional groups, perhaps the most common reaction (e.g. Kofoed, 1975a, b), but studies using such material being the formation of a cross-link of functional groups always suffer in extrapolation to the natural world. In with a methylene bridge (Pearse, 1968, pp. 70-73). this study we have developed methods which allow the Because preliminary experiments with phenoland heat-killed sediments suggested that microbial activContribution No. 318 from the Marine Sciences Research ity could ~ C C O U ~ ~ for most of the 14c-formaldehyde Center, State University of New York at Stony Brook uptake, this study was divided into 2 aspects: O Inter-Research/Printed in F. R. Germany 0171-8630/81/0008/0283/$ 02.00 Mar Ecol. Prog. Ser. 8: 283-289, 1982 (1) Development of a technique that inhibits microbial uptake of 14C-formaldehyde without interfering with physical/chemical uptake; (2) determining the relationship between physical/chemical uptake of 14C-formaldehyde and sedimentary organic matter. Microbial Inhibition with 30 % NaCl Distinguishing microbial from physical/chemical processes is a common problem in biogeochemical studies, and interpretation of results can hinge on the type of microbial control used (Brock, 1978). The probThe results of Experiment 1 suggested that microbial inhibition by 30 % NaCl was at least partly reversible. In Experiment 2, we investigated this apparent reversibility of inhibition. Sediment suspensions, prepared as in the previous experiment, were pre-incubated in either seawater or 30 % NaC1, then allowed to recover for 18 or 48 h before incubating with 14C-mixed amino acids. Incubation and sampling were performed as in the previous experiment. Experiment 3: Effect of Incubation Temperature and 30 % NaCl on 14C-formaldehyde Uptake lem is obvious in this study; clearly, formaldehyde An independent method of determining the effect of fixation is not suitable. Furthermore, because the pur30 % NaCl on microbial activity, in this case on 14Cpose of this study is to develop methods for use in formaldehyde uptake, is to compare uptake in 30 % feeding experiments, the labelling method should not NaCl and in seawater over a wide range of incubation leave toxic residues in the sediment. Therefore, we temperatures (Brock, 1978). If 30 % NaCl is an effechave investigated the efficacy of a concentrated solute, 30 % NaCl, as an inhibitor of microbial activity (Brock, tive inhibitor of microbial activity, the seawater sam1978). ple should exhibit higher uptake (microbial + physical/chemical) than the 30 % NaCl sample (physical/ chemical only) when incubated at non-lethal temperaExperiment 1: Effect of 30 % NaCl on 14C-mixed tures. At temperatures that inactivate or kill microbes, Amino Acid Uptake by Marine Sediment seawater and 30 % NaCl samples should display the same activity. Samples of a sediment suspension were This experiment was conducted to determine whether 30 % NaCl affects microbial activity associated with a marine sediment, as measured by incorporation of 14C-mixed amino acids (U-14C protein hydrolysate, 57 mCi mAtom-l, Amersham) from solution. A sediment suspension was prepared from the light fraction of an intertidal muddy sediment. Sediment suspensions (5 ml, 65 mg dry weight) were pre-incubated for 1.75 h in either seawater, 30 % NaClboth of which were filtered (0.45 pm) before use or a HgC1, solution (90 yg ml-l seawater). Samples were centrifuged and resuspended in one of the solutions and allowed to sit for 15 min. 14C-mixed amino acids (0.5 yCi) were then added to each sample. After l h incubation at 25 'C, subsamples were filtered onto Nuclepore filters (1 pm pore size, 25 mm diameter). Filters were placed in glass liquid scintillation vials and solubilized in 0.5 m1 tissue solubilizer (NCS, Amersham) for 16 h at 45 'C. After adding 10 m1 organic scintillant (OCS, Amersham), samples were counted on a Nuclear-Chcago Mark I1 liquid scintillation counter, and counts were corrected to dpm using the external standard ratio method with quench standards prepared with NCS-sediment quenching agent. Experiment 2: Effect of Varying Recovery Periods Following 30 % NaCl Treatment on Microbial Uptake of I4C-mixed Amino Acids centrifuged and resuspended in l 0 m1 seawater or 30 % NaC1, and then pre-incubated at experimental temperatures (2, 8, 23, 34, 60, and 75 "C) for 1 h before addition of 14C-formaldehyde. The stock solution of 14C-formaldehyde was prepared by diluting 500 pCi (10 mCi mmol-l, 1-3 % aqueous solution, Amersham) in 100 m1 distilled water (stock concentration 5 X l O P 4 M). After preincubation, 0.2 m1 (1 yCi) 14C-formaldehyde was added to each sample, which was then stoppered, shaken and incubated at the experimental temperatures for 44 h. After incubation, samples were centrifuged and resuspended in seawater or 30 % NaC1, according to treatment, to remove unincorporated '4C-formaldehyde. This step was important because 14C-formaldehyde binds with filters when the suspension is filtered during sampling. Subsamples were then filtered onto glass fiber filters (Whatman, GF/A, 25 mm), rinsed with 5 m1 seawater or 30 % NaCl, and prepared for liquid scintillation counting as described above. Relationship Between Physical/Chemical Uptake of '4C-formaldehyde and Sedimentary Organic Matter Experiment 4: Effect of Sedimentary Organic Matter on '4C-formaldehyde Seven sediments ranging from highly organic mud to clean sand were rinsed in distilled water and dried Lopez and Crenshaw: Radiolabelling of sedimentary matter 285 Table 1. Experiment 1: Effect of 30 % NaCl on I4C-mixed amino acid uptake by marine sediment Condition Pretreatment Treatment dpm % of I (1.75 h) (1 h) (X f s.d., n = 2) I seawater seawater 168,192 100 t 27,370 11 HgC1, HgC1, 2,145 1 3 2 l 1 I11 HgCI2 seawater 2,469 1.5 + 15 IV 30 % NaCl HgC12 1,647 1 .O + 199 V 30 % NaCl 30 % NaCl 3,125 1.9 f 383 V1 30 % NaCl seawater 60,496 36.0 f 3,618 at 105 "C. Portions of each sediment were then ashed at 500 "C for 2 d. Dried and ashed sediments (0.3 cm3) were then placed in 10 m1 30 % NaCl and allowed to rehydrate for 2 d. I4C-formaldehyde (0.5 pCi) was added, and samples were incubated at 25 "C for 45 h. Samples were then centrifuged and resuspended in 30 % NaCl. Muddy sediments (1, 2, 3 ,4 ) were subsampled by filtering portions onto preweighed Nuclepore filters. Sandy sediments were sampled with small, preweighed aluminium planchets (Rublee and Dornseif, 1978). After drying and weighing, subsamples were placed in scintillation vials, dampened with water, and then solubilized with tissue solubilizer and prepared for counting. Organic content of sediments was estimated by weight loss upon ashing. Experiment 5: Loss of I4C from Sediment Labelled with I4C-formaldehyde A fresh sediment suspension in 30 % NaCl was labelled for 44 h with I4C-formaldehyde, rinsed and transferred to seawater. Subsamples were then taken, beginning 5 h after transferring. Subsampling continued for 4 d. Sampling and counting procedures were similar to those used in Experiment 4.

Radiolabelling of Sedimentary Organic Matter with 14C-Formaldehyde: Preliminary Evaluation of a New Technique for Use in Deposit-Feeding Studies