Environ. Sci. Technol. 2000, 34, 4936-4942
Transfer of Benzo[a]pyrene and 2,2′,5,5′-Tetrachlorobiphenyl from Bacteria and Algae to Sediment-Associated Freshwater Invertebrates THOMAS L. BOTT* AND LAUREL J. STANDLEY Stroud Water Research Center, Avondale, Pennsylvania 19311
Feeding interactions between microorganisms and their grazers range from broad and general to very specific. Here we examined routes of transfer of [3H]benzo[a]pyrene (BaP) and 2,2′,5,5′[14C]tetrachlorobiphenyl (PCB-52) from microorganisms in freshwater sediments to oligochaetes (Lumbriculus variegatus) and chironomid larvae (either Stictochironomus sp. or a mix of smaller taxa) when exposed to the compounds added either directly to sediments or to bacteria or diatoms previously labeled and then added to sediments. The appearance of radiolabel in animals after a gut clearing step to differentiate between ingested and absorbed compound was followed in time course experiments. Relative to the added radiolabel, BaP concentrations were greater than PCB concentrations in L. variegatus and were greater in animals fed radiolabeled sediments or bacteria than those offered diatoms. In contrast, the chironomids accumulated more PCB than BaP. The mix of small chironomids bioaccumulated more PCB when fed prelabeled algae than when fed sediment or bacteria. However, Stictochironomus sp. bioaccumulated more from sediments and/or bacteria. Food selection influences pathways of contaminant transfer, even to small animals at the base of the food web. We also tested whether the bioaccumulation of BaP and PCB would be predicted by the Koc for the sediment (i.e., BCF/Koc ) 1). The quotients, averaged over experiments, were 1.08 and 1.53 for PCB-52 and BaP, respectively, but error terms were large, with coefficients of variation being 83% and 135%, respectively.
Introduction Sediments can be reservoirs for contaminants ranging from heavy metals to toxic organic compounds. Benthic in- and epifauna exposed to these substances in turn can be important links in the transfer of contaminants to higher organisms such as fish. The bioavailability of organic contaminants to benthic animals is influenced by physical and chemical properties of the compounds in question and of the sediment, particularly its organic matter content (1) and composition (2, 3), pH (4) and grain size distribution, and the degree of aging of the contaminant (5, 6). Organisms in aquatic environments acquire contaminants both from the bioconcentration of dissolved compounds and * Corresponding author phone (610)268-2153 (ext. 224); fax: (610)268-0490; e-mail:
[email protected]. 4936
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the ingestion of contaminated materials, either deliberately or incidentally, during feeding. Transfer of contaminants to top carnivore fish was related to the body burdens in prey (7, 8) and benthic feeding fish acquired PCBs from copepods, although even greater doses were obtained from sediment (9). Contaminant body burdens in benthic infauna were related to the size range of ingested particles which is determined by the mouth size of the organism (10) although feeding rate, absorption, and assimilation efficiencies all affect bioaccumulation from food resources (11). Landrum et al. (12) provide an excellent review of many of these considerations. In addition to the ingestion of bulk sediment, selective feeding on microbial food resources, e.g., bacteria and algae, occurs even among benthic organisms as small as oligochaetes (13-15), rotifers (16, 17), copepods (18, 19), and chironomids (20, 21). Much available information has been summarized by Bott (22). Just as for higher trophic levels, if food resources for these groups are contaminated, selective feeding will affect body burdens and pathways of contaminant transfer to them and their predators. Even for protozoa, 75% of the increase in bioconcentration of a hexachlorobiphenyl was attributed to grazing on bacteria (23). Here we examined the influence of two potential microbial food sources, bacteria and algae, on the bioaccumulation of benzo[a]pyrene (BaP) and 2,2′,5,5′-tetrachlorobiphenyl (PCB52) by two groups of sediment associated invertebrates, oligochaetes and chironomids. We focused on microorganisms and their consumers, which, while invisible to the naked eye and at the base of the food web, are extremely active metabolically and serve as points of entry for toxics into the food web where they can be transferred to higher organisms. In addition, we examined whether the bioaccumulation of BaP and PCB-52 could be accurately predicted from the Koc of the sediment: the premise of the equilibrium partitioning model (1).
Materials and Methods Study Animals. The oligochaete Lumbriculus variegatus, a common toxicity testing organism, was obtained from Aquatic Research Organisms, Hampton, NH. Chironomids were collected from White Clay Creek (Chester Co., PA). Two experiments were performed with Stictochironomus sp. and one with a mix of specimens belonging to the subfamilies Chironominae, Orthocladiinae, and Tanypodinae with estimated occurrences of approximately 55%, 35%, and 10%. Taxonomic resolution was based primarily on head capsule characteristics. L. variegatus is a burrowing organism, ingesting fine sediments and associated organic matter. Stictochironomus sp. is a collector-gatherer, feeding on fine particle detritus and associated microorganisms including algae, as are taxa belonging to Chironominae and Orthocladiinae; Tanypodinae are commonly predators (24). Surface sediments were aspirated from the streambed with a meat baster into an Erlenmeyer flask. Animals were eluted from the sediment using the swirl and decant method and then picked from associated fines into a holding vessel. Sediment Sources, Collection and Preparation. Surface sediments (∼1500 cm3) used in experiments were collected to a depth of ∼5 mm from White Clay Creek and from Cocalico Creek (Lancaster Co., PA). Sediments were sieved (2 mm mesh size) to remove macrofauna and large pebbles, picked free of macro- and meiofauna under a dissecting microscope, and allowed to settle overnight. After decanting overlying water, the sediment was mixed with an electric mixer and dough hook. A portion of the defaunated sediment was 10.1021/es001239i CCC: $19.00
2000 American Chemical Society Published on Web 10/25/2000
reserved for the gut clearance step of the feeding experiment. The remainder was amended either with radioactive compound or radiolabeled microbes for use in the feeding experiment. Labeling of Microorganisms and Sediments with [3H]BaP and [14C]PCB. [3H]Benzo[a]pyrene (sp. act. ) 268 mCi/ mg, Amersham Life Science, Arlington Heights, IL) and 2,2′,5,5′-[14C]tetrachlorobiphenyl (sp. act. ) 0.0418 mCi/mg, Sigma, St. Louis, MO) were transferred from toluene to dimethyl sulfoxide (DMSO) before use. The specific activity of the BaP was reduced to 0.0455 mCi/mg by adding nonradioactive compound. The nonradioactive BaP was dissolved in DMSO when small (e.g., µL) volumes were required for the labeling of bacteria or algae. However, sediments required larger BaP additions and were dry-spiked by dissolving the BaP in toluene, removing the toluene by rotary evaporation (which left the BaP adhering to the container walls), adding the sediment, and mixing thoroughly. This procedure eliminated excessive DMSO addition. Radiolabeling of Bacteria. A bacterial strain isolated from oil-contaminated soil was grown in 1 L of 0.05% tryptone yeast extract (TYE) broth containing 5 µCi [3H]BaP and 5 µCi [14C]PCB per L. After maximum turbidity was reached, the culture was pasteurized at 60 °C for 2 h. Cells were harvested by centrifugation (16 264g for 20 min), washed repeatedly (10 000g for 10 min) to remove unincorporated radioactivity, and resuspended in 5 mL of filter sterilized streamwater. The pellet was immersed for 20 min in a sonic bath to break it up after which three samples (250 µL each) were collected for radioactivity assay, and three were collected and fixed in 2% formalin for epifluorescence microscopic counts of cell number. Radiolabeling of Algae. Algal communities (dominated by diatoms and lesser amounts of desmids) were grown on roughened acrylic plates (5 cm × 5 cm) held in flowing streamwater under artificial illumination in the laboratory. Well-developed growths were treated with either a 1:4 or 1:6 dilution of a nematicide (Nemacur 3, Bayer, Agricultural Div., Kansas City, MO) which agitated invertebrates that infested the algal growths. After treatment, they were washed from the surface of the growth with a stream of water. Each treated plate was then put in a sterile dialysis bag with filter sterilized streamwater and placed in an indoor experimental stream for continued growth. The algal community was simplified by the treatment but was now free of invertebrates. Chlorophyll a concentrations on Nemacur-treated plates immediately following treatment were similar to those on untreated plates (9473 vs 8362 µg/g dry weight, respectively). Concentrations increased to 11 548 µg/g after 3 weeks of continued incubation in the ecosystem stream, indicating that a significant portion of the algal community remained viable. The regrown algal community was exposed to 7.5 µCi each of [3H]BaP and [14C]PCB in a Petri dish with 15 mL of filter sterilized streamwater. After 8-10 days of incubation in the laboratory the growth was scraped from plates, recovered by centrifugation (10 000g for 10 min), and washed repeatedly to remove unincorporated radiolabel. The algae were resuspended in 5 mL of sterile streamwater and treated in the sonic bath for 20 min to break up the pellet. Samples were collected for algal cell counts and radioactivity determinations. Radiolabeling of Sediment. [3H]BaP and [14C]PCB (13.64 µCi each) were mixed with a dough hook into ∼300 g of defaunated sediment to provide final concentrations of 1 µg/g wet weight (1 ppm) each. Earlier work with L. variegatus showed that this concentration was sublethal and yet resulted in measurable body burdens (4). The sediment was held for 10 days at 4 °C and remixed before use. The radiolabeled
sediments were amended with nonradioactive bacteria and algae as required to standardize microbial densities among treatments. Feeding Experiments with Lumbriculus variegatus. Radiolabeled sediments or sediments amended with either radiolabeled bacteria or diatoms were dispensed into vials to a depth of 2 cm. The bulk sediment was mixed continuously as the sediments were dispensed to ensure that each vial received sediment of similar size distribution. Six oligochaetes (L. variegatus) were added to each vial followed by 2-3 mm of white sand which was overlain with 1 mL of streamwater. Animals selected were in the mid-size range of those available because larger animals are known to stop feeding prior to reproduction by fragmentation and small animals refrain from feeding for the first 2-7 days following division (25). Fecal pellets produced by the oligochaetes were deposited on top of the sand, preventing their reingestion and facilitating their collection. Three vials of each treatment (sediments, bacteria, diatoms) were sampled after 2, 24, 48, 96, and 168 h (7 days) of incubation at room temperature. Three of the oligochaetes removed from each vial were put directly into water to release adhering sediment after which they were transferred to tared aluminum weigh boats. Three animals were transferred to clean defaunated sediment for gut clearance (16 or 4 h in experiments 1 and 2, respectively) after which animals were washed free of adhering sediment. Sediment samples from each vial were collected at each sampling time for counts of bacteria and algae and radioactivity determinations. Animal and sediment samples were dried, weighed, and analyzed for radioactivity. Fecal Pellet Collection. At selected times the fecal pellets produced by the feeding animals over a 24 h period were collected using a Pasteur pipet. Some of the sand was unavoidably picked up in the process. It was removed by transferring each sample to a conical centrifuge tube in which the sand settled to the bottom allowing the fecal pellets to be easily removed. Water removed in the process was added back to the experimental vial after filtering it through an Acrodisc filter. The pellets were dried, weighed, and analyzed for radioactivity. In a separate experiment with nonlabeled sediments, we collected fecal pellets from animals exposed to sediments
