Short-term lethality and sediment avoidance assays with endrin-contaminated sediment and two oligochaetes from Lake Michigan.

Mean 96-hr LCs0 values and standard deviations for the oligochaetes S. heringianus and L. hoffmeisteri exposed to endrin-contaminated sediment were 2,588 _ 1,974 txg/g dry weight sediment for 4 assays and 2,725 + 955 Cg/g for 2 assays, respectively. Mixed species testing data suggested that the toxicity to L. hoffrneisteri was reduced in the presence of S. heringianus, yet further testing is required. Ninety-six hour ECs0 burrowing avoidance values for both species (19 and 15.3 txg/g for S. heringianus and 59 ~xg/g for L. hoffmeisteri) were approximately 46 and 150 times lower than their respective mean 96-hr LCs0 values. Both S. heringianus and L. hoffmeisteri initially burrowed in contaminated sediment and then returned to the surface in numbers somewhat proportional to the sediment concentration and the length of exposure. Future use of oligochaete behavioral responses to sublethal sediment contamination for pollutant impact on benthic communities is promising. Toxic compounds with high partition coefficients that enter the Laurentian Great Lakes adsorb significantly to fine particles and settle to the bottom. The fate of these compounds and their interactions with the larger benthic organisms is largely unknown. Compound fates normally depend on complex combinations of sediment and xenobiotic chemical/physical properties and the activity pat1 To whom correspondence should be addressed (Univ. of Mich.) terns of the larger benthos (Petr 1977). Oligochaetes are of particular interest because they are frequently the dominant macrobenthic taxa, and because they rework (mix) sediments in a conveyorbelt fashion (Davis 1974; Krezoski et at. 1978; Robbins et al. 1979; McCall and Fisher t980; Robbins 1982; Krezoski and Robbins 1985; Robbins 1986), which can result in profound alterations of sediment characteristics (Robbins 1982; Fisher 1982; McCall and Tevesz 1982). Despite the importance of the oligochaetes, very few studies have examined contaminated sediment-oligochaete interactions. In previous freshwater oligochaete studies, contaminants were usually placed in solution, either without a sediment substrate or in the presence of uncontaminated sediment. The responses of several freshwater otigochaete species under these conditions have been determined for cadmium, mercury, pentachlorophenol, pulp mill effluent, and sewage sludge in water, and in water and uncontaminated sediment under a range of dissolved oxygen and temperatures (Chapman et al. 1982a, 1982b, 1982c). Death and changes in respiration were used as the measures of response. In most experiments, 96-hr LCs0 values increased (toxicity was decreased) when uncontaminated sediments were added. Survival was also enhanced and a decrease in community respiration was observed when Limnodrilus hoffmeisteri (Tubificidae) and Tubifex tubifex (Tubificidae) were exposed together to the above pollutants without sediment (Chapman et al. 1982a). Additionally, the toxicities of twentythree insecticides to Branchiura sowerbyi (Tubificidae) were generally reduced when uncontaminated sediment was added to test solutions (Naqvi 96 T.J . Keilty e t al. 1973), as were the toxicities of heavy metals and nitroaromatic compounds to Lumbriculus variegatus (Lumbriculidae) (Bailey and Lui 1980). Oligochaetes were exposed directly to contaminated sediments in only one study (Karickhoff and Morris 1985). The intent of their study, however, was to examine the role of tubificids in toxicant transport, and not to determine lethal and sublethal responses by the organisms. By virtue of their persistence and relative toxicity, compounds with high partition coefficients that sorb to the fine grained, organic fraction of the sediments pose serious potential health hazards to man. Although benthic organisms are an integral part of the aquatic food web, lethal testing of oligochaetes in contaminated sediments has not been reported. Contaminated sediments should afford a more natural testing medium than water (Chapman et al. 1982b) and provide an optimal medium for exposures to hydrophobic toxicants. Use of contaminated sediments also allows potential sublethal responses to be examined and quantified, such as changes in burrowing behavior at exposure levels approaching levels that have been found in the environment. The objective of this research was to compare the lethal and behavioral responses, including an ancillary examination of mixed species effects in lethality tests, to a sediment bound toxicant by two freshwater oligochaetes: Stylodrilus heringianus (Lumbriculidae) and Limnodrilus hoffmeisteri (Tubificidae). Materials and Methods The chlorinated pesticide endrin (1,2,3,4,10,I0, hexachloro-6,7epoxy-1,4,4a,5,6,7,8,8a-octahydro1,4-endo,endo-5,8-dimethanonaphthalene) was chosen because of its sorption characteristics (log kow = 5.6, Neely et al. 1974), its toxicity to other aquatic species (Grant 1976), and its availability in a commercially radiolabeled form (Pathfinder Laborator ies , Inc, St. Louis, MO). Although concentrations in the sediments of the Great Lakes today are negligible (Frank et al. 1981a, 1981b), endrin represents one of hundreds of highly-sorbed, potentially toxic compounds. Lake Michigan sediments were collected in October, 1983, with a Ponar grab approximately 10 km offshore from St. Joseph, Michigan, at a water depth of 42 m. To create a uniform sediment for experiments, sediments were dried at 60~ passed through a 0.25 mm mesh sieve, and thoroughly mixed. Sediments were reconstituted as needed with lake water and a few ml of fresh sediment to provide an active bacterial flora. Worms were collected at the sediment collection site in March or April of t984, 1985, and 1986 and maintained in the dark at 10~ in 200 L aquariums for a minimum of 1 month prior to use. Aquarium sediments were gently sieved (0.5 mm mesh) to concentrate worms. A fiber-optic light (to prevent unnecessary heating) and a dissecting microscope were used to sort and identify worms. For each exper imental concent ra t ion , endrin: 14C-endrin (1000:1) was added to reconstituted sediments via < 1 ml acetone carrier (< 1 ml acetone carrier also added to controls), and the mixture was stirred for 24 hr in 2 L Lake Michigan water. After settling 72 hr, overlying water, containing the acetone, was aspirated off and enough fresh aerated lake water was added back to the sediments (and restirred for approx. 10 min) to allow the resulting slurry to be poured equally into 50 ml Griffen beakers. The slurries were allowed to settle for 72 hr at 10~ Each beaker received approximately 25 g dry weight sediment and 25 ml water. Five to fifteen worms/beaker were added at this time. All experimental concentrations and controls were run in triplicate except where noted. Four 96-hr lethal and two 96-hr sediment avoidance assays were completed between October 1984 and August 1986. Two of the 96-hr lethal assays included both single and mixed species testing. Death was denned as absence of color and response to touch, and an unmistakable degree of body degeneration. Observations of burrowing behavior were made at 0.17, 0.5, 2, 8, 16, 24, 48, and 96 hr during one ECs0 assay for time series analysis. A worm was considered unburrowed if greater than an estimated 75% of its body was exposed on the sediment surface. In single species lethality tests (assays 1-4), 10 worms/beaker (30 total/concentration) were used. Five worms/species/beaker were used in one mixed species LCs0 assay and 10 worms/ species/beaker in a follow-up assay (run simultaneously with single species assays 3 and 4, respectively) to better define a potential mixed species response. Fifteen worms/beaker (45 worms total/concentration) were used in the first sediment avoidance ECs0 assay, and 10 worms/beaker (20 worms total/ concentration) were used in the second assay. Fewer worms/ beaker were used in the second avoidance assay to facilitate counting. Worms for the second sediment avoidance assay were added individually, and spaced out over the sediment surface. In all other assays, worms were temporarily placed in 20 ml vials of aerated lake water until sufficient numbers were obtained to begin an experiment. Typically, the worms formed a 'ball' which was gently added to each beaker. At higher concentrations, even though some worms burrowed and then emerged from the sediments, worms on the surface remained loosely entangled making exact counts difficult. The protocol of 10 worms/beaker in the second ECs0 assay eliminated this difficulty. Twenty worms (duplicate instead of triplicate replication) were used in the second assay. All experiments were conducted in an environmental chamber maintained at 10~ in the dark. Darkness was maintained to eliminate light-stimulated burrowing responses (White unpublished data). Overlying water in each beaker was not replaced during experiments. In sediment avoidance experiments, counts of worms on the sediment surface were made under a dissecting microscope when necessary, and beakers immediately returned to the chamber. Post experimental sediment endrin: 14C-endrin concentrations were determined by liquid scintillation, using a Packard 460 C counter. Endrin was removed from the sediment by 8-hr Soxhlet extraction in 240 ml hexane and 60 ml acetone (Sharom et al. 1980). Extraction volumes were reduced with a Buchler flash evapora tor to approximately 1 ml to concen t ra te samples. Liquid scintillation determinations were initially verified by gas chromatography (Varian Aerograph Series 1200, column temp. Oligochaete Lethality and Avoidance Assays 97 Table 1. Measured sediment endrin concentrations in lxg/g dry weight sediment for four 96-hr LCso assays and two 96-hr sediment avoidance ECso assays 96-hr LCso assay Sediment Concentrations 1 0, 72, 251,298,512, 829, 1092 2 0, 121,279, 421,982 3 0, 4.4, 91,432, 1352 4 98,414, 801, 1848 96-hr ECs0 assay Sediment Concentrations 1,2 0, 1.2, 10.7, 23.9, 52.4, 77.2, 104.1 210~ detector temp. 230~ Clean-up of samples for gas chromatographic analyses was with Florisil | and followed the procedure in the Pesticide Analytical Manual (1977). Prior to use, the radiopurity of the labeled endrin was determined by thin layer chromatography (Patil et al. 1970) and was found to be >98%. ECs0 arid LCso data with upper and lower 95% confidence limits were generated, using the Litchfietd and Wilcoxon (1949) log probit nomographic method. Table 2. Ninety-six hour LC5o values (~xg/g endrin dry wt. sediment) with upper and lower 95% confidence limits for Stylodrilus heringianus and Limnodrilus hoffmeisteri in single and mixed tests 95% Confidence Limits Species LCs0 (txg/g) Upper Lower S. heringianus Assay 1 140