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Archives of Environmental Contamination and Toxicology (v.47, #4)
Polar Organic Chemical Integrative Sampling and Liquid Chromatography–Electrospray/Ion-Trap Mass Spectrometry for Assessing Selected Prescription and Illicit Drugs in Treated Sewage Effluents by T. L. Jones-Lepp; D. A. Alvarez; J. D. Petty; J. N. Huckins (pp. 427-439).
The purpose of the research presented in this paper was twofold: (1) to demonstrate the coupling of two state-of-the-art techniques: a time-weighted polar organic chemical integrative sampler (POCIS) and microliquid chromatography–electrospray/ion-trap mass spectrometry and (2) to assess the ability of these methodologies to detect six drugs (azithromycin, fluoxetine, omeprazole, levothyroxine, methamphetamine, methylenedioxymethamphetamine [MDMA]) in a real-world environment, e.g., waste water effluent. In the effluent from three wastewater treatment plants (WWTPs), azithromycin was detected at concentrations ranging from 15 to 66 ng/L, which is equivalent to a total annual release of 1 to 4 kg into receiving waters. Detected and confirmed in the effluent from two WWTPs were two illicit drugs, methamphetamine and MDMA, at 2 and 0.5 ng/L, respectively. Although the ecotoxicologic significance of drugs in environmental matrices, particularly water, has not been closely examined, it can only be surmised that these substances have the potential to adversely affect biota that are continuously exposed to them even at very low levels. The potential for chronic effects on human health is also unknown but of increasing concern because of the multiuse character of water, particularly in densely populated, arid areas.
Use of Zeolite for Removing Ammonia and Ammonia-Caused Toxicity in Marine Toxicity Identification Evaluations by R. M. Burgess; M. M. Perron; M. G. Cantwell; K. T. Ho; J. R. Serbst; M. C. Pelletier (pp. 440-447).
Ammonia occurs in marine waters including effluents, receiving waters, and sediment interstitial waters. At sufficiently high concentrations, ammonia can be toxic to aquatic species. Toxicity identification evaluation (TIE) methods provide researchers with tools for identifying aquatic toxicants. For identifying ammonia toxicity, there are several possible methods including pH alteration and volatilization, Ulva lactuca addition, microbial degradation, and zeolite addition. Zeolite addition has been used successfully in freshwater systems to decrease ammonia concentrations and toxicity for several decades. However, zeolite in marine systems has been used less because ions in the seawater interfere with zeolite’s ability to adsorb ammonia. The objective of this study was to develop a zeolite method for removing ammonia from marine waters. To accomplish this objective, we performed a series of zeolite slurry and column chromatography studies to determine uptake rate and capacity and to evaluate the effects of salinity and pH on ammonia removal. We also assessed the interaction of zeolite with several toxic metals. Success of the methods was also evaluated by measuring toxicity to two marine species: the mysid Americamysis bahia and the amphipod Ampelisca abdita. Column chromatography proved to be effective at removing a wide range of ammonia concentrations under several experimental conditions. Conversely, the slurry method was inconsistent and variable in its overall performance in removing ammonia and cannot be recommended. The metals copper, lead, and zinc were removed by zeolite in both the slurry and column treatments. The zeolite column was successful in removing ammonia toxicity for both the mysid and the amphipod, whereas the slurry was less effective. This study demonstrated that zeolite column chromatography is a useful tool for conducting marine water TIEs to decrease ammonia concentrations and characterize toxicity.
The Effects of Sieving and Spatial Variability of Estuarine Sediment Toxicity Samples on Sediment Chemistry by D. J. Fisher; B. L. McGee; D. A. Wright; L. T. Yonkos; G. P. Ziegler; S. D. Turley (pp. 448-455).
In 1998, we conducted a field-validation study of the chronic 28-day whole-sediment toxicity test with Leptocheirus plumulosus in Baltimore Harbor, MD, an area where this amphipod is indigenous. This study included an evaluation of the effect of sieving on sediment chemical concentrations and the use of field replicates, or separate grabs from the same site, which provided an estimation of within-site chemical and toxicologic variability. Six stations in Baltimore Harbor, MD, were included in this evaluation. Chemical analysis of two separate unsieved field replicates from the six sites indicated that, overall, the chemical concentrations of replicates within each site were similar, especially for metals. Organic contaminants particularly total PCBs, had the highest variability between replicates. Chemical variability did not appear to be related to differences in organic carbon content or grain size or to variability in toxicologic end points. Results supported the use of composite samples in sediment toxicity tests. In addition, in most cases, sieving had little effect on sediment chemistry. For the metals and trace elements, only selenium showed a substantial change after sieving, with some samples increasing after sieving and others decreasing. Concentrations of acid-volatile sulfide (AVS) increased 194.6% at one station after sieving, although in most other cases, AVS and simultaneously extracted metals remained relatively unchanged. As expected, concentrations of organics generally decreased after sieving, but in the majority of cases this decrease was small (i.e., coefficient of variation ≤25%). Total benzene hexachloride and total chlordanes had the greatest changes, whereas polychlorinated biphenyl concentrations decreased at only two stations after sieving. Concentrations of polyaromatic hydrocarbons showed little change after sieving. These changes in sediment chemistry due to sieving must be viewed in the larger context of the potentially confounding effects that indigenous organisms may have on the interpretation of test results from whole-sediment toxicity tests.
Toxicity and Estrogenic Activity of a Wastewater Treatment Plant in Northern Italy by T. Schiliró; C. Pignata; E. Fea; G. Gilli (pp. 456-462).
Many xenobiotics, widely diffused in the environment, have the potential to disrupt the delicate endocrine system balance of wildlife and humans. Substances showing hormone-like actions in living organisms are defined endocrine-disruptor chemicals and they may mimic, block, or interfere with the synthesis, release, transport, elimination, and binding of natural hormones. Different ecotoxicologic studies were applied to screen for the discharge of estrogenic and toxic substances into the aquatic environment from a small industrial wastewater treatment plant based in Northern Italy. Samples of wastewater (3 L) were taken at three different periods during the year from different points of the plant’s process: three phases of treatment, and effluent. Untreated samples (1 L) were assayed with two toxicity tests: Microtox and Daphnia magna. A simplified proliferation test using human estrogen receptor–positive MCF-7 breast cancer cells (E-screen assay) was performed on 2-L samples after solid-phase extraction for the determination of total estrogenic activity; the presence of estrogenic substances was evaluated by measuring the proliferation rate in cells exposed to samples. The results of the Microtox assay employing the marine bacterium Vibrio fischeri were compared with data obtained from whole-effluent toxicity testing methods employing D. magna. Toxicity was found only for the influent point. The proliferative effect of the five points, relative to the positive control 17β-estradiol (relative proliferation effect), was between 2.4% and 39%, and the 17β-estradiol equivalent concentrations were between 0.06 and 3.94 ng/L. A first comparison between results from toxicity load and estrogenic activity was performed. At all process points, the effluent samples were considered acceptable (taking only toxicity into account), but we demonstrated that they had estrogenic activity in the river water indicating an input of estrogenic substances by way of the treatment plant.
Comparative Embryonic and Larval Developmental Responses of Estuarine Shrimp (Palaemonetes pugio) to the Juvenile Hormone Agonist Fenoxycarb by C. L. McKenney Jr.; G. M. Cripe; S. S. Foss; S. R. Tuberty; M. Hoglund (pp. 463-470).
Grass shrimp (Palaemonetes pugio) were reared separately through both embryonic and total larval development during exposure to fenoxycarb at measured concentrations of <2.2 to 888 μg L−1. A fenoxycarb concentration of 888 μg L−1 significantly (p < 0.05) inhibited embryonic development to larval hatching and extended the embryonic developmental period from 11.9 to 12.7 days. Exposure to fenoxycarb concentrations ≤502 μg L−1 had no significant (p > 0.05) effect on complete embryonic development. Significantly fewer shrimp successfully metamorphosed to postlarvae when exposed through complete larval development to fenoxycarb concentrations ≥4 μg L−1. Larval development of grass shrimp was therefore >2 orders of magnitude more sensitive to this juvenile hormone agonist than was embryonic development. Viability of larvae developing in fenoxycarb was concentration dependent. Development beyond third zoeal stage was significantly inhibited at fenoxycarb concentrations ≥190 μg L−1, whereas development beyond fourth zoeal stage was inhibited by a concentration of ≥45 μg L−1. Fenoxycarb exposure of developing larvae did not alter either the duration of total larval development or the total number of larval stages before metamorphosis. Rearing of fenoxycarb-exposed embryos through larval development without further exposure had no significant effect on number of larval stages, larval development rate, or metamorphic success of larvae. Similarities in the sensitivity of grass shrimp larvae and mosquito larvae to fenoxycarb suggests that the use of a bioassay protocol measuring the metamorphic success of crustacean larvae would be a valuable adjunct to the hazard assessment of newly developed pesticides that target endocrine control of metamorphosis in insects and possibly other endocrine-disrupting xenobiotics as well.
Effects of a Chronic Dietary Exposure of the Honeybee Apis mellifera (Hymenoptera: Apidae) to Imidacloprid by R. Schmuck (pp. 471-478).
Previous studies have shown that imidacloprid and insecticidally active imidacloprid plant metabolites are rapidly metabolized by honeybees. Accordingly, no striking differences were expected between the acute and the chronic dietary toxicity of imidacloprid to honeybees. More recently, however, an unexpectedly high chronic dietary toxicity to honeybees was reported for imidacloprid and imidacloprid plant metabolites, and a novel pharmacologic mechanism unrelated to the parent toxophor was postulated. In an extensive literature survey, no further evidence was found for the reported high difference between the acute and the chronic dietary toxicity of imidacloprid and its plant metabolites to honeybees. The majority of data indicated a dietary no observed lethal-effect concentration >0.04 and 0.02 mg/L 50% sucrose solution, respectively, for an acute and a chronic dietary exposure of honeybees to either imidacloprid or its plant metabolites. Findings of chronic feeding studies with those plant metabolites where the toxophor had already been cleaved did not support the hypotheses of a novel pharmacologic mechanism unrelated to the parent toxophor. No increased treatment-related mortality or behavioral abnormalities were recorded in four independent research facilities during a 10-day dietary exposure of honeybees of different ages to sucrose solutions spiked with the respective metabolites at 0.0001, 0.001, and 0.010 mg/L 50% sucrose solution.
Occurrence and Distribution of Pesticides in the Province of Bologna, Italy, Using Honeybees as Bioindicators by S. Ghini; M. Fernández; Y. Picó; R. Marín; F. Fini; J. Mañes; S. Girotti (pp. 479-488).
Samples of honeybees (Apis mellifera, n = 92) from 14 beehive monitoring stations located in 3 townships in the province of Bologna were analyzed from April to October 2000. The concentration of 32 organophosphorus pesticides and 5 carbamates was determined through liquid–liquid extraction followed by gas chromatography with a nitrogen–phosphorus detector and liquid chromatography coupled to mass spectrometry using atmospheric pressure chemical ionization in positive and negative ion modes. The most contaminated samples were from Granarolo Emilia where cereals (wheat, sorghum, and corn), sugar beets, and potatoes are the main agriculture products. Thirty-five pesticides were detected, with organophosphorus being the most abundant ones. Malathion was detected in 58% of the samples (mean level 0.360 mg/kg) followed by fenithrothion in 53% of the samples (mean level 0.544 mg/kg) and pirimiphos methyl in 48% of the samples (mean level 0.006 mg/kg). Temporal trends showed that the maximum detection frequency occurred in late spring and was associated with the use of treatment products and less rainfall. The obtained results demonstrated the feasibility of using honeybees for assessing pesticide exposure in agriculture settings.
Uptake and Effects on Detoxication Enzymes of Cypermethrin in Embryos and Tadpoles of Amphibians by K. Greulich; S. Pflugmacher (pp. 489-495).
A number of factors have been suggested for recently observed amphibian decreases, and one potential factor is pesticide exposure. We studied the uptake and effects of environmentally relevant concentrations of the pyrethroid insecticide cypermethrin on two different amphibian species, Bombina variegata and Rana arvalis. The uptake from water of 14C-labeled cypermethrin (0.4 μg/L) by eggs and tadpoles of B. variegata was investigated. After 24 hours of exposure, 153.9 ng cypermethrin/g fresh weight were found in embryos, thus indicating that the jelly mass of the eggs does not act as a sufficient physical barrier to protect embryos from exposure to this compound. Uptake of cypermethrin into tadpoles of both species and in all exposed individuals caused dose-dependent deformities; behavioral abnormalities such as twisting, writhing, and coordinated swimming; and mortality. In tadpoles of B. variegata and R. arvalis, the activity of microsomal and cytosolic glutathione S-transferase (mGST and sGST, respectively) were measured after treatment with cypermethrin. Activities of both GST systems increased significantly with increasing duration and concentration of cypermethrin exposure, with the reaction seeming stronger in B. variegata than in R. arvalis tadpoles. Alpha-cypermethrin—a racemic mixture of two cis isomers of cypermethrin—induced a stronger enzymatic response in the cytosolic fraction of R. arvalis tadpoles than cypermethrin at the same concentration. The observed physical and behavioral abnormities caused by environmentally relevant concentrations of cypermethrin indicate that despite detoxication of the chemical via GST-system contamination of ponds by cypermethrin could result in adverse effects on the development of amphibian embryos and tadpoles.
Toxicologic and Histopathologic Response of the Terrestrial Salamander Plethodon cinereus to Soil Exposures of 1,3,5-Trinitrohexahydro-1,3,5-Triazine by M. S. Johnson; H. I. Paulus; C. J. Salice; R. T. Checkai; M. Simini (pp. 496-501).
Red-backed salamanders (Plethodon cinereus) were exposed to four different concentrations of 1,3,5-trinitrohexahydro-1,3,5-triazine (RDX) in soil under controlled laboratory conditions for 28 days. Wild-caught P. cinereus (N = 20/treatment) were exposed to target concentrations of 5,000, 1000, 100, 10, and 0 mg RDX/kg soil (dry wt) using a microcosm design. Animals were fed 5 to 10 uncontaminated mutant Drosophila flies every 3 days and monitored daily. Animals were weighed 1 day before being placed in treatment and weekly thereafter. RDX concentrations in soil were analytically determined after the compound was added and mixed at the beginning, the midpoint, and the end of exposure. RDX soil concentrations were relatively stable throughout the exposure period. Signs of overt toxicity were observed primarily in the highest exposure group. Salamanders exposed to 5,000 mg RDX/kg soil exhibited signs of neuromuscular effects (lethargy, gaping, hypersensitivity, tremors) and exhibited significant weight loss. A single moribund animal from this group lost >20% of its original body weight and was killed. Animals in this exposure group also lost weight relative to animals in other treatments. Histopathologic evaluations, including an evaluation of melanomacrophage parameters, indicated no strong treatment-related findings. This study provided information regarding the effects from subchronic dermal exposure of a terrestrial amphibian species to RDX in soil and provides a microcosm approach to the evaluation of toxicity of contaminants in soil to a terrestrial vertebrate.
Organ-Specific Toxicokinetics and Dose–Response of Arsenic in Tilapia Oreochromis mossambicus by C. M. Liao; J. W. Tsai; M. P. Ling; H. M. Liang; Y. H. Chou; P. T. Yang (pp. 502-510).
We appraised organ-specific toxicokinetics and dose responses of arsenic burdens in tilapia Oreochromis mossambicus. We kinetically linked an Area-under-the-curve (AUC)–based acute toxicity model and a pharmacodynamic model to derive dose–response relationships between equilibrium organ-specific arsenic concentrations and mortality effects. The AUC-based acute toxicity model was also used to derive organ-specific internal effect concentration (IEC)–time-response relationships, which can also be applied to predict a time–mortality profile. We conducted a 7-day exposure experiment to obtain toxicokinetic parameters, whereas the AUC-based acute toxicity model was verified with LC50(t) data obtained from a 7-day acute toxicity bioassay. Our results demonstrated that 96-hour LC50 and incipient LC50 for tilapia exposed to arsenic are 28.68 (95% confidence interval to 24.92 to 32.44) and 25.55 mg L−1, respectively. Dose–response relationships followed the Hill equation, which could be expressed as organ-specific bioconcentration factors and incipient LC50. Organ-specific dose-response relationships showed that muscle, gill, and liver have a relatively steep sigmoid dose-response profile in that IEC50 were 26.6, 62.5, and 78.5 μg g−1 dry wt (dw), respectively. Organ-specific arsenic internal lethal burdens were the highest in the gill and the lowest in the muscle in waterborne-exposed tilapia. The IEC and target-organ concentrations derived in this study can be used in site-specific risk assessment.
Confirming the Species-Sensitivity Distribution Concept for Endosulfan Using Laboratory, Mesocosm, and Field Data by G. C. Hose; P. J. Van den Brink (pp. 511-520).
In Australia, water-quality trigger values for toxicants are derived using protective concentration values based on species-sensitivity distribution (SSD) curves. SSD curves are generally derived from laboratory data with an emphasis on using local or site-specific data. In this study, Australian and non-Australian laboratory-species based SSD curves were compared and the concept of species protection confirmed by comparison of laboratory-based SSD curves with local mesocosm experiments and field monitoring data. Acute LC50 data for the organochlorine pesticide endosulfan were used for these comparisons; SSD curves were fitted using the Burr type III distribution. SSD curves indicated that the sensitivities of Australian fish and arthropods were not significantly different from those of corresponding non-Australian taxa. Arthropod taxa in the mesocosm were less sensitive than taxa in laboratory tests, which suggests that laboratory-generated single-species data may be used to predict concentrations protective of semifield (mesocosm) systems. SSDs based on laboratory data were also protective of field populations.
Influence of a Mine Tailing Accident Near Doñana National Park (Spain) on Heavy Metals and Arsenic Accumulation in 14 Species of Waterfowl (1998 to 2000) by G. Gómez; R. Baos; B. Gómara; B. Jiménez; V. Benito; R. Montoro; F. Hiraldo; M. J. González (pp. 521-529).
This article presents the impact on waterbirds in Doñana National Park (Spain) of an accidental release of 5 million m3 acid waste produced by the processing of pyrite ore. Heavy metals (zinc, copper, cadmium, and lead) and arsenic were measured in several soft tissues (liver, kidney, and muscle) taken from 14 waterfowl species collected between April 1998 and May 2000. The main source of copper and zinc found in the waterfowl species examined was the spill waste, whereas cadmium, lead, and arsenic could also came from other sources. Kidney was the primary organ for cadmium and lead accumulation, whereas liver accumulated the most zinc and copper. Arsenic was concentrated in both muscle and liver tissue. The degree of contamination of the area where the birds lived, their age, their sex their size, and the time since the spill were found to have less influence than species and trophic level on the accumulation of metal in organs and tissues. Four species (Anser anser, Ciconia ciconia, Larus ridibundus, and Porphyrio porphyrio) were found to have the highest levels of the 5 elements.
Immune Function and Organochlorine Pollutants in Arctic Breeding Glaucous Gulls by J. O. Bustnes; S. A. Hanssen; I. Folstad; K. E. Erikstad; D. Hasselquist; J. U. Skaare (pp. 530-541).
Organochlorine contaminants (OCs) are known to affect the immune systems of wildlife, and in this study we assessed the relationship between blood concentration of different OCs and measurements relevant to immune status and function in arctic breeding glaucous gulls (Larus hyperboreus). In 1997 and 2001, we counted white blood cells (heterophils and lymphocytes) from blood smears, and in 2000 and 2001 we injected two novel nonpathogenic antigens (diphtheria and tetanus toxoids) into the pectoral muscle of gulls and measured the primary antibody responses. We then related these measurements to the blood concentrations of three pesticides (hexachlorobenzene [HCB], oxychlordane, and p,p′-dichlorodiphenyldichloroethylene) and seven different polychlorinated biphenyl congeners (PCB 101, 99, 118, 153, 138, 180, and 170). There were significant or near significant positive relationships (0.1 > p > 0.001) between most persistent OCs and the levels of heterophils in the blood for both sexes in 1997 and for male gulls in 2001. Similarly, levels of all persistent OCs and lymphocytes were positively related (0.1 > p > 0.001) in both sexes in 1997. This suggests that OCs are causing alterations to immune systems, which may decrease their efficiency and make the birds more susceptible to parasites and diseases. In female gulls, the antibody response to the diphtheria toxoid was significant and negative for HCB (p <0.01) and weaker, but significant, for oxychlordane (p <0.05), suggesting that OCs were causing an impairment of the humoral immunity. Various OCs have been linked to negative effects in our study population, including decreased survival and reproduction, and this study suggests that such compounds also affect immune status and function.
Polybrominated Diphenylethers and Methoxylated Tetrabromodiphenylethers in Cetaceans from the Mediterranean Sea by A. Pettersson; B. van Bavel; M. Engwall; B. Jimenez (pp. 542-550).
Eight tetrabrominated to hexabrominated diphenylethers were present at ppb levels in liver from cetaceans found stranded on the beaches of the Mediterranean Sea, Italy. The highest concentration was found in striped dolphin (sum polybrominated diphenyl ethers [PBDE] 8133 ng/g l.w.) and the lowest concentration in bottlenose dolphin (sum PBDE 66 ng/g lipid weight [l.w.]). The predominant congener in all samples was 2,2′,4,4′-tetraBDE (PBDE # 47) followed by, in decreasing order, the pentaPBDE # 99 and 100 and the hexaPBDE # 154 and 153. In 12 of the 14 analyzed samples, 3 different methoxylated PBDEs (MeO-PBDE # 1, 2, and 3) were detected at semiquantitatively calculated concentration ranges of 2 to 14 ng/g l.w.; 5 to 167 ng/g l.w.; and 7 to 628 ng/g l.w., respectively. In addition, several unidentified bromine compounds were seen when screening the samples in negative-chemical ionization (NCI) mode monitoring m/z 79 and 81, which illustrates the importance of running both electron-impact ionization and NCI when analyzing environmental samples. Electron-impact ionization is more specific for monitoring the molecular ion compared with NCI, which might overestimate the concentration of certain PBDE congeners.
Congener-Specific Polychlorinated Biphenyls in Cetaceans from Taiwan Waters by C. C. Chou; Y. N. Chen; C. S. Li (pp. 551-560).
During 2000 to 2001, a total of 73 blubber samples from 13 species of stranded or accidentally captured cetaceans were collected from Taiwan coastal waters for polychlorinated biphenyl (PCB) analysis. After homogenization, saponification, liquid–liquid extraction, and silica-gel solid-phase extraction, PCB concentrations were determined by gas chromatography/mass spectrometry. Total concentrations of 19 PCB congeners (ΣPCBs) were between 0.23 μg/g lipid weight of Risso’s dolphin to 33.73 μg/g lipid weight of rough-toothed dolphin. Pentachlorobiphenyls, hexachlorobiphenyls and heptachlorobiphenyls were the predominant PCB congeners species. PCB153 was the most abundant congener in all samples. The PCB153/ΣPCBs consistently comprised between 20% to 30% of all congeners. The toxicity measured as 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) were from 2.7 pg/g lipid weight of finless porpoise to 2,900 pg/g lipid weight of rough-toothed dolphin. PCB 118, a mono-ortho congener, was the largest contributor to TEQs. PCB concentrations and TEQs were higher in mature male than in immature male animals but were inconsistent in female animals because of a possible transferring of PCBs from maternal cetaceans to their offsprings during gestation and lactation. Stranded cetaceans had significantly higher PCB levels than by-catch cetaceans because of their higher lipid consumption during starvation or illness. From the collected samples, we also found that cetaceans from Taiwan waters had relatively lower PCB concentrations and TEQs than those from high-latitude areas.
Characterization of Soils Containing Adipocere by S. Fiedler; K. Schneckenberger; M. Graw (pp. 561-568).
The formation of adipocere (commonly known as grave wax), a spontaneous inhibition of postmortem changes, has been extensively analyzed in forensic science. However, soils in which adipocere formation occurs have never been described in detail. Therefore, this study is intended as a first step in the characterization of soils containing adipocere. Two grave soils (Gleyic Anthrosols) that prevent the timely reuse of graves due to the occurrence of adipocere and a control soil (Gleyic Luvisol) were selected from a cemetery in the Central Black Forest (Southwest Germany). Descriptions of soil morphology and a wide assay of physical, chemical, and microbiologic soil characteristics were accomplished. In contrast to the control soil, the grave soils were characterized by lower bulk density and pH. The degradation of the soil structure caused by digging led to a higher water table and the expansion of the reducing conditions in the graves where the prevalent absence of oxygen in range of the coffins inhibited decomposition processes. Although the formation of adipocere led to the conservation of the buried corpses, phosphorus, dissolved organic carbon, and cadavarine leaching from the graves was observed. Microbial biomass and microbial activity were higher in the control soil and hence reflected the inert character of adipocere. The study results clearly show the need for additional approaches in forensic, pedologic, and microbiologic research.
