Ecotoxicology, 13, 603–614, 2004 2004 Kluwer Academic Publishers. Manufactured in The Netherlands.
Assessment of Soil Toxicity from an Antitank Firing Range Using Lumbricus terrestris and Eisenia andrei in Mesocosms and Laboratory Studies* PIERRE YVES ROBIDOUX,1,* CHARLES DUBOIS,2 JALAL HAWARI1 AND GEOFFREY I. SUNAHARA1,* 1
Applied Ecotoxicology Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2 2 Defense Research Establishment Valcartier, Canadian Ministry of National Defense, 2459, Pie IX Boulevard, Val Be´lair, Quebec, Canada G3J 1X5 Accepted 22 August 2003
Abstract. Earthworm mesocosms studies were carried out on a explosives-contaminated site at an antitank firing range. Survival of earthworms and the lysosomal neutral red retention time (NRRT), a biomarker of lysosomal membrane stability, were used in these studies to assess the effect of explosives-contaminated soils on the earthworms Lumbricus terrestris and Eisenia andrei under field conditions. Toxicity of the soils samples for E. andrei was also assessed under laboratory conditions using the earthworms reproduction test and the NRRT. Results indicate that the survival was reduced up to 40% in certain explosive-contaminated soil mesocosms following 10 days of exposure under field conditions, whereas survival was reduced up to 100% following 28 days of exposure under laboratory conditions. Reproduction parameters such as number of cocoons and number of juveniles were reduced in many of the selected contaminated soils. Compared to the reference, NRRT was significantly reduced for E. andrei exposed to explosive-contaminated soils under both field and laboratory conditions, whereas for L. terrestris NRRT was similar compared to the reference mesocosm. Analyses showed that HMX was the major polynitro-organic compound in soils. HMX was also the only explosive detected in earthworm tissues. Thus, results from both field mesocosms and laboratory studies, showed lethal and sub-lethal effects associated to soil from the contaminated area of the antitank firing range. Keywords: contaminated soil; explosives; TNT; RDX; HMX; Neutral Red assay; biomarker
Introduction Neutral red retention time (NRRT) has been used as an earthworm biomarker for lysosomal membrane damage caused by environmental contaminants such as PCBs (Goven et al., 1993), pesticides (Booth et al., 2001) and heavy metals (Weeks and Svendsen, 1996; Svendsen and Weeks, 1997a; *To whom correspondence should be addressed: Tel: (+1) 514 283 6447; Fax: (+1) 514 496 6265; E-mail:
[email protected]
Scott-Fordsmand et al., 1998; Spurgeon et al., 2000). The NRRT assay was used for freshlyspiked soil studies under laboratory conditions (Svendsen and Weeks, 1997a; Scott-Fordsmand et al., 1998; Booth et al., 2001; Robidoux et al., 2002b), but also for contaminated-soils under laboratory and field conditions (Svendsen and Weeks, 1997a; Booth et al., 2001; Robidoux, et al. 2004). The earthworm species used for this assay includes Lumbricus castaneus (Svendsen et al., 1996), Aporrectodea rosea (Harreus et al., 1997), Eisenia veneta
604 Robidoux et al. (Scott-Fordsmand et al., 1998), A. caliginosa and L. terrestris (Spurgeon et al., 2000), as well as Lumbricus rubellus, E. andrei and E. fetida (Svendsen and Weeks, 1997a; Spurgeon et al., 2000). NRRT was also sensitive to 2,4,6-trinitrotoulene (TNT) concentrations in OECD artificial soil and on filter paper (Robidoux et al., 2002b). The field applicability of NRRT as a biomarker has been demonstrated in earthworms exposed at contaminated sites containing metals (Svendsen et al., 1996; Svendsen and Weeks, 1997b) or explosives (Robidoux et al., 2004). Polynitro-organic (PNO) contaminants such as TNT, 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) showed sub-lethal effects to earthworm Eisenia andrei in artificial (Robidoux et al., 2000; 2001) and sandy forest soils (Robidoux et al., 2002a). However, sub-lethal effects of mixed PNOs on earthworms under field or laboratory conditions are poorly known. This study assesses the lethal and sub-lethal effects of earthworm exposure to explosive-contaminated soils under field and laboratory conditions using the lysosomal membrane stability, measured as NRRT, in in situ mesocosm studies and earthworm reproduction test. Materials and methods Chemicals and reagents Neutral red was obtained from Sigma Chemical (St.Louis, MO). Other chemicals were ACS reagentgrades and were obtained from commercial suppliers. Deionized water (ASTM, type II) obtained using a Super-Q water purification system (Millipore) or Zenopure Mega-90 was used throughout the studies. Glassware was washed with phosphatefree detergent followed by rinses with acetone, nitric acid 10% (v v)1), and finally with deionized water. Balanced Salt Solution (BSS) was prepared as described earlier (Goven et al., 1994; Weeks and Svendsen, 1996; Robidoux et al., 2002b). Earthworms Mature adult individuals of Lumbricus terrestris were obtained from a commercial supplier (Appaˆts Saint-Gabriel, Saint-Gabriel de Brandon, QC, Canada). The laboratory worms (Eisenia andrei)
were obtained from Carolina Biological Supply (Burlington, NC, USA). The worms were maintained in bedding (Magic Products, Amherst Jct; WI, USA) supplemented with dry cereal (Magic Worm Food, Magic Products), at 20 1 C, 70– 80% humidity and a light/dark cycle (16 h ON/8 h OFF). Only adult E. andrei (ranging from 300 to 600 mg wet weight) having a well-developed clitellum were used for the mesocosm and laboratory experiments. Site description and Mesocosm experiments The contaminated site was located at the Range 13 of the Western Area Training Center (Wainwright, AB, Canada). This site was contaminated with different PNOs such as HMX, RDX, TNT and its by-products. The areas selected for the assessment were located front of the tanks used for the training (Fig. 1) corresponding to the contaminated area (Thiboutot et al., 1998). Concentrations of PNOs in soils were low or not detected in the zones immediately peripheral to the selected areas. Vegetation was low or absent in the selected contaminated areas because of the training activities. The non-contaminated reference area ‘‘R’’ (with healthy vegetation) at near proximity of the contaminated areas (53 m from firing point) was also used. Training activities were ceased during our experiments to avoid disturbance. Earthworms (L. terrestris and E. andrei) were exposed in situ in the selected areas of the site using field soil mesocosms. These mesocosms were made from 100 lm Nylon mesh, each folded and stitched from one piece and with lids sealed using Velcro tape to ensure containment. The dimensions of the mesh bags were 25 cm 25 cm 16 cm deep, and thus able to contain 10 l of soil and soil organisms exposed under field conditions. Blocks of soil were carefully removed from the ground using a square stainless steel tool (25 cm 25 cm 16 cm deep) and was transferred as whole blocks into the Nylon meshbags. Following acclimation (24 h) in clean artificial soil (70% silica, 20% clay, 10% peat moss; w w)1), indigenous earthworms (L. terrestris; n ¼ 10) and laboratory earthworms (E. andrei; n ¼ 10) were individually rinsed and then placed in their respective Nylon bags containing the soil block. Distilled water (1 l per mesocosm) and 10 g
Assessment of an Antitank Firing Range 605
Figure 1. Sampling plan, Range 13, Canadian Force Western Area Training Center, Wainwright (AB, Canada).
of dried cereal (Magic Worm Food, Magic Products) were added. The mesocosms (n ¼ 10) were closed, returned to their respective holes and left in place for 10 days. Thereafter the surviving earthworms were hand sorted from the mesocosms and counted. NRRT assays were carried out on survivors as described below (NRRT assay). Soil samples Soil samples were taken from the mesocoms after the experiment and transferred in a 10-l polyethylene pail. The soil samples were transported and homogenized by hand at the laboratory. Explosives were extracted from the soil using acetonitrile, and concentrations of PNOs (HMX, RDX, TNT, Tetryl) and some of their degradation products in soil samples were determined by HPLC as described below (Physical and chemical analyses). Earthworm reproduction test At the beginning of each experiment, 500 g dry weight (DW) of soil was rehydrated to 60% of the water-holding capacity method described in section (Physical and chemical analyses). The effects of explosives-spiked soils on growth and reproduction of E. andrei were assessed using the ISO (1998) method. Survival and growth of adult earthworms were determined after 28 days of exposure, whereas
reproduction parameters, including cocoon production and hatching, and juveniles survival and growth were measured after 56 day. Following acclimation (from 1 to 4 days) in OECD (1984) artificial soil, 10 E. andrei were individually rinsed, weighed and added to each replicate jar containing the test substance in soil. Glass jars were closed using lids with 1.6 mm air holes. Food (2 g of dry cereal) was added to the surface of the test matrix at the beginning of the experiment and then once weekly. Water loss was compensated weekly by adding deionized water. The earthworms were hand sorted from the soil and the number of surviving E. andrei (per replicate) as well as the individual weight (wet biomass) were recorded at 28 days. The survivors were purged for 24 h and frozen at )85 C for later chemical analysis of PNO in tissues. The remaining soil (containing cocoons) was then returned to their respective jars and incubated for another 28 days in order to continue the exposure of cocoons and juveniles to the test soil. At 56 days of the test, the number of hatched and non-hatched cocoons, the number of juveniles and their biomass were recorded. Results of the treatment groups were compared statistically to control groups (negative control and reference) to determine the sub-lethal effects on earthworms following 28 days (weight change or growth) and 56 days (reproduction) of exposure. The pH and the water content were
606 Robidoux et al. measured before and after each test as described below (Physical and chemical analyses). The positive control results using the reference toxicant (EC50 for the number of juveniles was 18.9 mg kg)1 dry soil of 2-chloracetamide) were consistent with in-house data and previous studies (Robidoux et al., 2000, 2001). NRRT assay Surviving earthworms from a single treatment were taken after exposure for the biomarker measurements. For the mesocosms experiments, five survivors of each species (L. terrestris or E. andrei) were taken after the 10-day exposure in mesocosms. For the earthworm reproduction test, a maximum of five surviving adult individual E. andrei were taken after 28 days of exposure in soil. The determination of the NRRT was done after the exposure to the contaminated soils using the conditions described earlier (Weeks and Svendsen, 1996; Robidoux et al., 2002b). Briefly, a working NR solution (80 lg ml)1) was prepared in BSS. A small sample of coelomic fluid (approx. 50 ll) was taken from each live worm using an hypodermic needle preloaded with 50 ll BSS. The coelomic fluid and BSS mixed sample (25 ll of the 100 ll) was placed on a microscopic slide. After 60 s (to allow the cells to adhere, 25 ll of NR solution was added and mixed on the slide. Each slide was scanned continuously for 1 min at 5 min intervals under a light microscope (400). The NRRT is the time of the first interval where the ratio of coelomocytes with fully stained cytoplasms exceeds 50% of the total number of coelomocytes counted. Physical and chemical analyses Soil concentrations of TNT and its degradation products, RDX, HMX and Tetryl concentrations were extracted from soil samples, as described previously (Robidoux et al., 2000) using the sonication–acetonitrile HPLC method (USEPA, 1997). Briefly, acetonitrile (10 ml) was added to 2 g soil sample and then vortexed for one minute. The sample is then placed in an ultrasonic bath for 18 h at 8 C followed by adding 5 ml of a CaCl2 solution (5 g l)1) to an equal volume of soil extract. The mixture was then filtered through a 0.45 lm membrane (Millex-H, Millipore). The filtered
solution was analyzed using a Waters HPLC chromatographic system composed of a Model 600 pump, Model 717 Plus sample injector, a Model 996 Photodiode-Array detector and a temperature control module, or a Thermo Separation Products chromatographic system composed of Model P4000 pump, a Model AS1000 injector, including temperature control for the column, and a Model UV6000LP PhotodiodeArray Detector. For TNT analyses, a Supelcosil C8 column (25 cm 4.6 mm ID, 5 lm particles) and a mobile phase composed of water (82%, v v)1) and 2-propanol (18%, v v)1) were used. The flow rate was 1 ml min)1 and the column temperature was held at 35 C. The sample volume injected was 50 ll with a run time of 40 min. The analytes were detected by UV absorption at 254 nm. The limit of detection (LOD) in the extract was 25 ppb for TNT, and 50 ppb for 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6nitrotoluene (2,4-DANT) and 2,6-diamino-4nitrotoluene (2,6-DANT). For RDX and HMX analyses, the column used was a Supelcosil LC-CN (25 cm 4.6 mm, 5 lm). Temperature of the column was held at 35 C. The initial solvent composition was 30% methanol and 70% water, which was held for 8 min. A linear gradient was run from 30% to 65% methanol over 12 min. This solvent ratio was changed to the initial conditions over 5 min. These initial conditions were then held for a extra 5 min. The detector (LOD ¼ 50 and 100 ppb for RDX and HMX, respectively) was set to scan from 200 to 350 nm. RDX, HMX, TNT and the TNT metabolites were used as reference standards (>99% pure) and were obtained from Supelco (Oakville, ON) and Accustandard (New Haven, CT). The injection volume was 50 ll. Results were expressed on a dry weight (DW) basis. The limit of quantification of TNT and its metabolites, RDX and HMX in the soil were 0.25 mg kg)1 DW and the precision was >95%. Water content of soil was obtained by heating soil to 103 2 C for 16 h. The water holding capacity was determined by saturating the soil with water and measuring the water content as described elsewhere (Robidoux et al. 2000). At the end of the mesocosm experiments, the pH values were measured at the laboratory using a 1:5 (v v)1) suspension of soil in water (ISO, 1994). Organic
Assessment of an Antitank Firing Range 607 matter content were estimated from weight loss after 1 h ignition at 600 C. Body burdens of explosives and degradation products were estimated in the surviving earthworms from the reproduction test. Explosives were extracted from individual earthworms using the acetonitrile technique as described by Renoux et al. (2000). Briefly, 7 ml of acetonitrile was added to the frozen tissue in a Teflon tube. Samples were prepared using a Polytron tissue homogenizer, vortexed, and transferred in an ultrasonic bath for 18 h. Then, the tubes were centrifuged (10,000 g, 10 min, 4 C) and 5 ml of supernatent of extract is added to 5 ml of CaCl2 (10 g l)1) solution in 20 ml vials. Samples were left 2 h at 4 C, then filtered on a 0.45 lm membrane prior to HPLC analysis (USEPA, 1997) described above. Data analysis The NRRT data were analyzed with a non-parametric Kruskall–Wallis ANOVA to identify differences among the mesocosms or the soil samples. When differences among the means were found, a Mann–Whitney U test for independent samples was used to determine the differences (p 0:05) between specific mesocosms or soil samples. Statistical methods to determine differences between reference and other soil samples for lethal and sub-lethal endpoints included parametric hypothesis tests (e.g., Dunnett’s Multiple Comparison, Bonferroni T-Test; p 0.05) and non-parametric hypothesis test (e.g., Steel’s Many-One Rank Tests; p 0.05). Data were expressed as the average standard deviation (SD) or standard error (SE). Results Physical analysis of soils The moisture, water holding capacity and pH of soil mesocosms are presented in Table 1 (data collected after exposure). The water content of soil in mesocosms ranged from 10.0% to 18.2% (w w)1) whereas the water holding capacity of soil ranged from 47.8 to 64.2 (ml 100 g)1 dry soil). The water content relative to water holding capacity ranged from 17.6% to 28.7% (data not shown). The pH of soils ranged from 6.48 to 8.70 (Table 1). Organic matter
content in soil ranged from 2.1 to 7.6% (w w)1; Table 1). Relations between these parameters (water content, water holding capacity, pH and organic matter) and the responses (lethality, growth reproduction and NRRT) of the earthworms (E. andrei and L. terrestris) were not observed. HMX concentrations in soil Soil concentrations of HMX in the mesocosms ranged from
