Environ. Sci. Technol. 2000, 34, 1865-1870
Methods for Toxicity Assessment of Contaminated Soil by Oral or Dermal Uptake in Land Snails. 1. Sublethal Effects on Growth A N N E T T E G O M O T - D E V A U F L E U R Y * †,‡ A N D ANTONIO BISPO§ Laboratoire Biologie et Ecophysiologie, Universite´ Franche-Comte´, Place Leclerc, 25030 Besanc¸ on Cedex, France, I.U.T. Besanc¸ on-Vesoul, Avenue des Rives du Lac, 70000 Vaivre et Montoille, France, and I.R.H. Environnement, 11 bis rue Gabriel Peri, 54000 Vandoeuvre, France
Among the terrestrial invertebrate fauna, snails are primary consumers. Sublethal (4 weeks) bioassays using Helix aspersa aspersa (Haa) and Helix aspersa maxima (Ham) have been established to evaluate the potential impact of pollutants present in the soil via oral and dermal exposure through measurement of a biologically important, sublethal endpoint: growth. In both subspecies, trace elements (Cd, Cr, Pb, and Zn) in a contaminated soil (S1) administered orally, exerted dose dependent inhibition of growth. On dermal uptake (spraying the snails with leachate of soil S1), however, Haa appeared unaffected, whereas Ham showed a slight inhibition of growth. After contamination with organic substances (phenanthrene, trichlorophenol, pentachlorophenol), soil S2 reduced the growth of Haa (oral: +++; dermal: ++), whereas Ham were not inhibited by dermal exposure and only affected by dietary exposure at very high doses. The methods proposed represent a new tool for soil risk assessment by two distinct exposure routes: oral and dermal.
Introduction The purpose of ecological risk assessment for terrestrial ecosystems is to identify species-specific uptake pathways and contaminant exposure levels of the habitats concerned. In general, risk assessment is based on a triad of risk assessment information (1), which, as recommended by Suter (2), involves an association of chemical and ecological testing and toxicity evaluation so as to establish a relationship between the toxic contaminants and the ecological effects. The tests used to evaluate the toxicity of the pollutants reaching the soil use various species of microorganisms, plants, and a few species of invertebrate animals (3). However, in the tests making up the current standards, only two trophic levels are represented. It is thus urgent to develop further easy-to-use tests based on other land animals which must be key indicator species playing an important role in the ecosystems, offering the possibility to perform tests in both experimental and natural conditions while remaining readily available (4). Concerning the fauna, the species of inverte* Corresponding author phone: 03 81 665788; fax: 03 81 665797; e-mail:
[email protected]. † Universite ´ Franche-Comte´. ‡ I.U.T. Besanc ¸ on-Vesoul. § I.R.H. Environnement. 10.1021/es9907212 CCC: $19.00 Published on Web 03/28/2000
2000 American Chemical Society
brates used for the tests standardized so far are all part of the saprophytic decomposer trophic level (tests ISO/DIS 11268-1 (5) and ISO/DIS 11268-2 (6) with Eisenia fetida and test ISO/TC 190 SC4 WG4 (7) with Folsomia candida). Other experimental test systems, described in specialist reviews, concern various species of the main taxonomic groups of invertebrates (8, 9) and should broaden the possibilities for the prediction of the environmental impact of chemicals on soil ecosystems. As a complement to the tests based on decomposer invertebrates, land snails (pulmonate gastropods) are very suitable since they fully satisfy the criteria for bioindicators (10). They live at the surface of the soil where they act by burrowing, ingesting soil and plants (both alive and decomposing), and breathing air. Test snails can be exposed to the whole environment as in field tests or can be subjected to laboratory contamination to explore the relative contribution of different exposure pathways (oral, dermal, respiratory) which is not possible to do with species that must spent their lives entirely in water or in the earth. Contamination of terrestrial environments has been assessed with several species of snails (Helix aspersa, H. pomatia, Arianta arbustorum, Cepaea nemoralis) [see review in ref 11]. For our experiments, we chose the “garden snail” Helix aspersa aspersa, which occurs in many parts of the world (12, 13). Its rearing is now controlled (14), and it is well suited to tests in the laboratory (15) and in the field (16). We also used a closely related but larger subspecies Helix aspersa maxima which is rather found in hotter countries (Algeria), which is easy to rear and which presents biological characteristics suited to laboratory testing (15). In earlier works, we reported that (15) oral contaminant intake is a suitable route to show the inhibitory influence of metals or mixtures of contaminated soil and food (16) since snails have a regular intake of soil which, depending on its composition, can affect growth (17). However, absorption of toxins by skin contact cannot be ignored since it has been reported that calcium, indispensable for snail reproduction and growth, can be taken up across the foot epithelium of H. aspersa (18). So, after having analyzed the effects of contaminants associated with food (oral administration) (16), the purpose of this study was to compare the effects of the ingestion of soils contaminated with metals or organic chemicals with the effects of two aqueous leachates of the same soils sprayed on and around the snails. The bioassays were performed in order to evaluate the toxicity of, first, the soil (ingestion) and second the leachate (skin contact) for monitoring soil pollution in any season independently of all environmental restriction with two biological models.
Materials and Methods Animals. Two subspecies of snail, Helix aspersa aspersa (Haa) and Helix aspersa maxima (Ham), were used. The Haa snails were bred from specimens collected in the Cavaillon area of France, and the Ham snails were from stock originally collected in Algeria. Both subspecies were reared in the same optimum conditions enabling a full biological cycle in 16 weeks whatever the season: temperature: 20 ( 2 °C; photoperiod 18h L/6h D; relative humidity: 80-90%; feed: meal for snails (Helixal, Le´pine Co., Clairvaux les Lacs, 39130, France). Soils Sample Preparation. Soil S0 was taken in an agricultural area (La Bouzule, 54, France). The 15-cm top layer of soil was sampled and dried at 40 °C until constant weight. It was then crushed and sieved at 4 mm. Soil characteristics are as follows: pH 6, carbon content 16.53 VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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by contact with moist supports on which the snails move or in which they bury themselves. The toxic impact of the contamination therefore varies with the daily and seasonal fluctuations of weather which also determine the activity of the animals. To evaluate the toxic capacity of a soil independently of the rainfall, temperature, etc., samples of the soil are taken to the laboratory where the sublethal toxicity of the soil as such and of its leachates are measured. Study of the Action of Soil as Such (Oral Intake). We compared the growth of the snails fed with the mixtures containing increasing proportions of uncontaminated or contaminated soil (Table 2). The growth of snails fed with Helixal only (controls) is used to observed the effect of the ingestion of increasing percentage of control soil S0. In these experiments, the surfaces across which the snails move were not contaminated. The concentrations of contaminants in the feed mix (soil-Helixal) are reported in Table 3. The food was supplied in excess. Study of Substances Eluted from Soil by Water (Uptake by Contact). Snails and their surroundings were sprayed with the aqueous leachates of the soils (10 mL for each box), while the feed (Helixal) was left uncontaminated. As with the tests based on soil ingestion, a comparison was made between the snails sprayed with leachate of control soil S0 and those sprayed with leachates of the contaminated soil (soils S1 and S2); any differences in growth rates were noted. Experimental Groups. The experiments were carried out with 1-month-old, juvenile snails with a mean weight of 1 ( 0.2 g, at the beginning of their rapid growth phase. All experiments were carried out in duplicate. Each group was composed of five snails housed in clear Pyrex boxes with a volume of 1.6 dm3 (24 × 10.5 × 8 cm) with a clear glass lid for the first 2 weeks. From the third week, to take the size of the snails into account, the volume of the cage was doubled by replacing the flat cover with a second box placed upside-down on the first. The floor of the housing was covered with blotting paper dampened with water in the series contaminated orally and dampened with soil leachate for the series using contamination by skin contact. The food was given in excess in a 5-cm Petri dish on the floor of the cage. Growth Monitoring. The experiments lasted for 4 weeks during which the cages were cleaned three times a week. At each cleaning, the food was replaced, as was the blotting paper floor covering which, like the snails, was again sprayed with water or leachate. One weighing a week enabled us to calculate the mean weight progression (( SD) of the various groups. The mean weights of the treated and control groups were compared by Fisher’s test (ANOVA). Growth was also
TABLE 1. Soil and Leachate Analysis for Soil Samples S0, S1, and S2 soil concn soil sample soil S0 Cd Cr Pb Zn phenanthrene 2,4,6-trichlorophenol pentachlorophenol soil S1 Cd Cr Pb Zn soil S2 phenanthrene 2,4,6-trichlorophenol pentachlorophenol a
theoretical, mg‚kg-1
measd, mg‚kg-1
20 800 800 2000 800 80 80
0.38 88.6 49.5 131
