SPENT CARBIDE WASTE RETAINS TOXICITY LONG TERM AFTER DISPOSAL IN CAVES AND MINES toksičnost karbidnih odpadkov V JAMAH IN RUDNIKIH Andrew A. SEMIKOLENNYKH1*, Anna A. RAHLEEVA1 & Tatjana B. POPUTNIKOVA1 Abstract UDC 546.261:551.435.84 Andrew A. Semikolennykh, Anna A. Rahleeva & Tatjana B. Poputnikova: Spent carbide waste retains toxicity long term after disposal in caves and mines we studied the environmental impact of wastes derived from calcium carbide, which is widely used for generating acetylene in industry and speleology. It was shown that spent carbide is toxic for biota and harmful to cave ecosystems and the surrounding environment. The toxic components of spent carbide waste were found to include calcium hydroxide, strontium and polycyclic aromatic hydrocarbons. The 50% lethal doses (LD 50%) of fresh spent carbide waste were calculated as 0.28-0.32 g/l in biotests with daphnia, infusoria, and fishes. The toxicity of spent carbide declined only slowly over time, with toxicity still present in 13-year-old samples. Spent carbide should be disposed of with great care to ensure that it cannot be disseminated into natural water systems. Spent carbide de-activation could be provided within isolated bowls filled with water (micro sediment bowls) or within water-proof storage containers, and complete recycling could be achieved through the addition of deactivated waste to solid building materials. Keywords: carbide, cave, mine, waste, toxicity, impact. Izvleček UDK 546.261:551.435.84 Andrew A. Semikolennykh, Anna A. Rahleeva & Tatjana B. Poputnikova: Toksičnost karbidnih odpadkov v jamah in rudnikih Proučevali smo okoljski vpliv odpadkov kalcijevega karbida, ki se uporablja v jamarstvu in industriji. Izkaže se, da so karbidni ostanki so toksični in škodljivi za jamsko okolje. Glavne toksične sestavine so calcijev hidroksid, stroncij in policiklični aromat-ski ogljikovodiki. 50% smrtne doze (LD 50%) izračunane iz biotestov na vodnih bolhah, infuzorijah in ribah so v območju 0.28-0.32 g/l. Toksičnost karbidnega apna s časom počasi upada, tudi 13 let stari vzorci so še vedno nevarni. Porabljen karbid ne sme priti v naravna vodna okolja. Pogosto ga odlagajo v betonske posode delno napolnjene z vodo (mikrosedimentne posode), še boljši način pa je odlaganje v popolno vodotesne hranilnike. Popolno reciklažo pa bi lahko dosegli z dodajanjem deaktiviranih ostankov v gradbene materiale. Ključne besede: karbid, jama, rudnik, toksičnost, vpliv na okolje. INTRODUCTION A number of karst caves and depleted mine sites contain waste previously disposed by tourist groups and speleological research expeditions. Disposed of by tourist equipment such as old ropes, fuel, and unused food are often accumulated in considerable amounts. Such wastes left behind by former and, occasionally, recent speleological expeditions can present a serious negative anthropogenic impact on the subterranean ecosystems, new cave visitors, and a whole local population due to the risk of ground water pollution often affecting the regional drinking water supply. In this article we present the results of our research into the negative effects of spent calcium carbide as a hazardous waste. 1 Soil Science Faculty, Moscow State University, Russia, e-mail: aasemik@list.ru Received/Prejeto: 13.9.2010 Calcium carbide is widely used in industrial acetylene production for welding tools and in chemical synthesis. It is also used in caving to fuel acetylene lamps. Spent carbide is usually left by some cavers anywhere in caves where the recharging of gas-generators takes place, and over many years this can result in substantial accumulations of such wastes (carbide dumps). The problem of carbide disposal has a long history. By the present time the use of carbide lamps is highly restricted in some countries (Australia, USA, New Zealand, etc.). However, such lamps are still used in many other countries, and large carbide dumps are present in many caves. Pure calcium carbide (synonyms: acetylenogen, calcium acetylide, and calcium dicarbide) - CaCj - is a white crystalline substance. The modern industrial synthesis of CaCj involves high-temperature combustion of a mixture of calcium oxide and coke (A dictionary^ 2004). CaO + 3C ^ CaC2 + CO Industrial calcium carbide is usually produced from impure raw materials (ignited crystalline limestone, ash-rich coke, coal, etc.). A technique for solid organic wastes utilization by reaction with calcium oxide has also been developed. Such technological processes result in the production of dark-grey carbide colored by coallike substances. Impurities present in carbide can also include calcium phosphide and sulphide, which account for the unpleasant smell of carbide and the acetylene produced. The reaction of calcium carbide with water creates acetylene: CaC2 + 2H2O ^ C2H2 + Ca(OH)2 For many years cavers believed that the use of calcium carbide was harmless for human health and the environment. That commonly-held view was substantiated by the fact that the waste's main chemical compound is calcium hydroxide - Ca(OH)2, which reacts over time with atmospheric carbon dioxide to form non-toxic calcium carbonate. The first experiments to investigate spent carbide toxicity for microorganisms were conducted by Agagab- yan (1955). An aquatic solution of the waste at 0.5% concentration (waste:water = 1:200) caused bacterial cell death within 10 minutes. It was also shown that grampositive bacteria tend to be more resistant to spent carbide than gram-negative bacteria. The key research into the toxic effects of spent calcium carbide on Ptomaphagus hirtus cave beetles was conducted by Peck (1969). Here, the toxic effect of carbide on beetle eggs was shown to result in the death of all hatched larvae. At the same time, the control population developed normally. A comprehensive analysis of the spent carbide toxicity was carried out by Lavoie (1980) who conducted a series of in situ and in vitro experiments. It was shown that calcium carbide waste causes the death of most microorganisms within 10 minutes, even if applied at concentrations as low as 0.1% (waste:water = 1:1000). However, the number of microorganisms found under the waste in the cave floor was comparable with that occurring in the non-waste control area. This phenomenon was interpreted by Lavoie to be a result of the neutralization of spent carbide toxicity upon contact and reaction with atmospheric carbon dioxide. The main cause of such toxicity was a high pH (about 11.2) of the waste. The experiments on neutralizing the waste demonstrated that its pH reduced from 11.2 to 6.3 within 25 days in a natural cave environment and within 10 days under forced additional aeration. The replicated experiment with the carbide waste neutralized to pH 6.3 demonstrated an absence of toxic effects to E. coli within a period of 60 minutes. The general conclusion by Lavoie was that the waste is highly toxic, but loses its toxicity within a short period of time, and that the toxic effect is manifested only within small areas of a cave. The aims of our study were to investigate the negative impact of calcium carbide waste by analyzing the following key points: - chemical composition of different treatments of technical carbide; - transformation of the waste in time under cave conditions and upon treatment with water; - toxic effects of the waste on different organisms; - possible path-ways of ecologically safe waste utilization. STUDY SAMPLES Two series of samples were used in our experiments to identify the degree of toxicity and spatial migration of the toxic substances present within spent carbide. Sampling procedures included collection of 100 g samples of mate- rial in plastic bags. Samples were stored until processing in a refrigerator at +4°C in closed bags. Series I. Sample freshly prepared in laboratory and exposed in a mine for a month. Fresh waste of calcium carbide (sample 1a) was prepared for our experiment by reaction of calcium carbide with water inside a gas generator cylinder designed to produce acetylene for illumination. The fresh waste appeared as a moist powder (water content was 25% by mass), weakly aggregated (crumbly), light grey with occasional small dark and white mottles, with an acrid odor. The experiment using the above sample was set up in the Nikitskaya Mine, south-west of Moscow, Russia (abandoned underground limestone mine, also known as Nikitskaya Quarry). A level patch of a clay ground was used as a treatment plot. After taking a clay sample for microbiological analysis, a 100 g of the fresh waste (sample 1a) was distributed within a 10 x 10 cm area at the surface of the treatment plot. After one month, the 0-1 cm layer of ground just below the waste deposit was sampled for microbiological analysis; a control sample was taken from random points more than 1 m away from the waste deposit. A sample of the remaining waste (sample 1b) was also collected for determination of its chemical composition and toxic properties after the exposure. A portion of sample 1b was pre-treated with water (see methods section), and then the solid residue (sample 1c) was analyzed for its chemical composition and toxicity. Series II: Samples of wastes exposed for different time periods in caves and quarries. The sampling strategy was aimed at collecting spent carbide samples that were exposed under the cave environment for different time periods. The samples were taken from waste piles left by cavers in separate underground locations at specified times (information from personal communications). It should be mentioned that, inevitably, such samples originated from different calcium carbide manufacturers. Sample 2 - from the aforementioned Nikitskaya Mine (Moscow area, Russia), the 'Amphitheatre' Cavern, 150 m from the entrance; exposed for a period of 1-2 years. Sample 3 - from the Nikitskaya Mine, a tunnel about 300 m from the entrance; exposed for a period of 3-4 years. Sample 4 - from Snezhnaya, the Snow Cave (Bzyb-skiy Ridge, Abkhazia), near the Gremyachiy (Roaring) Cavern; exposed for a period of 2 weeks. Sample 5a - from the Barenschacht Cave (Switzerland), a semi-siphon area, the depth of 550 m; exposed for a period of 13 years. Sample 5b - water-treated portion of unground 5a sample. Sample 5c - water-treated portion of the 5a sample ground using a porcelain mortar and pestle. The certified chemically pure fresh calcium hydroxide (Ca(OH)2) was used as a pH control sample. MATERIALS AND METHODS Bulk chemical composition of solid wastes was determined by the energy-dispersive x-ray fluorescence method (Fitton 1997). The instrument "TEFA" produced by OPTEK Ink. was used. Determination of organic and carbonate carbon was done by the method of dry combustion in oxygen flow at 1,000°C (Nelson & Sommers 1996) followed by measuring the emitted carbon dioxide by coulometric titration using an automatic analyzer AN-7529 with combusting device US-7077 (produced by PO "Izmeritel", Gomel, Belorussia). Samples for organic carbon determination were pre-treated with hydrochloric acid. Untreated samples were used for determining the total carbon. The carbonate (inorganic) carbon was calculated as the difference between the total and organic carbon. Polyaromatic compounds were analyzed using the high performance liquid chromatography method and methylated spirit extraction. Polyaromatic compounds were divided in the C18 type column with liquid phase "acetonitrile/water" and measured by a "Stayer" chromatograph with ultraviolet detector. The water-treated samples and water extractions were obtained by mixing the waste with DDI water at the solid-to-liquid ratio of 1:10 and subjecting to four hours rotation at 240 turns per minute. Biotesting was carried out on three different organisms: infusoria, daphnia, and fishes. The cultures of organisms were supported in the "LETAP" collection of the Laboratory of Ecotoxicology (Soil Science Faculty, Moscow State University). Biotesting on infusoria (Paramecium caudatum) Ehrenberg for toxicity determination was performed in a micro-aquarium with individual compartments. Ten to twelve individual cells were placed in each compartment using a capillary pipette. Then 0.6 ml of pure drinking water was added to control compartments, and 0.6 ml of test extractions was added to test compartments. All tests were done with five replications, i.e., each test solution was dropped into five compartments containing in total at least 50 infusorians. The exact number of individuals in each compartment was counted under a microscope. Once the start time of biotesting had been recorded, the micro-aquarium with the filled compartments was placed for 24 hours into an incubator set at 22-24°C. In acute toxicity tests and their diluted versions we identified the mean lethal concentration of pollutant, i.e., the dose which would be fatal to 50% of the test-objects over a 24-hour exposure. Biotesting on Daphnia magna Straus was carried out in three-replications using one-day-old daphnia. Glass cylinders were filled with 100 ml of either test extraction or control solution, and then 10 daphnia individuals were placed into each glass cylinder. After 96-hours of exposure the numbers of surviving crustaceans were recorded. Biotesting on fish (Danio rerio Hamilton-Buchanan) was conducted. Two-week old fish were placed in tanks at a density of one individual per one liter of pure drinking water (control) or the same volume of test solution at the temperature of 23°C, aerated using an air-pump for two hours daily. The experiment was done with five replications. The mortality of fish over a period of 96 hours indicated the degree of toxicity. The fish were not fed during the experiment. The LD50 values were determined using a Probit-analysis (type of regression analysis). A Probit (probability unit) is a probability value corresponding to the percentage of response. The statistical significance of each experimental value depends on its deviation from standard toxic doses. Regression lines in the 'LgD-Pro-bit' coordinates allow for a graphical determination of LD values (Finney 1971). Seed germination tests were conducted using the seed of oats (Avena sativa) 100-ml pots, factory-produced seedling compost (with pH of 5.5-6.0 and nutrient concentrations as follows: N - 150-250 mg/l, P2O5 - 120-150 mg/l and K2O - 150-300 mg/l) and spent calcium carbide. The four experimental treatments, each replicated twice, had different volume proportions of the waste mixed with the compost: 1) 100% compost (control treatment); 2) 90% of compost and 10% of waste, 3) 75% of compost and 25% of waste and 4) 50% of compost and 50% waste (by volume). Ten oat seeds were planted at a depth of 3-5 mm in each pot, watered to reach the soil moisture of 60% and incubated at 23°C. The seed germination time and seedling number and height were recorded on the 3'd, 7'h and 11'h day of experiment. Microbiological analyses of the samples of cave floor soil involved the preparation of soil suspensions at different degrees of dilution (different soil-to-water ratios) that were inoculated on nutrient agar (hydrolyzed power of fish meat - 24 g/l, NaCl - 4 g/l, agar - 12 g/l; pH - 7.3; produced by State Research Center for Applied Microbiology & Biotechnology (Obolensk, Russia) and incubated at 27°C for five days. Then the number of colony forming units (CFU) was counted in tree replications and the average number was calculated. RESULTS AND DISCUSSION The data on the bulk chemical composition of calcium carbide waste samples are presented in Tab. 1. The waste samples were composed mainly of calcium and, to a lesser extent, magnesium compounds (probably hydroxides) that made up about 90% of the sample mass. The content of stable mineral impurities from silica, aluminum, and iron was about 5-7%. The concentrations of heavy metals were generally insignificant. However, the concentrations of sulfur and strontium were high. We can suggest that some part of strontium is bound with sulfur in a stable form. The variation in strontium concentrations is probably associated with the samples origin from different calcium carbide manufacturers. The bulk chemical composition of water-treated samples 1c (fresh waste exposed for 1 month), 5b (un-ground 13-year-old waste) and 5c (grounded 13-year-old waste) was characterized by slightly lower concentrations of potassium and strontium and slightly higher concentrations of aluminum, silica and magnesium. Generally, the assemblage of chemical elements did not change upon contact with water. The data on the organic and carbonate carbon distribution in the samples are presented in Tab. 2. The organic carbon content in most samples was about up to 2%, except for the old waste sample 5a that contained 3.55% of organic carbon. 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