EARTHWORMS AS BIOINDICATORS OF MERCURY POLLUTION

Jennifer Hinton and Marcello M. Veiga

Department of Mining and Mineral Process Engineering, University of British Columbia,

hinton@interchange.ubc.ca, veiga@mining.ubc.ca

ABSTRACT

Mercury (Hg) pollution can be evidenced by high levels in soils, water and biota. As the toxicological impacts of mercury are largely dependent upon speciation, understanding its transformations and impacts of the various chemical forms are vital to preventing harmful human and environmental health effects. Bioindicators play an important role in identifying the factors controlling Hg toxicity and bioavailability and can ultimately be used to evaluate hazardous situations. A simple, cost-effective methodology would be particularly beneficial in regions of the world such as developing countries, where mercury pollution can be extensive, yet resources limited. Earthworms are simple, well-studied creatures that can quickly provide indications of bioavailability at relatively low costs. A methodology using the earthworm Eisenia foetida has been developed to evaluate the bioavailability of Hg in mining tailings and aqueous solutions. Results indicate that E. foetida do accumulate Hg and a positive correlation exists between Hg concentrations in worm tissues, the substrate they consume and the length of exposure. To investigate the effect of natural organic acids as mediators of Hg bioavailability, metallic Hg was dissolved in tannic acid and “fed” to the worms in a substrate of paper and silica sand. Total Hg and MeHg were analyzed to determine whether methylation of Hg was occurring in the substrate, directly within the worms (e.g. in the intestines), or in the tannic acid-Hg solution. The MeHg:Total Hg ratio was up to 160 times higher in worm tissues than both the tannic acid-Hg solution and the substrate. This result is particularly significant in darkwater systems, where naturally occurring organic acids may be facilitating methylation internally within organisms. The reaction of metallic mercury with organic acids from sediments and darkwater systems is definitely an important pathway for mercury bioavailability.

Keywords: mercury bioaccumulation, bioindicators, earthworm, darkwater, biogeochemical cycling

Introduction

It is well known that dissolved organic matter, such as humic and fulvic acids (HA and FA), forms more stable and predominant mercury complexes than any inorganic species (Duinker, 1980; Xu and Allard, 1991). As well, HA and FA enhance solubility of organic matter and associated mercury. Over a large pH range (4 to 9), Hg becomes very soluble when more than 20 ppm of FA is added to solution (Schnitzer and Kerndorff, 1981). Melamed et al. (1999) experimentally demonstrated that humic acid solutions increase the solubility of metallic Hg. When organic acids contact metallic Hg, in interstitial waters for example, soluble complexes are formed at lower Eh levels than those observed in the Eh-pH diagram for inorganic soluble Hg species (Tromans et al., 1996). As oxygen is likely the main electron donor in this complex formation, Hg oxidation is controlled by oxygen diffusion in water. So, when Hg-contaminated sediments ("hot spots") exist in shallow creeks with high dissolved oxygen levels or atmospheric Hg is deposited on top of organic soils, the possibility for formation of Hg-rich soluble complexes is high. In deep sediments, available oxygen is likely to be extremely low and non-replenished (Meech et al., 1998). How Hg associated with organics transforms into methylmercury (MeHg) is unclear. As fulvic acids are known to be methyl-group donors, methylation of these complexes seems to be feasible through either biotic or abiotic processes (Mannio et al., 1986; Verta et al., 1986).

Interestingly, Hg-organic complexes may be subject to direct bioaccumulation. Since most Hg found in fish flesh is already methylated, Hg-organic complexes consumed by invertebrates or fish may be methylated in intestines of the organisms. Rowland et al (1977) showed that Hg(II) ingested as a chloride can be methylated in less than 20 hours by intestinal bacteria. They estimated that the total MeHg synthesized from ingested inorganic Hg in man is approximately 0.4 mg/day. No information is currently available on intestinal methylation of Hg-organic complexes.

Bioindicators can aid identification of factors controlling Hg toxicity and bioavailability and can ultimately be used to evaluate hazards where Hg pollution is present. Metal bioavailability is dependent upon a number of geochemical and biological factors. The presence of organic matter (Gagnon and Fisher, 1997, Standley, 1997), colloidal particles and certain minerals, such as sulfides (Melamed, 1997) or Fe, Mn oxides (Gagnon and Fisher, 1997), influence speciation and/or sorption mechanisms and thus bioavailability of various metal compounds (Benoit et al., 1999; Wen-Xiong et al., 1998). Organism physiology, internal solubilization capabilities (Gagnon and Fisher, 1997), food quality (i.e. nutrients) and feeding behavior also affect the metal assimilation efficiency (Lawrence et al., 1999, Wen-Xiong et al., 1998). Thus, an appropriate bioindicator organism must be reasonably well understood in terms of biological qualities and responses and be broadly applicable to various external (e.g. geochemical) conditions.

Substantial evidence indicates that earthworms accumulate heavy metals from various media and are particularly suitable for the assessment of contaminant bioavailability (Edwards and Bohlen, 1996; Goats and Edwards, 1988; Rhett et al., 1988; Neuhauser et al., 1985; Ireland, 1983). They ingest large quantities of soil and are in full contact with the substrate they consume. They constitute up to 92% of the invertebrate biomass of soils and participate in many food chains, acting as a food source for a wide variety of organisms including birds, fish, insects, various mammals, and reptiles (Ireland, 1983; ASTM E1676-95). In addition, they are easily bred, extensively studied, and are approved for use in toxicity testing by the US EPA, the European Economic Community and the Organization for Economic Cooperation and Development (ASTM E1676-95). Despite these factors, little information exists concerning Hg and MeHg uptake in these organisms. Few studies (Braunschweiler, 1995; Rhett et al., 1988; Marquenie and Simmers, 1988; Martin and Coughtrey, 1982) have documented Hg concentrations in earthworm tissues and even fewer (Lawrence et al, 1999; Yongcan et al, 1998; Beyer et al., 1985) have addressed the biological and physiological elements that influence Hg bioavailability in these organisms.

Methodology

Organism Culturing and Selection. E. foetida were initially acquired from a local composting cooperative and cultured in a dark plastic, ventilated bin on a diet of either alfalfa pellets. Worms were hand-selected for testing on the basis of sexually maturity, as evidenced by the presence of a clitellum, size (0.25 to 0.3 g wet weight), and liveliness. Prior to use, chosen worms were stored for 24 hours on damp filter paper to void their gut contents.

Tannic Acid-Hg Jar Tests. Two separate series of these tests were conducted. Metallic mercury (TA1: 3.03 g and TA2: 6.18 g) was added to 0.005M tannic acid (0.3 and 1.0 L volumes) and vigorously stirred for one (TA1) and three (TA2) days to promote dissolution. Total Hg concentrations in the tannic acid solutions were 696 ppb (TA1) and 1150 ppb (TA2). Prior to full scale testing, 3 to 5 worms were exposed to the pure tannic acid solution (pH 4.1) in Petri dishes to provide some indication of acute responses. Most specimens died within two hours. Subsequent adjustment of the pH (to 5.85 and 6.02, respectively) enabled long term habitability for the worms.

Shredded, kaolin-based paper (25g) and 175g (TA1) or 100g (TA2) of fine silica sand were added to nine 500mL acid washed, glass jars. The pH adjusted tannic acid-Hg solution (80 mL) was added and jars were manually shaken to homogenize. Groups of 25 to 30 worms were weighed and added to each jar. To minimize stress, populations were left relatively undisturbed for the duration of the tests (14 or 28 days). Silica not only retains some moisture, but is also used by worms for grinding during the digestion process. At the conclusion of the exposure period, worms were removed from each jar, carefully washed and dried, counted and weighed. Observations such as motility, light sensitivity and physical qualities (e.g. discolouration) were documented to provide some indication of toxic responses. Cleaned worms were then placed in Petri dishes with damp filter paper for either 24 or 72-hour depuration periods, then re-washed and re-weighed. Worms from two jars (OCT99-7, OCT99-8) were kept in mixtures of clean paper towel (15g) and silica (50g) saturated with 50 mL of distilled water for a period of 5 days. Prior to analysis these worms were also depurated for 24 hours. Experimental specifications for each jar are noted in Table 1.

Sample Preparation and Analysis. Following post-depuration washing and drying, worms were placed in 250ml, acid washed Erlenmeyer flasks and digested in 20 mL of 0.7M nitric acid to be analyzed by CVAA. Samples submitted for MeHg analysis were not digested but frozen immediately following post-depuration washing and drying. Methylmercury was analyzed by Cebam Analytical Inc, Seattle, WA by aqueous phase ethylation, purging, Tenax trap pre-collection, GC separation and CVAFS detection. Total Hg and MeHg in the tannic acid-Hg solution was analyzed directly by the aforementioned methods. At the conclusion of the 28-day test period, 178g of substrate material (i.e. tannic acid saturated paper and silica sand) was leached with distilled water (300mL). The combined material was shaken vigorously for several minutes. Leachate (198 mL) was subsequently extracted using a vacuum filter and submitted for MeHg and total Hg analysis.

Results

Results of this research program indicate that E. foetida can bioaccumulate Hg and other metals from the substrate they consume. In the tannic acid-Hg jar tests, Hg concentrations in worm tissues ranged from 0.7 to 7.3 ppm (Table 1). Bioconcentration factors (the ratio of Hg concentrations in worm tissues to the substrate) averaged 3.7. The most bioconcentration occurred in populations that exhibited toxic responses to their environment (e.g. discolouration, impaired mobility). Conversely, lowest Hg concentrations were detected in the healthiest populations. Variability between populations was likely due to insufficient homogenization of jars contents.

Although 35 to 40% more Hg was, on average, accumulated during the 28-day tests, populations were generally healthier and more responsive than the 14-day jar tests. Average worm weight actually increased for 28-day populations. This overall improved condition could be due to reduced stress associated with the extended acclimatization period. As healthier, less stressed organisms are supposed to be more indicative of natural uptake, 28-day tests are recommended. Depuration times (24h, 72h, 5days plus 24h) were also compared during this study.

Sample No.	Test Description	Hg in Tissues (ppb)	Hg in Substrate (ppb)	Bioconcentration Factor
OCT99-1	14d, 24hr dep	2837	828	3.4
OCT99-2	14d, 24hr dep	2449	828	3.0
OCT99-5	14d, 72hr dep	3695	828	4.5
OCT99-6	14d, 72hr dep	1614	828	1.9
CAM99-1	28d, 24h dep	861	278	3.1
OCT99-3	28d, 24hr dep	2499	828	3.0
OCT99-4	28d, 24hr dep	749	828	0.9
OCT99-7	28d, 5d feed plus 24h dep	7305	828	8.8
OCT99-8	28d, 5d feed plus 24h dep	4055	828	4.9
14d Average	-	2649	828	3.2
28d Average	-	3094	718	4.1
Overall Average	-	2896	767	3.7

Table 1 - Results of Tannic Acid-Hg Jar Tests

The average worm weight loss was about 30% greater in the worms fed clean paper for 5 days following the test; despite this, Hg concentrations were up to 40% higher in these populations. It is possible that during the additional 5 days, Hg containing gut contents were more thoroughly assimilated than other tests. The overall weight decrease may be indicative of more complete purging. Hg concentrations in tissues generally increased with purging time (Figure 1). Physiological explanations for these responses are currently being studied in greater detail. A 5-day “clean” substrate feeding (plus 24 hour depuration) following a 28 day test provides the most reliable results.

Fig. 1 – Total Mercury Concentrations in Worm Tissues versus Purging Time

Total Hg and MeHg were analyzed to assess whether methylation of Hg was occurring in the substrate, directly within the worms (e.g. in the intestines), or in the tannic acid-Hg solution. The MeHg:Total Hg ratio was up to 2400 times higher in worm tissues (33.3 ppb) than both the tannic acid-Hg solution (0.059 ppb) and the substrate (0.007 ppb). This result is particularly important in darkwater systems, where naturally occurring organic acids may be facilitating methylation internally within organisms. However, MeHg (32.2 ppb) constituted only around 1% of the total Hg in worm tissues (from a 28-day test), which is considerably lower than measured values in higher organisms. It is possible that, as earthworms are consumed (i.e. as the Hg moves up the food chain), it is subject to further methylation internally within other organisms.

Discussion

The reaction of metallic mercury with organic acids is definitely an important pathway for mercury bioavailability. The mechanisms influencing bioaccumulation or methylation of organic complexes require further study. This testing program revealed that the MeHg:Total Hg ratio was 2400 times higher in worm tissues than the substrate and 125 times the tannic acid-Hg solution. This result is particularly important in darkwater systems, such as in the Amazon, where naturally occurring organic acids may be facilitating methylation internally within organisms.

Bioindicators play an important role in identifying the factors controlling Hg toxicity and bioavailability and can ultimately be used to evaluate hazards where Hg pollution is present. Easily implemented, low-cost methods, such as the one presented, can be highly beneficial for rapid diagnosis of potentially hazardous situations, particularly in regions such as the Amazon where resources are limited and pollution is widespread. Earthworms (E. foetida) are capable of accumulating Hg and other metals and a positive correlation exists between Hg concentrations in worm tissues, the substrate they consume and the length of exposure (a dose-response relationship). The methodology presented can be effectively used as a tool for the assessment of Hg and other metal bioavailability in polluted soil, sediments and tailings.

Acknowledgements

We would like to thank BC Research, particularly Janet Piccard and Rick Vos, for both laboratory support and helpful consultations throughout the earthworm bioaccumulation study. Many thanks to research sponsors: a Natural Science and Engineering Research Council of Canada (NSERC) operating grant #217089-99.

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