1. Eisenia fetida as a bioindicator of arsenic rich soils in Hawaii
Russell D. Auwae
Sponsors: Dr. Theodore Radovich and Dr. Nguyen Hue
University of Hawaii at Manoa
Department of Tropical Plant and Soil Sciences (TPSS)
IS 489: Environmental Practicum
12/14/2010

2. Eisenia fetida as a bioindicator of arsenic rich soils in Hawaii
Dr. Nguyen Hue, Dr. Theodore Radovich, Russell Auwae
Abstract:
Arsenic-based pesticides were prevalent in Hawaii during the 1920s to 1950s to help
manage weeds in sugarcane fields. Since then, Hawaii soils have accumulated high amounts of
arsenic, which have the potential to damage human health. Health risks include: skin lesions and
hardenings, cancers, and neurological disorders. Many of these sugarcane lands are being
converted to commercial and residential areas, which pose potential health risks to those who
live and work on these lands. Eisenia fetida, an epigeic earthworm, was used during a microcosm
experiment to observe its responses to different concentrations of arsenic in the soils. The
phenomenon known as hormesis was observed during the duration of this study. Low doses of
bioaccessible arsenic (~2 mg kg¯¹) acted as a tonic, which stimulated the appetite, digestion, and
reproduction of the earthworms.
Introduction:
Arsenic (As) is one of the most important global environmental and health toxicants (Das
et al., 2004; Ortiz-Escobar et al., 2006; Fowler et al., 2007; Ravenscroft et al., 2009). As is
known to be detrimental to human health, causing skin hyperpigmentation, lesions and hardening
(keratosis), a variety of cancers, cardiovascular disease, diabetes, and anemia as well as
reproductive, developmental, immunological, and neurological effects (Klemmer et al., 1975; US
EPA, 2003, Brown, 2008; Ravenscroft et al.,, 2009). Arsenic accumulation in soils is often due
to the production or application of arsenic-based pesticides (fungicides, herbicides, and
insecticides) (Matera and Le Hécho, 2001). In Hawaii, pesticides containing sodium arsenite
(NaAsO2) were widely applied to sugarcane fields in the early 20th
century (Hanson, 1962;
Klemmer et al., 1975; Hallecher et al., 1985; Hunter et al., 1995).
Almost all of the former sugarcane lands in Hawaii, where high amounts of As have
accumulated, are no longer under sugarcane cultivation. These lands are rapidly being converted
to residential, community (school, civic center), small business farming, and various commercial
and industrial uses (Cox et al., 1994). High levels of As in these soils presents a potential direct
contact risk to those residing or working on these lands, and is of great concern for those
growing or consuming produce in family and community gardens or commercial plantations, as
been voiced by local citizens (Harden, 2006).
In this study, we were interested in the performance of living organisms in arsenic
contaminated soils. According to Van Hook (1974), earthworms could serve as useful
bioindicators of contamination because of their fairly consistent exposure to the contaminant.
Thus our objectives were to determine: 1) the performance (mortality/survival/growth) rate of
Eisenia fetida in arsenic rich soils 2) the amount of bioaccessible As in the soils.
Materials and Methods:
Two soils from Waimanalo on Oahu and Kea’au on Hawaii were used in this study. Soils
from Waimanalo represent low arsenic soil (~20 mg kg¯¹ As) and soils from Kea’au represent
high arsenic soil (~300 mg kg¯¹ As).

3. Soil samples were crushed to pass a 4mm sieve and placed into 8 in. diameter nursery
pots to monitor the mortality rate of the earthworms. Each nursery pot was filled with 600g of
soil total. Five treatment types were established: 100% Waimanalo soil, 75% Waimanalo soil +
25% Kea’au soil, 50% Waimanalo and Kea’au soil, 75% Kea’au soil + 25% Waimanalo soil, and
100% Kea’au soil. Each treatment type was replicated three times.
Each replicate was inoculated with eight non-clitellated litter worms (Eisenia fetida)
each. These earthworms were taken from a compost bin maintained by TPSS staff. Scraps of
lettuce sustained the earthworms during this experiment. The duration of this study was six
weeks. All microcosm pots were stored indoors to control soil moisture and temperature.
The weight of the earthworms includes live weight and gut contents. To measure
earthworm biomass, all earthworms were cleaned and placed into paper bags. They were later
lyophilized for two days at -40 °C and weighed.
Total, and bioaccessible As was measured with an inductively coupled plasma (ICP)
spectrometer (Hue et al., 2000). Bioaccessible As was measured as follows. One gram of soil
was equilibrated with 100 ml of HCl, pH 1.5, for 1 hour at 37 °C, the suspension was then
filtered through a Whatman no. 42 filter paper. Arsenic in the filtrate was subsequently measured
with an ICP.
Results:
Measured bioaccessible As ranged from 1.13-9.44 mg kg¯¹ As (Table 1). Relative worm
weight ranged from ~50-129 %. Growth of Eisenia fetida peaked at ~2 mg kg¯¹ As and decreases
with increasing amounts of bioaccessible As (Figure 1).
Table 1: Measured bioaccessible arsenic corresponding with the estimated total amount of arsenic and the amount of
Kea’au in each pot.
Numberof Trials Waimanalo(g) Kea'au (g) EstimatedTotal As(mg/kg) Bioaccessible As(mg/kg) Initial EW(g) 2wksEW(g) 4wksEW(g) 6wksEW(g)
1 600 0 20 1.45 2.1 2.2 3.4 3.4
2 450 150 90 3.04 2.2 3.0 4.4 4.2
3 300 300 160 5.87 2.3 4.0 5.2 5.1
4 150 450 230 6.33 2.0 4.5 5.4 5.4
5 0 600 300 6.86 2.1 4.4 5.4 6.0
6 600 0 20 1.13 1.6 2.5 3.9 4.4
7 450 150 90 2.34 1.9 4.0 5.8 6.0
8 300 300 160 4.70 2.4 5.4 6.1 5.8
9 150 450 230 4.95 1.7 3.5 5.0 4.9
10 0 600 300 8.17 1.7 3.6 4.6 4.2
11 600 0 20 1.22 1.6 4.4 5.5 5.1
12 450 150 90 3.53 1.4 3.7 4.5 4.4
13 300 300 160 5.64 1.9 4.6 5.6 4.9
14 150 450 230 6.76 1.7 4.4 5.4 5.0
15 0 600 300 9.44 1.8 4.9 6.1 6.1

4. Figure 1: Growth peak of E. fetida at ~2 mg kg¯¹ As.
Discussion:
Low doses of As seem to have acted as a tonic, which stimulated appetite and growth.
Previous studies have shown that low doses of As have caused a noticeable increase in growth,
also known as the hormetic effect (Neuhauser et al., 1984; van Gestel et al., 1989, 1991; Fischer
and Molnar, 1996; Zhang et al., 2009). The concentrations of As used in this study seem to be
adequate enough for E. fetida to show a noticeable response.
The duration of this study was relatively short compared to other studies (Fischer and
Molnar, 1997; Garg and Kaushik, 2005), which might have yielded different outcomes if given
more time. The small population of worms in each pot, which meant less competition for food,
may have also played a role in the growth increase (Spurgeon et al. 1994). Less competition for
food might have given the earthworms more energy to overcome the toxic effects of As
(Langdon et al., 2003; Holmstrup et al., 2011).
To reduce their growth, food scraps were reduced by half, but the earthworms still seem
to increase in weight and number. Another factor may be due to the fact that majority of the
earthworms used at the beginning of this study were in their juvenile stage, which might explain
this steady growth increase toward maturity. Furthermore, live weight of an earthworm is
strongly influenced by its gut content and hydration status (Frund et al., 2010).
RelativeWormWeight(%)
Bioaccessible Arsenic (mg kg¯¹)

5. If given more time and resources to conduct this experiment, we might have been able to
measure the amount of As in the earthworm tissue with an x-ray fluorescence spectrometer
(XRF). Due to time constraints, we were also not able to measure earthworm cocoon production.
Further Directions for Research:
It would be interesting to conduct a similar As microcosm experiment with the use of
endogeic and/or anecic earthworms to see if they respond differently to As contaminated soils.
As mentioned before, does different concentrations of As affect earthworm cocoon production?
And how much As accumulated in the body tissue of these earthworms? In addition, finding
more economical ways of soil remediation would greatly help contaminated areas like Kea’au,
HI.
Conclusion:
The bioaccessible arsenic measured in this study exceeds the daily exposure amount of
0.004-0.007 mg kg¯¹. This poses serious threats to residents, works, schools, and civic centers in
the Kea’au area. Our results show that low doses of As are acting as a tonic for Eisenia fetida and
there is evidence of a hormetic effect.
Acknowledgements:
Special thanks to Dr. Theodore Radovich and Dr. Nguyen Hue for providing the
necessary materials and facilities to conduct this experiment. We also thank Dr. John Cusick for
facilitating this collaboration.
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