BIOREMEDIATION OF SOIL CONTAMINATED WITH HEXACHLOROBENZENE
Abstract: The ability of four plants to bioaccumulate hexachlorobenzene (HCB) from
contaminated soil was examined. Two grasses (rye- Lolium multiflorum- and corn - Zea mays L.
var. rugosa Bonaf), a tuber (cherry belle radish - Raphanus sativus), and squash (yellow crookneck - Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir) were germinated in HCB
contaminated soil (2% HCB). Growth above the soil level (excluding the root growth) was used
to assess the plant’s bioaccumulating ability. Of the four plant types, only squash indicated the
presence of HCB in the plant growth. In a separate study, the level of HCB in 200 ppm
contaminated soil was reduced to 160 ppm after 4 weeks germination of squash seedlings.
Juliana De Santis*; Rachel Snider*; Robert M. Granger, II†.
Department of Chemistry, Sweet Briar College, Sweet Briar, VA 24595. †(434) 381-6403
e-mail: rgranger@sbc.edu.
Keywords: Environmental Chemistry, Bioremediation, Bioaccumulation, Hexachlorobenzene,
Biomagnification, Bioconcentrate
Introduction:
Hexachlorobenzene (HCB) was first introduced in 1945 as a fungicide, and was widely used
until 1965 to protect the seeds of onions and sorghum, wheat, and other grains against fungus
while in storage. HCB has also been used to make fireworks, ammunition, and synthetic rubber1.
It is a byproduct of the manufacture of industrial chemicals including carbon tetrachloride,
perchlorethylene, trichloroethylene, and pentachlorobenzene. It is also a known impurity in
several pesticide formulations.2 Other continuing sources of this persistent chemical include
application of contaminated pesticides, incomplete incineration of chlorine-containing wastes,
and release from old dump sites.3
HCB is a white crystalline solid that is soluble in organic solvents and insoluble in water. It is
semi-volatile and therefore partitions into the atmosphere as a result. HCB is very resistant to
breakdown, and is known to bioconcentrate in the fat of living organisms. Estimated half lives in
soil from aerobic and anaerobic degradation range from 2.7 to 22.9 years. Low water solubility,
high stability, and semi-volatility favor its long range transport throughout an ecosystem2. A
discussion of the environmental behavior of HCB cites properties, including its resistance to
degradation, its high mobility, and it high lipid solubility. These factors help explain its
detection in all environmental compartments and its presence in the adipose tissues of virtually
all members of the general population.3
HCB undergoes significant bioaccumulation and biomagnification in the food chain.
Bioaccumulation is the intracellular accumulation of environmental pollutants such as heavy
metals and organic pollutants by living organisms. Bioaccumulation could provide a method of
removing HCB from contaminated soils. Removal and incineration of the plant material that has
bioaccumulated HCB could provide an economically preferable means of cleaning super-
contaminated sites. The method most commonly employed is the removal and incineration of
the topsoil. This leaves the site unsuitable for any agricultural use.
Food is determined to be the principal route of exposure for the general population. HCB in
experimental animals has been found to be readily absorbed by the oral route and poorly
absorbed via the skin. Because HCB metabolizes slowly and elimination of it is slow, it is often
present in breast-milk and poses risks to lactating mothers. HCB has also been shown to
accumulate in lipid-rich tissues and it crosses the placental barrier. The evaluation of the effects
on human health draws on numerous reviews,3-6 most notably a study of an accidental poisoning
incident in Turkey3 that occurred in 1955-1959, when HCB-treated wheat grain was ground into
flour and used to produce bread. This resulted in more than 600 cases of porphyria cutanea tarda
with a high mortality rate. In this incident, clinical manifestations of poisoning included
disturbances in porphyrin metabolism, dermatological lesions, hyperpigmentation,
hypertrichosis, enlarged liver, enlargement of the thyroid gland and lymph nodes, and
osteoporosis or arthritis. Most nursing infants of exposed mothers died within a year. To date,
there is no strong evidence that HCB contamination causes cancer in people, although the U.S.
Department of Health and Human Services has determined that hexachlorobenzene may
reasonably be expected to be a carcinogen.1
Experimental:
Materials
Rye (Lolium multiflorum), Corn (Zea mays L. var. rugosa Bonaf), Radish (Raphanus sativus)
and Squash (Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir) seeds were all
purchased from Southern States Cooperative Inc. Potting Soil was supplied by Conrad Farad,
Inc. Hexachlorobenzene and hexane were purchased from Aldrich Chemical Company and used
as received. Naphthalene was purchased from Eastman Organic Chemicals. Chloroform was
purchased from Burdick and Jackson.
Instruments
Gas chromatographs and mass spectra were obtained on a Hewlett Packard 5890 Series II Gas
Chromatograph coupled to a Hewlett Packard 5972 Series Mass Selective Detector. The GC/MS
method employed a Hewlett Packard GC capillary column Part No. 19091S-433 for the study
that compared the bio-uptake of HCB of various plant species and a Supelco MDN-1 Fused
Silica Capillary column for the study that examined the bioremediation of soil using squash. In
both studies, the GC method contained an inlet valve temperature of 300ºC. The detector
temperature was 280ºC. The initial oven temperature was 40ºC and after 6 minutes the oven
temperature increased at a rate of 5ºC per minute to a final temperature of 200ºC. The run then
remained at 200ºC for an additional 5 minutes. The total run time was 38 minutes.
Soil Contamination and Germination:
For the study that compared the bio-uptake of HCB of various plant species, 2,460 grams of dry
potting soil were weighed and to this soil 50.6 grams of HCB were added and mixed thoroughly
giving 2% HCB in the soil.
For the study that examined the bioremediation of soil using squash, a mass of 0.328 grams of
HCB was dissolved in hexane and used to moisten 1,640 grams of dry soil. The hexanemoistened soil was mixed thoroughly and the hexane was allowed to evaporate, giving the soil a
concentration of HCB of 200 ppm8.
For both studies, the seeds were planted in the starter kits and allowed to germinate.
Instrument Calibration:
For the study comparing the bio-uptake of HCB of various plant species, an eight-point
calibration curve was made (see figure 1). Naphthalene was chosen as an internal standard for
the calibration. Standard dilutions were made from a stock solution of 1515.15 ppm HCB in
hexane and a stock solution of 4531.72 ppm naphthalene in hexane. Standards 0 through 8 each
had a 45.3 ppm internal standard (spike) of naphthalene and a range of concentrations of HCB
from 0.0 ppm to 106.5 ppm [ 0.0 ppm (1) 15.2 ppm (2), 30.3 ppm (3), 45.5 ppm (4), 60.8 ppm
(5), 76.0 ppm (6), 91.2 ppm (7); 106.5 ppm (8)].
For the study examining the bioremediation of soil using squash, a five-point calibration curve
was made (see figure 2). Again, naphthalene was chosen as an internal standard for the
calibration. Five standard soil samples (standards 9 to 13) were made with concentrations
ranging from 50 ppm to 500 ppm HCB and extracted to adjust for any interference from the
matrix (soil) and any operational error introduced from the extraction process [50 ppm (9), 100
ppm (10), 200 ppm (11), 300 ppm (12), and 500 ppm (13) HCB]. An 18.00 gram sample of soil
from standard 9 was placed on a sintered glass Büchner funnel. A 50 mL aliquot of chloroform
spiked with 15 ppm naphthalene as an internal standard was added to the 18.00 grams of soil and
stirred on the funnel. The solvent was then pulled through the funnel by vacuum filtration. This
process was repeated two more times and all three 50 mL aliquots of chloroform were combined
and filtered through Celite® to remove any particulate matter. The filtered chloroform extract
was then reduced in volume to 5 mL. This process was repeated for each standard 10 – 13.
Each standard 1 through 8 and 9 through 13 was analyzed by GC-MS. From the gas
chromatograph of each standard, the ratio of the area under the HCB peak to the area under the
naphthalene peak was calculated and plotted versus the concentration of HCB in each standard
(see figures 1 & 2). The use of the internal naphthalene standard helps correct for any
operational error in injection volume.
Harvesting Plant Growth:
For the study comparing the bio-uptake of HCB of various plant species; Ten days after initially
planting the rye grass, squash, and radish seeds, plant growth was cut at the soil level, excluding
the root structure from harvest. The corn was harvested in the same manner after 22 days. Each
plant was rinsed to remove any soil remaining on the plant, and then weighed. The total
harvested mass of each plant type is given in table 1.
For the study examining the bioremediation of soil using squash, the squash plants were gently
removed from the soil and excess soil removed from the root structure. The soil from the
planting tray was spread out in a hood and allowed to dry over a four-day period.
Extraction of HCB:
For the study comparing the bio-uptake of HCB of various plant species, each plant was pureed
in an Oster Osterizer 14-speed blender with 100 ml of hexane to extract any HCB that might be
present in the plant. The extract was filtered through Celite® to remove any particulate matter
and the volume was reduced on a rotary evaporator to concentrate any HCB present in the
extract. Each extract was spiked to 45.3 ppm naphthalene from the stock solution mentioned
above and tarred to 10 mL hexane in a volumetric flask. One L of extract for each sample was
then injected onto the GC/MS.
For the study examining the bioremediation of soil using squash, soil from the planting trays was
extracted in exactly the same manner used to extract the standard soil samples.
Results:
For the study comparing the bio-uptake of HCB of various plant species, the standards showed
HCB with a retention time of approximately 24 minutes and the parent ion (284) was easily
observed. The presence of six chlorine atoms on HCB provided an easily identifiable isotope
breakdown pattern in the MS. The naphthalene spike had a retention time of approximately 6
minutes and the parent ion (128) was easily observed. Each plant extract was analyzed by
GC/MS using the same method as the standards. The HCB peak had a retention time of 24
minutes and the parent ion (284) was easily observed. The squash extract was the only one in
which we were able to identify HCB. The concentration of HCB, in ppm (g/g) for the squash
plants was 810 ppm.
For the study examining the bioremediation of soil using squash, the standards showed HCB
with a retention time of approximately 28 minutes and the parent ion (284) was easily observed.
The naphthalene spike had a retention time of approximately 15 minutes and the parent ion (128)
was easily observed. The soil that had squash cultivated for 4 weeks was analyzed by GC/MS in
the same manner as the standards. Strong peaks were observed in the GC trace at approximately
15 minutes for naphthalene and at approximately 28 minutes for HCB and their identities were
confirmed by the mass spectra. The soil contained 158.6 ppm ( 160 ppm) HCB. This indicates
an approximate 40 ppm reduction in the concentration of HCB after 4 weeks from the time the
squash seeds were planted.
Discussion
Since HCB tends to bio-concentrate in fatty tissue, it may seem a bit surprising that corn was not
the plant species that most readily accumulated HCB as corn has a higher fat and lipid content
than the other plants studied7. Nonetheless, our results demonstrated that squash clearly had the
highest ability to bio-accumulate hexachlorobenzene from contaminated soil and the
accompanying reduction in HCB in the soil was measurable after only 4 weeks.
The ability of squash to sequester HCB could provide a means of cleaning sites of high HCB
contamination. Successive generations of squash planted on a contaminated site could be used to
bio-accumulate the HCB from the soil, and then the plants could be incinerated to destroy the
HCB. Further research on the bioaccumulation of HCB could compare bioaccumulation ability
of different species of squash. The next logical study would be a pilot plot on an outdoor
“garden” and would allow the squash to reach maturity. This type of study would require a
significant investment to ensure containment and to cover clean up costs. This sort of study is
presently beyond the abilities of our labs.
Another area of interest would be to determine how squash sequesters HCB from the soil. An
understanding of the biochemical mechanism of how HCB is sequestered might prove useful in
the development of bioremediation strategies.
Note: This study did not involve an examination of the biochemical breakdown of HCB that
might have occurred in the process of bioaccumulation. But no evidence of chlorine-containing
compounds, other than HCB, was noticed in the GC/MS analyses of each plant extract.
References
1. Agency for Toxic Substances and Disease Registry. Hexachlorobenzene. US Department
of
Health
and
Human
Services,
Georgia,
USA,
September
1997.
http://www.atsdr.cdc.gov/tfacts90.html. Accessed 27 March 2002.
2. IMOC, POPs Assessment Report. http://www.chem.unep.ch/pops/indxhtms/asses6.html
December 1995. Accessed 1 November 2001.
3. Environmental Health Criteria No. 195. World Health Publications. 1997, xviii. ISBN
92 4 157195 0. Order number 1160195.
4. G. M. Lackmann, Biology of the Neonate 2002, 81(2), 82-85.
5. G. M. Lackmann; J. Angerer; U. Salzberger; U. Tollner. Biology of the Neonate, 1999,
76(4), 214-219.
6. M. L. S. Queiroz; R. C. R. Perlingeiro; M. R. Quadros; C. A. Souza. Human &
Experimental Toxicology 1998, 17(3) 172.
7. Total lipid content of sweet corn is 1.18% and that of yellow crook-neck squash is 0.1%.
Values obtained form the USDA Nutrients Database for Standard Reference.
www.nal.usda.gov/fnic/cgi-bin/nut_search.pl . Accessed 15 March 2002.
8. Concentrations in units of parts per million were calculated as (grams analyte / grams
matrix)106.
Table 1: Mass of each plant type
harvested in comparison study.
Plant
Grams
Type Haravested
Rye Grass
4.06
Corn
2.99
Radish
6.03
Squash
8.86
Calibration Curve for HCB with Napthalene Spike
[HCB]/[Spike]
y = -0.015x – 0.1327 R2 = 0.9774
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-10
10
30
50
70
90
110
[HCB], in ppm
Figure 1: Calibration curve for the study that compared the biouptake of HCB of various plant
species. The y-axis is the ratio under the GC peaks for HCB/Naphalene (hexane) for standards 1
 8. The x-axis is ppm HCB for standards 1  8. Standards 1 through 8 increased in HCB
from 0 ppm (1) to 15.2 ppm (2), 30.3 ppm (3), 45.5 ppm (4), 60.8 ppm (5), 76.0 ppm (6), 91.2
ppm (7); 106.5 ppm (8). Each standard had a 45.3 ppm naphthalene spike as an internal
standard.
Calibration Curve for HCB with Napthalene Spike
y = 0.0077x – 0.0578 R2 = 0.9996
4.0000
[HCB]/[Spike]
3.5000
3.0000
2.5000
2.0000
1.5000
1.0000
0.5000
0.0000
0
100
200
300
400
500
600
[HCB], in ppm
Figure 2: Calibration curve for the biremediation of soil study. The y-axis is the ratio under the
GC peaks for HCB/Naphalene (chloroform) for standards 9  13. The x-axis is ppm HCB for
standards 9  13. Standards 9 through 13 increased in HCB, 50 ppm (9), 100 ppm (10), 200
ppm (11), 300 ppm (12), and 500 ppm (13) HCB. Each standard had a 15 ppm naphthalene
spike as an internal standard.