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The effects of high levels of polycyclic aromatic hydrocarbons on sediment physicochemical properties and benthic organisms in a polluted stream
Chemosphere, Vol.16, No.5, P r i n t e d in G r e a t B r i t a i n
pp
1053-1063,
1987
0045-6535/87 $ 3 . 0 0 + .OO P e r g a m o n J o u r n a l s Ltd.
THE EFFECTS OF HIGH LEVELS OF POLYCYCLIC AROMATIC HYDROCARBONS ON
+ SEDIMENT PHYSICOCHEMICAL PROPERTIES AND BENTHIC ORGANISMS IN A POLLUTED STREAM
William J. Catallo III
(I) and Robert P. Gambrell (2)
(I) Division of Chemistry and Toxicology, Virginia Institute of Marine Science, Gloucester Point, VA
(2) Laboratory for Wetland Soils and Sediments, Center for Wetland Resources, Louisiana State University, Baton Rouge LA
ABSTRACT The effects of polycyclic aromatic hydrocarbons on benthic micro- and meiofaunal biomass was determined and related to selected sediment properties such as redox potential and organic matter content in a contaminated environment. INTRODUCTION
Bayou Bonfouca is a palustrlne, mlxed-forested, ecosystem in the Lake Pontchartrain Basin near Slidell, Louisiana (I).
As a result of a fire at a wood products treatment plant
in 1970, approximately 9 hectares of the bayou and surrounding drainage areas have been heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) in a coal tar mixture known as creosote.
PAHs in the environment, food, water, and the workplace are of concern
because of the mutagenlcity and/or carclnogenlclty of several of the compounds in this class (2-5).
+ VIMS
Contribution
No.
1355 1053
105 ~
Creosote
is formed as a by-prOduct
of coal and is characterized
with CI-C 4 alkyl homologs also present used as a sealant and "non-leachable" railroad
during the high temperature
by concentrated
ties, and pilings.
(> 900°C)
mixtures of predominantly
in limited amounts
(6-8).
carbonization
unsubstituted
This material commonly
bioclde for wood products such as telephone
Normally,
PAHs is
poles,
wood is pressure treated at elevated temperatures
to
ensure that organisms already present are killed and that the creosote has thoroughly permeated
surface layers.
arsenates
may be employed when severe fouling
encountered
(7,8).
Adjunct treatments
Creosoting
operations
with pentachlorophenol
is expected or when creosote
tend to be spill-prone
PAHs, PCP, and various metals have been detected preservation
facilities
(9,10).
in environments
PAH compounds organisms
there is growing biochemical
and mixtures
and biota near wood-
to neoplasia
and proliferative
(11-14).
However,
literature
disorders
effects and the physicochemical
properties
in workers and human populations
there is little work dealing specifically
of sediments
The purpose of this work was to examine properties
biomass estimates hypothesized
in contaminated
of benthic bacteria,
control.
properties
protozoans,
Superfund
It was
PAH toxicity would be observed in blomass reductions
in all trophlc and changes
in
such as redox potential and organic matter content relative
the reader is referred to references
is the only published Louisiana
and meiofauna.
For more extensive reviews of PAHs, creosoting operations,
Bayou Bonfouca,
between these
in natural systems.
the effects of PAHs on selected sediment
levels examined and that this would be manifested related sediment
with
areas of Bayou Bonfouca and to relate these with
fungi,
that concentration-dependent
linking various
in exposed aquatic
the effects of PAHs on benthic micro- and melofauna and the relationships
physicochemlcal
of
poles (9).
and epidemiological
as well as to increases of certain cancers
exposed to these materials
"bleed" is
and high concentrations
Smaller amounts of these substances have been identified
near point sources such as wharf structures and telephone At present
(PCP) and copper
6-9, and 15-17.
and the situation
to a at
To our knowledge,
this
study of the Bayou Bonfouca spill site, which was placed on the
Cleanup List in 1982.
MATERIALS AND METHODS
Four sites were established pollution
at Bayou Bonfouoa along an apparent gradient of creosote
and were assigned numbers as follows:
2 = "moderately
contaminated",
contaminated".
This gradient
were characterized
site I - control or "uncontaminated",
slte 3 = "heavily contaminated",
was later verified by analysis of sediment samples.
by soft bottoms,
m), similar vegetation
and shading,
and similar proximity
to the Slidell
gentle surface-water approximately
flows,
equivalent
site
site 4 = " extremely
shallow depths
litter input
urban area and its associated
All sites
(0.6 - 1.2
(visual estimate),
sources of pollution.
Sediment Characterization:
Bulk surface
(-10 cm) sediment samples for particle size analysis were collected
each site wlth a shovel and were stored
in plastic buckets with llds.
Sediments
to be
from
1055
analyzed for metals were collected with acid-rinsed teflon scrapers and placed in glass jars with teflon caps.
Redox potential (Eh) measurements were taken in situ using commercially
available platinum electrodes and saturated calomel reference electrodes.
Sediment pH was
determined using a calibrated combination pH electrode and values for both pH and Eh were obtained with a portable pH/millivolt meter (Orion Model 399A). Particle size distributions were determined via a standard hydrometer method (18), and readily oxidizable organic matter content by the potassium chromate/acidlc oxidation method of Jackson (19).
Sediment metals were extracted using the hot HCI/HNO 3 method of Anderson
(20) and analyzed by inductively coupled argon plasma emission spectrometry (ICAP).
PAH Extractions:
All PAH extractions were accomplished using highly purified reagents of "HPLC grade" (Curtin Matheson, Houston, TX), "Pesticide Grade" (Sargent Welch, Dallas, TX), or "Nanograde"
(Malllnekrodt, St. Louis, MO).
reagent grade.
Anhydrous Na 2 SO 4 and KOH were of analytical
The PAH standards phenanthrene, pyrene, l-methyl fluoranthene,
benzo(k)fluoranthene,
benzo(e)pyrene,
dibenzo(a,h)anthracene,
dlbenzo(a,i)pyrene were
donated by the NCI Chemical Carcinogen Reference Repository (NIH, Bethesda, MD). Naphthalene, anthracene, chrysene, pyrene, and benzo(a)pyrene Scientific) were of 98 % purity or better.
(Aldrich Chemical and Fischer
A mixture of 16 PAH standards
(Supelco Inc.,
Bellefonte, PA) also was used for standardization. All solvents, column packings, drying agents, and standards were assayed via high pressure liquid chromatography (see below) for impurities eluting in the range of interest. All sediment and water samples for PAN analyses were collected using solvent-rinsed glass or metal equipment and were stored in glass or metal containers with foil-lined caps. Care was taken to avoid contamination and exposure of standards, samples, and extracts to light was minimized.
Sediments were extracted using the method of Farrington et al. (21)
with the addition of ultrasonic disruption prior to alkaline reflux extraction, and the substitution of cyclohexane for benzene as the primary solvent.
Other modifications
included the use of centrlfugation instead of filtration, with extract volume reductions being accomplished where necessary by evaporation under a dry stream of nitrogen while sample containers were held in a controlled-temperature water bath (< 4°C) rather than in a rotary evaporator.
Surface water samples were extracted via the method of Sorrell et al
(22). PAHs were identified and quantified using a Waters Associates Model 480 Isocratic HPLC with the following specifications:
6000a pump; U6KA injector; a 480 L.C. detector; and a
Waters Associates RCM Bondapak column with a precolumn filter. acetonltrile:water at I ml/mln.
The solvent system was 86:14
Seperated compounds were scanned at 254 nm and absorbance
was recorded on a Hewlett Packard 3390a integrator with quantification accomplished by referencing sample peak heights to standard curves for each compound identified. The following criteria were utilized for PAH identification: compound was required to match that of a standard, upon the addition of an appropriate "spike",
I) retention time of the
2) detector response had to increase
3) the compound had to exhibit its documented
absorbance maximum as well as several lesser absorbance peaks.
The ratios of the absorbance
1056
maximum to the corresponding
lesser peaks were required
to match those determined
using
standards.
Microbiological Assays:
ATP was determined
photometrically
At each site, approximately scintillation
vials containing
following
the method of Portier
(pers. comm., 23).
one gram of sediment was placed in preweighed,
9 ml of pH 7.4
phosphate buffer.
sterile,
These were stored on ice
until analysis
which occurred no more than 12 hours after collection.
to equilibrate
at room temperature
Samples were allowed
for three hours prior to analysis at which time the
sample vials were dried and weighed to determine
the actual weight of sediment collected.
100 ~i of sample was reacted with 100 ~i of a releasing agent in a 5 ml glass tube and placed
in an ATP phtometer.
specific
for bacterial
The releasing agents
(Lumac Systems,
(23).
100 ~i of luciferin/luciferase
ATP-dependent
and the resultant
detected and recorded as relative standard and background to calculate
corrected
protoctists,
The mixture was then treated with evolution of light was
For each run, "quench"
determinations
in the results.
The corrected
was conferred
culture exclusively
to the linear range of an ATP standard curve and were Conversion
of RLU to cell
is neither required nor usually employed.
(24) and Jensen's
(25) media were used in pour plate procedures
fungi and bacteria respectively.
~g/ml to Jensen's media to inhibit overgrowth Meiofauna
community
by the releasing agents.
for the purposes of biomass comparison between sites.
Both Martin's
RLU values
of total ATP of a specific microbial
fungi) and this specificity
number or cell density
of a known ATP
These values as well as sample weight were used
RLU which are reported
All RLU values were referenced appropriate
light units (RLU).
RLU were recorded.
direct photometric
(bacteria,
FL) were
or somatic cells and allowed for permeable release of nucleotides
without cell lysis or action by cellular enzymes
represent
Titusville,
Cycloheximide
by filamentous
to
(Sigma) was added at 40
fungi
(23).
(sediment biota retained on a 500 ~m sieve after passing through a I mm
sieve) were collected
from each site using a 3-cm corer.
cm which was expected
to contain over 95 % of the total meiofauna
treated with a formalin/rose
bengal solution,
wet-sieved,
all meiofauna were counted with a Nikon dissecting
Samples were taken to a depth of 8 (26).
Core samples were
rinsed with distilled water,
and
microscope.
RESULTS AND DISCUSSION
The concentrations presented
in Table I.
of PAHs in sediment and water samples These data are in general agreement
analyses of Bayou Bonfouca creosote Sediment presented
characterization
in Tables
by Laseter
from Bayou Bonfouca are
with the GC-mass spectrometric
(27).
data are presented
in Tables 2 and 3.
2, 4, and 5 were generated by Duncan's Multiple
a 0.05 experimentwise
error.
indicated highly significant
All statistics
Range Tests
(DMRT) with
The DMRTs were conducted after analysis of variance differences
(P < 0.01) between the reference
(ANOVA)
(site I) and the
1057
spill areas (sites 2-4).
All non-significant ANOVAS (P > 0.05) were reflected by non-
significance in the DMRTs which are presented in the tables.
Table
1.
Concentrations o f PAHs i n S e d i m e n t s a n d W a t e r f r o m B a y o u Bonfouca, Slidell, Louisiana (Parts per million)
Site
PAH Compound
Surface Water
Sediments
I
............
3 unknowns
3-5 unknowns
2
Naphthalene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(J)fluoranthene/ benzo(k)fluoranthene*
3
4
Naphthalene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(J)fluoranthene/ benzo(k)fluoranthene Benzo(a)pyrene
Naphthalene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(j)fluoranthene/ benzo(k)fluoranthene Benzo(a)pyrene
* Isomers not resolved.
8.3 7.6 28.7 14.7 27.1 18.3
1380 358 435 107 608 178
4.9
75
0.7 0.6 2.3 0.4 1.2 2.1
7720 7720 17670 1110 3360 800
nd** 0.3
940 40
14.1 12.3 155.O 39.7 110.0 85.0
7117 29310 1650 6580 1660
5.5 6.6
2280 610
** Not detected.
As can be seen from examination of Tables I-3, the most prominent differences between sites were creosote content, organic matter (OM), redox potential (Eh), pH, Zn, and Ca concentrations.
The particle size distributions indicated closely related sediment types
with sites I and 2 bordering between "silt loam" and "sandy loam", and sites 3 and 4 being "silt loam" (Table 3).
The OM contents varied considerably between sites and the presence
of poorly degraded plant material in the vertical sediment profiles of sites 2-4 suggested that this was the result of low saprophytic activity.
Since site I had low OM and no
detected creosote, the increasing presence of OM in sites 2-4 was likely the result of creosote toxicity to saphrophytlc organisms.
1058
T a b l e 2.
Sediment C h a r a c t e r i s t i c s Site
1
2
3
4
Organic Matter (%)
x- 3.4a si 0.2
9.5b 0.06
16.4c 0.7
32.3d 0.66
pH
x- 7.4a sI 0.2
7.6ab 0.2
8.2b 0.3
I0.4c 0.7
Eh (mV)
xi -214a s- 115
-I04b 52
-99b 63
-35b 57
NI 3, s- standard deviation. Means (x) followed by the same letter on a horizontal line are not significantly different at pI 0.05.
The Eh values for each site appeared to be related to the creosote concentrations as well.
At site I, Eh values showed strongly reduced conditions resulting from limited oxygen
flux to the sediments (as a result of flooding) and metabolically active microbial communities.
At sites 2, 3, and 4, the redox potentials were progressively less reducing as
creosote levels increased.
This Eh trend may be interpreted in two ways: (I) microbial
metabolic processes were limited in the spill areas, or (2) higher energy redox systems (ie., NO 3_ -> NH3/NH4 + , or Mn(III, IV) -> Mn(II)) predominated in these areas.
Since all
sites were permanently flooded, well supplied wlth OM, similar in grain size, and comparable in terms of Mn and Fe concentrations,
T a b l e 3.
the latter interpretation is unlikely.
S e l e c t e d Sediment P r o p e r t i e s Site
I
Clay (%) Silt (%) Sand (%) Creosote (%) * Cu (~g/g) Zn Cd Pb Ni As Fe Mn Ca Mg Mo A1
10 68 22 0 2 30 4 30 4 35 5590 51 714 900 8 9700
2
3 49 48 0.8 10 80 6 80 2 25 6200 73 1900 580 6 10300
3
4
4 66 30 10
5 46 49 25
12 115 10 52 16 135 7320 193 23400 3080 8 10600
8 80 13 32 4 37 4300 117 49000 1340 5 6660
Estimated from phenanthrene concentrations in samples and source creosote.
In the absence
1059
of biolimiting factors it would be expected that all sites would demonstrate strongly reducing conditions similar to site I. Sediment pH values at some sites were surprisingly high initially.
Upon further
investigation, the trend of increasing pH at sites 2-4 was thought to be related to the the progressively higher levels of Ca, probably introduced by runoff of lime from a local concrete plant (Table 3).
It is also probable that the high levels of creosote in the
sediments of sites 3 and 4 caused electrode interference and artificially high pH values. Further, although the pH values were in some cases "significantly different" from the control, there was a difference of less than I pH unit between sites I, 2, and 3, and this was not considered to be biologically important.
Site 4, however, had higher pH values and
this may have had an impact on benthic organisms, but relative to the high creosote concentrations also found at this site, the pH was thought to be of minor importance, particularaly with respect to microorganisms. soils and sediments are influenced by pH.
It should be mentioned that measured Eh in
A pH correction on measured Eh is difficult to do
in natural environments though it is known that increasing pH is accompanied by decreasing Eh values.
Thus it is important to point out that if appropriate pH correction factors
could be applied to the Eh values presented in Table 2, the more contaminated sites would indicate an even greater difference relative to the control area. The metals concentrations reported in Table 3 represent "potentially bioavailable', levels, ie., not including metals contained within the lattice structure of clay minerals. These metals were most likely tightly bound to sediment constituents and not readily available, hence not significantly bioinhibitory relative to other sources of toxicity. The results of the ATP analysis of the mlcrobenthos and direct counts of meiofauna are presented in Tables 4 and 5 respectively.
These data show that the abundances of viable
bacteria, fungi, and protozoa at sites 2, 3, and 4 were much lower than site I.
DMRTs of
all communities from site I against sites 2, 3, and 4, showed significant decreases in these communities with the means of each site decreasing with increasing creosote.
Meiofauna
counts show analogous reductions in response to increasing levels of creosote and this may be related to depressed microbenthic activity as well.
Table 4.
Microbenthic Community Analysis RLU/g sediment
I
Bacteria
x-
Site 2
3
4
s-
22619a 7768
9117b 2710
5501bc 6686
95c 28
Protozoa
xs-
19911a 5731
5857b 1715
4951bc 5690
78c 31
Fungi
~s-
6983a 6792
327b 552
136b 207
N= I0, s- population std. dev. at P- 0.05.
3b 6
Means (2) with same letter are not significantly different
1060
Table 5. Meiobenthic Community Analysis Organisms/10 cm Site
1
2
3
4
Nematodes
x= s=
91a 20
14b 21
Ob 0
Ob 0
Oligochaetes
x= s=
20a 13
16ab 15
Ob 0
Ob 0
Other
x= s=
7b 8
Ob 0
Ob 0
38b 38
Ob 0
Ob 0
Total Melofauna
22a 7
x= s=
133a 20
N=3, s= population
std. dev.
Means wlth the same letter are not significant
at P= 0.05.
Viable plate count results are given in Table 7 and support the interpretations preceedlng
sections
in that there is a creosote-dependent
fungal colony forming units
decrease
of
in both bacterial
and
(CFU).
Table 6.
Summary of P l a t e Count Results CFU/Plate Slte
I
Bacteria
xs=
TNTC --
Fungi
x= s=
58 9
2
3
351 118
116 47
40 6
39 6
4
71 22 0.25 0.40
N= 12, s= std. dev. Colonies
too numerous
to count. Count stopped at CFU= 500 in all samples.
CONCLUSIONS
The results of this study indicate that the high levels of PAHs in the sediments of large areas of Bayou Bonfouca adversely was manifested creosote levels more oxidizing.
at all of the trophic increased,
detrital
affected micro- and melobenthlc
communities
and this
levels studied as well as in sediment properties. accumulations
also increased and redox potentials
These phenomena were related to a reduction of saprophytic
As became
biomass relative
106 1
to the control area.
The removal of fungi in polluted sites was probably the major factor
in detrital accumulation since fungi are of primary importance in terms of litter degradation (28).
The reduction of bacterial biomass was thought to be the primary cause of
increasing redox potentials.
The association of measured high levels of PAHs with
alterations of the physical-chemical characteristics of the sediments is considered to be the primary finding of this research.
Attention is drawn to the biochemical findings in
hopes that the methods described in this work will be applied and expanded in future research and that the concept of employing trophic biomass measurements for the evaluation of pollutant impacts in natural systems will be subjected to further study.
ACKNOWLEDGEMENTS
The authors thank Walter B. Sikora, Jean P. Sikora, and Samuel P. Meyers for advice, materials, and critical review of this research.
Thanks are extended to Michael Bender,
Richard Wetzel, John Zeigler, and Ruth Hershner for further review and support as well as to the Virginia Institute of Marine Science for extending facilities and editorial services for the final preparation of this manuscript.
REFERENCES
I)
U.S. Fish and Wildlife Service (1979).
Habitats of the United States.
2)
Epstein, S. S. (1983).
Classification of Wetlands and Deepwater
U.S. Department of Energy, Washington D.C., pp. 9-93
Environmental determinants of human cancer. Cancer Res. 34, pp.
2425-2435
3)
World Health Organization (1972).
Health Hazards of the Human Environment. Special
Problems: Mutagens, Carcinogens, Teratogens. Wrld. Hlth. Org. 213, Geneva, Switzerland. 387 PP.
4)
LaVole, E., V. Bendenko, N. Hirota, S. Hecht, and D. Hoffman (1980). A Comparison of
the mutagenlclty, tumor-lnltiatlng activity, and complete carcinogenicity of polynuclear aromatic hydrocarbons. Symposium.
5)
Edwards, N. T. (1983).
environment - A Review.
6)
In: Polynuclear Aromatic Hydrocarbons, Third International
Jones/Leber (eds.). Ann Arbor Science Publishers, MI, pp. 705-722
Polycycllc aromatic hydrocarbons (PAHs) in the terrestrial
J. Environ. Qual.
12(4)
National Institute for Occupational Safety and Health (1970).
Department of Health, Education, and Welfare,
7)
pp. 427-41
Wilson, P. J., and J. H. Wells (1950).
NY, pp. 364-374
Washington D.C.,
Coal Tar Products.
U.S.
pp. 1-29
Coal, Coke, and Coke Chemicals. McGraw Hill,
1062
8) Library of Congress (1981).
Handbook of Wood Technology and House Construction.
Research and Education Association, Library of Congress #81-50949, pp. 320-345
9) Neff, J. M. (1980).
Polycyclic Aromatic Hydrocarbons in the Aquatic Environment.
Applied Science Publishers.
10)
Zitko, V. (1978).
Toxicol. 14
11)
262 pp.
Aromatic hydrocarbons in aquatic fauna.
Bull. Environ. Contam.
pp. 621-631
Meyers T. P., and J. D. Hendrlcks (1982).
A summary of tissue lesions in aquatic
animals induced by controlled exposures to environmental contaminants, chemotherapeutic agents, and potential carcinogens.
12)
Marine Fisheries Review 44 (12)
Schultz, M. E., and R. J. Schultz (1981).
pp. 1-17
Effects of dimethyl benz(a)anthracene and
dimethylnitrosamlne on viviparous fish Poeciliopsis sp.
1981NCI Symposium on the Use of
Small Fish Species in Carcinogenesis Testing. National Cancer Institute, Bethesda Md
13)
Redmond, C. K., B. R. Stroblno, and R. H. Cypress (1976).
by-product workers.
14)
Cancer appearance among coke
Annals of the New York Academy of Sciences 271
Blumer M., W. Blumer, and T. Reich (1977).
pp. 116-23
Polycyclic Aromatic Hydrocarbons in soils
of a mountain valley: Correlation with highway traffic and cancer incidence.
Environ. Sci.
and Tech. 11 pp. 1082-1084
15) Catallo, W. J. (1984). on a Benthic Ecosystem.
The Effects of High Levels of Polycycllc Aromatic Hydrocarbons
Masters Thesis, Louslana State University, Baton Rouge LA 70808
16) National Academy of Science (1972). Washington D.C.
17)
Particulate Polycyclic Organic Matter.
NAS,
361 pp.
Guerin, M. R. (1978).
Energy sources of polycyclic aromatic hydrocarbons.
In:
Polycyclic Aromatic Hydrocarbons and Cancer. Academic Press, NY, pp. 3-42.
18)
Miller, B. J. (Unpublished paper).
Particle size analysis by the hydrometer method.
method of analysis for field soil survey laboratories. Department of Agronomy, Louisiana State University, Baton Rouge LA 70808.
19)
Jackson, M. L. (1967).
20)
Anderson, J. (1974).
HCI procedures.
Sell Chemical Analysis.
Prentiss Hall, NJ, pg. 221
A study of the digestion of sediment by the HNO3-H2SO 4 and HNO 3-
Atomic Absorption Newsletter 13 (I), pp. 31-32.
A
1063
21)
Farrlngton, J. W., and B. W. Trlpp (1975).
hydrocarbons in surface sediments. Environment,
22)
A comparison of analysis methods for
ACS Symposium Series in Marine Chemistry in the Coastal
pp. 267-84
Sorrell, K. C., R. C. Dressman, and E. F. McFarren (1977).
High pressure liquid
chromatography measurement of polynuclear aromatic hydrocarbons in water. Prepublication copy, U.S. EPA Water Supply Research Division,
23)
Cincinnati, Ohio.
Portler, R. J., and S. P. Meyers (1982).
pp. 2-39
Monitoring blotransformatlon and
biodegradation of xenoblotlcs in simulated aquatic mlcroenvlronmental systems. Developments in Industrial Microbiology 23 Chap. 42
24)
Martin, J. P. (1950).
In:
pp. 459-75
The use of acid, rose bengal, streptomycin in the plate count
method for estimating soil fungi.
Soil Sci. 69
pp. 215-33
25)
Jensen, H. Z. (1930) Actinomycetes in Danish soils.
26)
Sikora, W. B., and J. P. Sikora (1982).
Soil Sci. 30 pp. 59-77
Ecological implications of the vertical
distributions of melofauna in salt marsh sediments.
In: Ecological Comparisons,
1982
Academic Press, NY, pp. 269-282.
27)
Laseter, J. (Unpublished report).
Captain of the Port of New Orleans.
The Bayou Bonfouca creosote spill.
Report to the
1981 Center for Bio-Organic Studies, University of New
Orleans, New Orleans LA
•28)
Reice S. R. (1974).
woodland stream.
(Received
Environmental patchiness and breakdown of leaf litter in a
Ecology 55 pp. 1271-82
in G e r m a n y
8 November
1986)
pp
1053-1063,
1987
0045-6535/87 $ 3 . 0 0 + .OO P e r g a m o n J o u r n a l s Ltd.
THE EFFECTS OF HIGH LEVELS OF POLYCYCLIC AROMATIC HYDROCARBONS ON
+ SEDIMENT PHYSICOCHEMICAL PROPERTIES AND BENTHIC ORGANISMS IN A POLLUTED STREAM
William J. Catallo III
(I) and Robert P. Gambrell (2)
(I) Division of Chemistry and Toxicology, Virginia Institute of Marine Science, Gloucester Point, VA
(2) Laboratory for Wetland Soils and Sediments, Center for Wetland Resources, Louisiana State University, Baton Rouge LA
ABSTRACT The effects of polycyclic aromatic hydrocarbons on benthic micro- and meiofaunal biomass was determined and related to selected sediment properties such as redox potential and organic matter content in a contaminated environment. INTRODUCTION
Bayou Bonfouca is a palustrlne, mlxed-forested, ecosystem in the Lake Pontchartrain Basin near Slidell, Louisiana (I).
As a result of a fire at a wood products treatment plant
in 1970, approximately 9 hectares of the bayou and surrounding drainage areas have been heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) in a coal tar mixture known as creosote.
PAHs in the environment, food, water, and the workplace are of concern
because of the mutagenlcity and/or carclnogenlclty of several of the compounds in this class (2-5).
+ VIMS
Contribution
No.
1355 1053
105 ~
Creosote
is formed as a by-prOduct
of coal and is characterized
with CI-C 4 alkyl homologs also present used as a sealant and "non-leachable" railroad
during the high temperature
by concentrated
ties, and pilings.
(> 900°C)
mixtures of predominantly
in limited amounts
(6-8).
carbonization
unsubstituted
This material commonly
bioclde for wood products such as telephone
Normally,
PAHs is
poles,
wood is pressure treated at elevated temperatures
to
ensure that organisms already present are killed and that the creosote has thoroughly permeated
surface layers.
arsenates
may be employed when severe fouling
encountered
(7,8).
Adjunct treatments
Creosoting
operations
with pentachlorophenol
is expected or when creosote
tend to be spill-prone
PAHs, PCP, and various metals have been detected preservation
facilities
(9,10).
in environments
PAH compounds organisms
there is growing biochemical
and mixtures
and biota near wood-
to neoplasia
and proliferative
(11-14).
However,
literature
disorders
effects and the physicochemical
properties
in workers and human populations
there is little work dealing specifically
of sediments
The purpose of this work was to examine properties
biomass estimates hypothesized
in contaminated
of benthic bacteria,
control.
properties
protozoans,
Superfund
It was
PAH toxicity would be observed in blomass reductions
in all trophlc and changes
in
such as redox potential and organic matter content relative
the reader is referred to references
is the only published Louisiana
and meiofauna.
For more extensive reviews of PAHs, creosoting operations,
Bayou Bonfouca,
between these
in natural systems.
the effects of PAHs on selected sediment
levels examined and that this would be manifested related sediment
with
areas of Bayou Bonfouca and to relate these with
fungi,
that concentration-dependent
linking various
in exposed aquatic
the effects of PAHs on benthic micro- and melofauna and the relationships
physicochemlcal
of
poles (9).
and epidemiological
as well as to increases of certain cancers
exposed to these materials
"bleed" is
and high concentrations
Smaller amounts of these substances have been identified
near point sources such as wharf structures and telephone At present
(PCP) and copper
6-9, and 15-17.
and the situation
to a at
To our knowledge,
this
study of the Bayou Bonfouca spill site, which was placed on the
Cleanup List in 1982.
MATERIALS AND METHODS
Four sites were established pollution
at Bayou Bonfouoa along an apparent gradient of creosote
and were assigned numbers as follows:
2 = "moderately
contaminated",
contaminated".
This gradient
were characterized
site I - control or "uncontaminated",
slte 3 = "heavily contaminated",
was later verified by analysis of sediment samples.
by soft bottoms,
m), similar vegetation
and shading,
and similar proximity
to the Slidell
gentle surface-water approximately
flows,
equivalent
site
site 4 = " extremely
shallow depths
litter input
urban area and its associated
All sites
(0.6 - 1.2
(visual estimate),
sources of pollution.
Sediment Characterization:
Bulk surface
(-10 cm) sediment samples for particle size analysis were collected
each site wlth a shovel and were stored
in plastic buckets with llds.
Sediments
to be
from
1055
analyzed for metals were collected with acid-rinsed teflon scrapers and placed in glass jars with teflon caps.
Redox potential (Eh) measurements were taken in situ using commercially
available platinum electrodes and saturated calomel reference electrodes.
Sediment pH was
determined using a calibrated combination pH electrode and values for both pH and Eh were obtained with a portable pH/millivolt meter (Orion Model 399A). Particle size distributions were determined via a standard hydrometer method (18), and readily oxidizable organic matter content by the potassium chromate/acidlc oxidation method of Jackson (19).
Sediment metals were extracted using the hot HCI/HNO 3 method of Anderson
(20) and analyzed by inductively coupled argon plasma emission spectrometry (ICAP).
PAH Extractions:
All PAH extractions were accomplished using highly purified reagents of "HPLC grade" (Curtin Matheson, Houston, TX), "Pesticide Grade" (Sargent Welch, Dallas, TX), or "Nanograde"
(Malllnekrodt, St. Louis, MO).
reagent grade.
Anhydrous Na 2 SO 4 and KOH were of analytical
The PAH standards phenanthrene, pyrene, l-methyl fluoranthene,
benzo(k)fluoranthene,
benzo(e)pyrene,
dibenzo(a,h)anthracene,
dlbenzo(a,i)pyrene were
donated by the NCI Chemical Carcinogen Reference Repository (NIH, Bethesda, MD). Naphthalene, anthracene, chrysene, pyrene, and benzo(a)pyrene Scientific) were of 98 % purity or better.
(Aldrich Chemical and Fischer
A mixture of 16 PAH standards
(Supelco Inc.,
Bellefonte, PA) also was used for standardization. All solvents, column packings, drying agents, and standards were assayed via high pressure liquid chromatography (see below) for impurities eluting in the range of interest. All sediment and water samples for PAN analyses were collected using solvent-rinsed glass or metal equipment and were stored in glass or metal containers with foil-lined caps. Care was taken to avoid contamination and exposure of standards, samples, and extracts to light was minimized.
Sediments were extracted using the method of Farrington et al. (21)
with the addition of ultrasonic disruption prior to alkaline reflux extraction, and the substitution of cyclohexane for benzene as the primary solvent.
Other modifications
included the use of centrlfugation instead of filtration, with extract volume reductions being accomplished where necessary by evaporation under a dry stream of nitrogen while sample containers were held in a controlled-temperature water bath (< 4°C) rather than in a rotary evaporator.
Surface water samples were extracted via the method of Sorrell et al
(22). PAHs were identified and quantified using a Waters Associates Model 480 Isocratic HPLC with the following specifications:
6000a pump; U6KA injector; a 480 L.C. detector; and a
Waters Associates RCM Bondapak column with a precolumn filter. acetonltrile:water at I ml/mln.
The solvent system was 86:14
Seperated compounds were scanned at 254 nm and absorbance
was recorded on a Hewlett Packard 3390a integrator with quantification accomplished by referencing sample peak heights to standard curves for each compound identified. The following criteria were utilized for PAH identification: compound was required to match that of a standard, upon the addition of an appropriate "spike",
I) retention time of the
2) detector response had to increase
3) the compound had to exhibit its documented
absorbance maximum as well as several lesser absorbance peaks.
The ratios of the absorbance
1056
maximum to the corresponding
lesser peaks were required
to match those determined
using
standards.
Microbiological Assays:
ATP was determined
photometrically
At each site, approximately scintillation
vials containing
following
the method of Portier
(pers. comm., 23).
one gram of sediment was placed in preweighed,
9 ml of pH 7.4
phosphate buffer.
sterile,
These were stored on ice
until analysis
which occurred no more than 12 hours after collection.
to equilibrate
at room temperature
Samples were allowed
for three hours prior to analysis at which time the
sample vials were dried and weighed to determine
the actual weight of sediment collected.
100 ~i of sample was reacted with 100 ~i of a releasing agent in a 5 ml glass tube and placed
in an ATP phtometer.
specific
for bacterial
The releasing agents
(Lumac Systems,
(23).
100 ~i of luciferin/luciferase
ATP-dependent
and the resultant
detected and recorded as relative standard and background to calculate
corrected
protoctists,
The mixture was then treated with evolution of light was
For each run, "quench"
determinations
in the results.
The corrected
was conferred
culture exclusively
to the linear range of an ATP standard curve and were Conversion
of RLU to cell
is neither required nor usually employed.
(24) and Jensen's
(25) media were used in pour plate procedures
fungi and bacteria respectively.
~g/ml to Jensen's media to inhibit overgrowth Meiofauna
community
by the releasing agents.
for the purposes of biomass comparison between sites.
Both Martin's
RLU values
of total ATP of a specific microbial
fungi) and this specificity
number or cell density
of a known ATP
These values as well as sample weight were used
RLU which are reported
All RLU values were referenced appropriate
light units (RLU).
RLU were recorded.
direct photometric
(bacteria,
FL) were
or somatic cells and allowed for permeable release of nucleotides
without cell lysis or action by cellular enzymes
represent
Titusville,
Cycloheximide
by filamentous
to
(Sigma) was added at 40
fungi
(23).
(sediment biota retained on a 500 ~m sieve after passing through a I mm
sieve) were collected
from each site using a 3-cm corer.
cm which was expected
to contain over 95 % of the total meiofauna
treated with a formalin/rose
bengal solution,
wet-sieved,
all meiofauna were counted with a Nikon dissecting
Samples were taken to a depth of 8 (26).
Core samples were
rinsed with distilled water,
and
microscope.
RESULTS AND DISCUSSION
The concentrations presented
in Table I.
of PAHs in sediment and water samples These data are in general agreement
analyses of Bayou Bonfouca creosote Sediment presented
characterization
in Tables
by Laseter
from Bayou Bonfouca are
with the GC-mass spectrometric
(27).
data are presented
in Tables 2 and 3.
2, 4, and 5 were generated by Duncan's Multiple
a 0.05 experimentwise
error.
indicated highly significant
All statistics
Range Tests
(DMRT) with
The DMRTs were conducted after analysis of variance differences
(P < 0.01) between the reference
(ANOVA)
(site I) and the
1057
spill areas (sites 2-4).
All non-significant ANOVAS (P > 0.05) were reflected by non-
significance in the DMRTs which are presented in the tables.
Table
1.
Concentrations o f PAHs i n S e d i m e n t s a n d W a t e r f r o m B a y o u Bonfouca, Slidell, Louisiana (Parts per million)
Site
PAH Compound
Surface Water
Sediments
I
............
3 unknowns
3-5 unknowns
2
Naphthalene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(J)fluoranthene/ benzo(k)fluoranthene*
3
4
Naphthalene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(J)fluoranthene/ benzo(k)fluoranthene Benzo(a)pyrene
Naphthalene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(j)fluoranthene/ benzo(k)fluoranthene Benzo(a)pyrene
* Isomers not resolved.
8.3 7.6 28.7 14.7 27.1 18.3
1380 358 435 107 608 178
4.9
75
0.7 0.6 2.3 0.4 1.2 2.1
7720 7720 17670 1110 3360 800
nd** 0.3
940 40
14.1 12.3 155.O 39.7 110.0 85.0
7117 29310 1650 6580 1660
5.5 6.6
2280 610
** Not detected.
As can be seen from examination of Tables I-3, the most prominent differences between sites were creosote content, organic matter (OM), redox potential (Eh), pH, Zn, and Ca concentrations.
The particle size distributions indicated closely related sediment types
with sites I and 2 bordering between "silt loam" and "sandy loam", and sites 3 and 4 being "silt loam" (Table 3).
The OM contents varied considerably between sites and the presence
of poorly degraded plant material in the vertical sediment profiles of sites 2-4 suggested that this was the result of low saprophytic activity.
Since site I had low OM and no
detected creosote, the increasing presence of OM in sites 2-4 was likely the result of creosote toxicity to saphrophytlc organisms.
1058
T a b l e 2.
Sediment C h a r a c t e r i s t i c s Site
1
2
3
4
Organic Matter (%)
x- 3.4a si 0.2
9.5b 0.06
16.4c 0.7
32.3d 0.66
pH
x- 7.4a sI 0.2
7.6ab 0.2
8.2b 0.3
I0.4c 0.7
Eh (mV)
xi -214a s- 115
-I04b 52
-99b 63
-35b 57
NI 3, s- standard deviation. Means (x) followed by the same letter on a horizontal line are not significantly different at pI 0.05.
The Eh values for each site appeared to be related to the creosote concentrations as well.
At site I, Eh values showed strongly reduced conditions resulting from limited oxygen
flux to the sediments (as a result of flooding) and metabolically active microbial communities.
At sites 2, 3, and 4, the redox potentials were progressively less reducing as
creosote levels increased.
This Eh trend may be interpreted in two ways: (I) microbial
metabolic processes were limited in the spill areas, or (2) higher energy redox systems (ie., NO 3_ -> NH3/NH4 + , or Mn(III, IV) -> Mn(II)) predominated in these areas.
Since all
sites were permanently flooded, well supplied wlth OM, similar in grain size, and comparable in terms of Mn and Fe concentrations,
T a b l e 3.
the latter interpretation is unlikely.
S e l e c t e d Sediment P r o p e r t i e s Site
I
Clay (%) Silt (%) Sand (%) Creosote (%) * Cu (~g/g) Zn Cd Pb Ni As Fe Mn Ca Mg Mo A1
10 68 22 0 2 30 4 30 4 35 5590 51 714 900 8 9700
2
3 49 48 0.8 10 80 6 80 2 25 6200 73 1900 580 6 10300
3
4
4 66 30 10
5 46 49 25
12 115 10 52 16 135 7320 193 23400 3080 8 10600
8 80 13 32 4 37 4300 117 49000 1340 5 6660
Estimated from phenanthrene concentrations in samples and source creosote.
In the absence
1059
of biolimiting factors it would be expected that all sites would demonstrate strongly reducing conditions similar to site I. Sediment pH values at some sites were surprisingly high initially.
Upon further
investigation, the trend of increasing pH at sites 2-4 was thought to be related to the the progressively higher levels of Ca, probably introduced by runoff of lime from a local concrete plant (Table 3).
It is also probable that the high levels of creosote in the
sediments of sites 3 and 4 caused electrode interference and artificially high pH values. Further, although the pH values were in some cases "significantly different" from the control, there was a difference of less than I pH unit between sites I, 2, and 3, and this was not considered to be biologically important.
Site 4, however, had higher pH values and
this may have had an impact on benthic organisms, but relative to the high creosote concentrations also found at this site, the pH was thought to be of minor importance, particularaly with respect to microorganisms. soils and sediments are influenced by pH.
It should be mentioned that measured Eh in
A pH correction on measured Eh is difficult to do
in natural environments though it is known that increasing pH is accompanied by decreasing Eh values.
Thus it is important to point out that if appropriate pH correction factors
could be applied to the Eh values presented in Table 2, the more contaminated sites would indicate an even greater difference relative to the control area. The metals concentrations reported in Table 3 represent "potentially bioavailable', levels, ie., not including metals contained within the lattice structure of clay minerals. These metals were most likely tightly bound to sediment constituents and not readily available, hence not significantly bioinhibitory relative to other sources of toxicity. The results of the ATP analysis of the mlcrobenthos and direct counts of meiofauna are presented in Tables 4 and 5 respectively.
These data show that the abundances of viable
bacteria, fungi, and protozoa at sites 2, 3, and 4 were much lower than site I.
DMRTs of
all communities from site I against sites 2, 3, and 4, showed significant decreases in these communities with the means of each site decreasing with increasing creosote.
Meiofauna
counts show analogous reductions in response to increasing levels of creosote and this may be related to depressed microbenthic activity as well.
Table 4.
Microbenthic Community Analysis RLU/g sediment
I
Bacteria
x-
Site 2
3
4
s-
22619a 7768
9117b 2710
5501bc 6686
95c 28
Protozoa
xs-
19911a 5731
5857b 1715
4951bc 5690
78c 31
Fungi
~s-
6983a 6792
327b 552
136b 207
N= I0, s- population std. dev. at P- 0.05.
3b 6
Means (2) with same letter are not significantly different
1060
Table 5. Meiobenthic Community Analysis Organisms/10 cm Site
1
2
3
4
Nematodes
x= s=
91a 20
14b 21
Ob 0
Ob 0
Oligochaetes
x= s=
20a 13
16ab 15
Ob 0
Ob 0
Other
x= s=
7b 8
Ob 0
Ob 0
38b 38
Ob 0
Ob 0
Total Melofauna
22a 7
x= s=
133a 20
N=3, s= population
std. dev.
Means wlth the same letter are not significant
at P= 0.05.
Viable plate count results are given in Table 7 and support the interpretations preceedlng
sections
in that there is a creosote-dependent
fungal colony forming units
decrease
of
in both bacterial
and
(CFU).
Table 6.
Summary of P l a t e Count Results CFU/Plate Slte
I
Bacteria
xs=
TNTC --
Fungi
x= s=
58 9
2
3
351 118
116 47
40 6
39 6
4
71 22 0.25 0.40
N= 12, s= std. dev. Colonies
too numerous
to count. Count stopped at CFU= 500 in all samples.
CONCLUSIONS
The results of this study indicate that the high levels of PAHs in the sediments of large areas of Bayou Bonfouca adversely was manifested creosote levels more oxidizing.
at all of the trophic increased,
detrital
affected micro- and melobenthlc
communities
and this
levels studied as well as in sediment properties. accumulations
also increased and redox potentials
These phenomena were related to a reduction of saprophytic
As became
biomass relative
106 1
to the control area.
The removal of fungi in polluted sites was probably the major factor
in detrital accumulation since fungi are of primary importance in terms of litter degradation (28).
The reduction of bacterial biomass was thought to be the primary cause of
increasing redox potentials.
The association of measured high levels of PAHs with
alterations of the physical-chemical characteristics of the sediments is considered to be the primary finding of this research.
Attention is drawn to the biochemical findings in
hopes that the methods described in this work will be applied and expanded in future research and that the concept of employing trophic biomass measurements for the evaluation of pollutant impacts in natural systems will be subjected to further study.
ACKNOWLEDGEMENTS
The authors thank Walter B. Sikora, Jean P. Sikora, and Samuel P. Meyers for advice, materials, and critical review of this research.
Thanks are extended to Michael Bender,
Richard Wetzel, John Zeigler, and Ruth Hershner for further review and support as well as to the Virginia Institute of Marine Science for extending facilities and editorial services for the final preparation of this manuscript.
REFERENCES
I)
U.S. Fish and Wildlife Service (1979).
Habitats of the United States.
2)
Epstein, S. S. (1983).
Classification of Wetlands and Deepwater
U.S. Department of Energy, Washington D.C., pp. 9-93
Environmental determinants of human cancer. Cancer Res. 34, pp.
2425-2435
3)
World Health Organization (1972).
Health Hazards of the Human Environment. Special
Problems: Mutagens, Carcinogens, Teratogens. Wrld. Hlth. Org. 213, Geneva, Switzerland. 387 PP.
4)
LaVole, E., V. Bendenko, N. Hirota, S. Hecht, and D. Hoffman (1980). A Comparison of
the mutagenlclty, tumor-lnltiatlng activity, and complete carcinogenicity of polynuclear aromatic hydrocarbons. Symposium.
5)
Edwards, N. T. (1983).
environment - A Review.
6)
In: Polynuclear Aromatic Hydrocarbons, Third International
Jones/Leber (eds.). Ann Arbor Science Publishers, MI, pp. 705-722
Polycycllc aromatic hydrocarbons (PAHs) in the terrestrial
J. Environ. Qual.
12(4)
National Institute for Occupational Safety and Health (1970).
Department of Health, Education, and Welfare,
7)
pp. 427-41
Wilson, P. J., and J. H. Wells (1950).
NY, pp. 364-374
Washington D.C.,
Coal Tar Products.
U.S.
pp. 1-29
Coal, Coke, and Coke Chemicals. McGraw Hill,
1062
8) Library of Congress (1981).
Handbook of Wood Technology and House Construction.
Research and Education Association, Library of Congress #81-50949, pp. 320-345
9) Neff, J. M. (1980).
Polycyclic Aromatic Hydrocarbons in the Aquatic Environment.
Applied Science Publishers.
10)
Zitko, V. (1978).
Toxicol. 14
11)
262 pp.
Aromatic hydrocarbons in aquatic fauna.
Bull. Environ. Contam.
pp. 621-631
Meyers T. P., and J. D. Hendrlcks (1982).
A summary of tissue lesions in aquatic
animals induced by controlled exposures to environmental contaminants, chemotherapeutic agents, and potential carcinogens.
12)
Marine Fisheries Review 44 (12)
Schultz, M. E., and R. J. Schultz (1981).
pp. 1-17
Effects of dimethyl benz(a)anthracene and
dimethylnitrosamlne on viviparous fish Poeciliopsis sp.
1981NCI Symposium on the Use of
Small Fish Species in Carcinogenesis Testing. National Cancer Institute, Bethesda Md
13)
Redmond, C. K., B. R. Stroblno, and R. H. Cypress (1976).
by-product workers.
14)
Cancer appearance among coke
Annals of the New York Academy of Sciences 271
Blumer M., W. Blumer, and T. Reich (1977).
pp. 116-23
Polycyclic Aromatic Hydrocarbons in soils
of a mountain valley: Correlation with highway traffic and cancer incidence.
Environ. Sci.
and Tech. 11 pp. 1082-1084
15) Catallo, W. J. (1984). on a Benthic Ecosystem.
The Effects of High Levels of Polycycllc Aromatic Hydrocarbons
Masters Thesis, Louslana State University, Baton Rouge LA 70808
16) National Academy of Science (1972). Washington D.C.
17)
Particulate Polycyclic Organic Matter.
NAS,
361 pp.
Guerin, M. R. (1978).
Energy sources of polycyclic aromatic hydrocarbons.
In:
Polycyclic Aromatic Hydrocarbons and Cancer. Academic Press, NY, pp. 3-42.
18)
Miller, B. J. (Unpublished paper).
Particle size analysis by the hydrometer method.
method of analysis for field soil survey laboratories. Department of Agronomy, Louisiana State University, Baton Rouge LA 70808.
19)
Jackson, M. L. (1967).
20)
Anderson, J. (1974).
HCI procedures.
Sell Chemical Analysis.
Prentiss Hall, NJ, pg. 221
A study of the digestion of sediment by the HNO3-H2SO 4 and HNO 3-
Atomic Absorption Newsletter 13 (I), pp. 31-32.
A
1063
21)
Farrlngton, J. W., and B. W. Trlpp (1975).
hydrocarbons in surface sediments. Environment,
22)
A comparison of analysis methods for
ACS Symposium Series in Marine Chemistry in the Coastal
pp. 267-84
Sorrell, K. C., R. C. Dressman, and E. F. McFarren (1977).
High pressure liquid
chromatography measurement of polynuclear aromatic hydrocarbons in water. Prepublication copy, U.S. EPA Water Supply Research Division,
23)
Cincinnati, Ohio.
Portler, R. J., and S. P. Meyers (1982).
pp. 2-39
Monitoring blotransformatlon and
biodegradation of xenoblotlcs in simulated aquatic mlcroenvlronmental systems. Developments in Industrial Microbiology 23 Chap. 42
24)
Martin, J. P. (1950).
In:
pp. 459-75
The use of acid, rose bengal, streptomycin in the plate count
method for estimating soil fungi.
Soil Sci. 69
pp. 215-33
25)
Jensen, H. Z. (1930) Actinomycetes in Danish soils.
26)
Sikora, W. B., and J. P. Sikora (1982).
Soil Sci. 30 pp. 59-77
Ecological implications of the vertical
distributions of melofauna in salt marsh sediments.
In: Ecological Comparisons,
1982
Academic Press, NY, pp. 269-282.
27)
Laseter, J. (Unpublished report).
Captain of the Port of New Orleans.
The Bayou Bonfouca creosote spill.
Report to the
1981 Center for Bio-Organic Studies, University of New
Orleans, New Orleans LA
•28)
Reice S. R. (1974).
woodland stream.
(Received
Environmental patchiness and breakdown of leaf litter in a
Ecology 55 pp. 1271-82
in G e r m a n y
8 November
1986)