United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas NV 89114
Research and Development
EPA/600/S4-85/059 Jan. 1986
Project Summary
Application of Chemical
Fractionation/Aquatic Bioassay
Procedure to Hazardous Waste
Site Monitoring
V. Lopez-Avila, W. D. McKenzie, W. W. Sutton, R. Kaminsky, U. Spanagel,
T. A. Olsson, and J. H. Taylor
The chemical fractidnation/aquatic
bioassay test basically involves biologi-
cal testing, first using a given collection
of leachate, surface water, or liquid
waste, and then using fractions and
subtractions of the original sample ma-
terial. The final test result, derived from
a compilation of these different
bioassay responses, is used to identify
bioactrve fractions of the original sam-
ple material, to assess some of the ad-
ditive, synergistic and/or antagonistic
effects caused by the component waste
chemicals, and to provide a preliminary
(or screening) hazard evaluation for the
aquatic ecosystem. While chemical
analysis of sample material is not a part
of the procedure, a combination of bio-
logical test data and chemical analytical
data will allow for the identification of
compounds and groups of compounds
that present the greatest environmen-
tal hazard.
While the procedure has been suc-
cessfully used for monitoring industrial
pollutants, the overall technique was
not considered to be ready for use at
hazardous waste sites. Preliminary
testing had indicated some potential
problems with the chemical fractiona-
tion phase; therefore, an evaluation
was conducted using a laboratory pre-
pared waste leachate sample. Since the
results from this initial evaluation indi-
cated that procedural revisions were
necessary, a series of experiments were
then conducted to improve the chemi-
cal fractionation phase. When these
procedural revisions had been made,
another evaluation was conducted
using samples taken from actual haz-
ardous waste sites.
In spite of the complex matrix en-
countered when using liquid waste ma-
terial, the fractionation technique was
reasonably effective at partitioning the
neutral organics. Partitioning of inor-
ganics was also reasonably efficient.
However, partitioning of polar organics
was not particularly impressive, and
several of the alcohols and acids were
recovered in both inorganic and organic
fractions. Further studies will perhaps
improve the fractionation efficiency,
and therefore improve the overall pro-
cedure's usefulness as a monitoring
method.
This Project Summary was devel-
oped by ERA's Environmental Monitor-
ing Systems Laboratory, Las Vegas,
NV, to announce key findings of the re-
search project that is fully documented
in a separate report of the same title
(see Project Report ordering informa-
tion at back).
Introduction
Assessing current and potential prob-
lems at uncontrolled waste sites has
been very difficult. The problem of eval-
uating complex chemical mixtures
rather than specific chemical com-
pounds causes part of the difficulty. An-
other problem is that toxicity data and
environmental transport data are lim-
ited for many of the waste compounds,
especially those that are byproducts of
organic synthesis as opposed to com-
mercial products. Biological monitoring
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techniques can often be very useful
when dealing with such problems, and
the biological procedures are particu-
larly effective when combined with
chemical analysis of the sample mate-
rial.
The chemical fractionation/aquatic
bioassay procedure is used to identify
bioactive fractions of a particular sam-
ple, to assess some of the additive, syn-
ergistic, and/or antagonistic effects
caused by the component chemicals,
and to provide a preliminary (or screen-
ing) hazard evaluation for the aquatic
environment. The procedure basically
involves biological testing, first using a
given collection of leachate, surface
water, or liquid waste, and then using
chemical fractions and subtractions of
the original sample material. The final
test result is derived from a compilation
of these different bioassay responses.
Skeletonema costatum (saltwater al-
gae), Selenastrum capricornutum
(freshwater algae), Mysidopsis bahia
(saltwater crustacean), and Daphnia
magna (freshwater crustacean), are
used as test organisms during the as-
say. However, the saltwater algae and
saltwater crustaceans tests are not con-
ducted if an inland location of a particu-
lar waste site precludes any possibility
of estuary contamination. There is a
good data base available for these fre-
quently used biological procedures that
indicate assay responsiveness to many
pollutant compounds, and some prog-
ress has been made toward method
standardization.
In spite of the procedure's successful
application for monitoring industrial
pollutants, the overall technique was
not considered to be ready for use at
hazardous waste sites. Preliminary test-
ing had indicated some potential prob-
lems with the chemical fractionation
phase; therefore, an evaluation was
conducted using a synthetic (laboratory
prepared) waste leachate sample. Since
the results from this initial evaluation
indicated that procedural revisions
were necessary, a series of experiments
were conducted to improve the chemi-
cal fractionation phase of the overall
technique. When these procedural revi-
sions had been made, another evalua-
tion was conducted using samples
taken from actual hazardous waste
sites.
Chemical Fractionation/Aquatic
Bioassay Procedure
During each separate analysis, a por-
tion of the original sample material is
first tested for toxicity using the differ-
ent bioassay procedures. If a toxic re-
sponse (to include either stimulation or
inhibition of the algal populations) does
not occur in any of the component as-
says, no further testing is conducted
using this particular waste or leachate
collection. If a toxic response does oc-
cur for any of the component tests, a
second portion of the waste material is
then chemically fractionated, and each
fraction is separately tested using the
crustacean and algal assays. The result-
ing fractions or test samples are the
(1) total organics, (2) base/neutrals,
(3) organic acids, (4) organic residuals,
(5) recombination of 2,3, and 4, (6) total
inorganics, (7) cations, and (8) anions.
The fractionation procedure can proc-
ess up to a 2-liter sample, but additional
fractionations are frequently necessary
before biological testing begins. While
replicate fractionation runs are being
completed, those fractions and subtrac-
tions collected from the initial fractiona-
tion are stored at approximately 4°C
prior to compositing the respective final
samples. When the fractionation phase
is complete, each of the final test sam-
ples mentioned above should contain a
sufficient volume (i.e., volume/concen-
tration) to provide sample material for
the four bioassay tests. Obviously, it is
better to have an excess of sample ma-
terial than to discover that there is insuf-
ficient material to complete the compo-
nent assays.
The sample is initially filtered through
a 0.45 IL glass fiber filter. The filtrate is
then loaded on a resin column. An XAD-
4 and an XAD-8 resin column are used
in tandem with the eluate from the XAD-
4 resin being passed through the XAD-8
column. The aqueous eluate that passes
through the resin columns, and the ad-
ditional water (HPLC grade) that is
added to remove any remaining inor-
ganics, are combined and designated as
the total inorganic fraction. Organic
compounds are eluted from the XAD-4
and XAD-8 resins with acetone and di-
ethylether respectively.
The total organics fraction is concen-
trated (to remove the acetone and di-
ethylether) and is then extracted with
methylene chloride at a pH > 12 to iso-
late the base/neutrals. The remaining
aqueous phase is extracted with
methylene chloride at a pH < 1 to iso-
late the organic acids. A solvent ex-
change step (dimethylsulfoxide) com-
pletes the fractionation process for
these subsamples. The remaining
aqueous phase (i.e., organic residuals
subtraction) is concentrated to dryness
and resuspended in dimethylsulfoxide.
A proportionate amount of base/neu-
trals, organic acids, and organic residu-
als are combined for the recombination
fraction. This reconstituted total organ-
ics fraction provides a separate sample
for biological testing.
The aqueous phase from the initial
column separation (XAD-4 and XAD-8
resins) contains the total inorganics
fraction. Separate subsamples of the in-
organic fraction are then fractionated,
using ion exchange resins, to provide
the respective cation and anion sam-
ples. A Dowex 1-X8 column is used to
provide the cation subtraction and a
Dowex 50 W-18 column is used to pro-
vide the anion subfraction. Both
columns are eluted with deionized
water.
Of the nine separate sample types
(i.e., original sample, total organics,
base/neutrals, organic acids, organic
residuals, recombination of organic
subtractions, total inorganics, cations,
and anions), only the original material is
used for biological testing prior to com-
pleting the fractionation phase. If multi-
ple fractionations are required, these
would be completed and the resulting
fractions composited (with the previous
runs) before any additional samples are
biologically tested. Eight concentrations
or dilutions of each fraction can be
tested using each of the four compo-
nent bioassays, i.e., 0.01, 0.1, 1.0, 10.0,
25, 50, 75, and 100 percent. However,
fewer dilutions will frequently provide
sufficient information, i.e., 0.01,1.0, and
100 percent. The 100 percent or original
concentration is based on the approxi-
mate concentration that existed in the
original sample material. Dilution me-
dia must be prepared for the respective
algal assays, and dilution water must be
prepared for the crustacean tests. In
some cases, the testing laboratory
might not wish to return a sample to the
original concentration and would in-
stead biologically test the more concen-
trated fraction or subfraction.
The biological test species are all of
ecological importance to the respective
freshwater and estuary environments;
they are available commercially, and
stock populations or cultures are fairly
easy to maintain at the testing labora-
tory. However, it should be emphasized
that none of the biological assays are
conducted as definitive tests in which
range finding evaluations precede as-
says to characterize the concentration
response curve. The four component i
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assays are, in fact, being used as
screening procedures to test samples of
unknown chemical composition, and
for each of the respective assays, a min-
imum number of test organisms is
used. The crustacean assays are both
conducted under static conditions
where test solutions are not renewed
during the testing period. The static test
obviously requires less sample material
than the flow-through procedure which
is sometimes a critical factor when plan-
ning assays for a limited amount of frac-
tion and subtraction sample material.
Procedure Evaluation
Preliminary Testing
The current demonstration used both
hazardous waste site sample material
and a synthetic (laboratory prepared)
leachate which contained a limited
number of compounds, i.e., 2, 4-
dimethylphenol, cadmium sulfate, 4-
chlorophenol, and triethanolamine. The
synthetic material was obviously not as
complex as the actual hazardous waste
and was used as sample material during
the first part of the evaluation. Chemical
analysis of the resulting fractions and
subfractions indicated a need for further
testing and revision of the fractionation
procedure, i.e., the individual com-
pounds were not efficiently partitioned
during fractionation. This need for pro-
tocol revision was also reflected in
some of the bioassay test data which
were, of course, acquired from testing
the poorly partitioned fractions. This ini-
tial demonstration also indicated the
need for some minor revisions to the
bioassay portion of the protocol, espe-
cially in the instructions for preparing
the various fraction samples immedi-
ately prior to biological testing (e.g., sol-
vents, nutrient supplements, suggested
amounts of sample material, number of
dilutions, etc.). Several problems were
encountered when preparing the labo-
ratory cultures of Skaletonema
costatum, and specific protocol revi-
sions have been made for the required
salts, metals, and vitamins used in the
culture media.
Several preliminary studies were then
conducted in an effort to improve the
fractionation scheme prior to fractionat-
ing the waste material. These prelimi-
nary efforts included (1) a pretest frac-
tionation of a waste site sample, (2) an
attempt to separately elute the organic
acids and base/neutrals from the XAD-4
resin by sequential elution, (3) a test
where phenol and pentachlorophenol
concentrations were determined in a
modified mass balance study using two
different waste sample volumes, (4) a
study of compound recovery (at two dif-
ferent spiking concentrations) where
fractionations using the XAD-4 resin
were compared with fractionations
using the XAD-4/XAD-8 resin column
sequence, and (5) a brief study which
examined potential compound losses
during sample concentration.
A complete method protocol for the
chemical fractionation/aquatic bioassay
procedure is given in the project report.
An outline of the fractionation proce-
dure is shown in Figure 1. The project
report protocol is the most complete
version of the procedure and is one that
includes (1) the revisions made follow-
ing the synthetic sample fractionation
and associated biological testing, and
(2) the revisions made following the
separate experiments mentioned
above. However, based on the subse-
quent fractionations using actual haz-
ardous waste site material, additional
method revisions, beyond those al-
ready incorporated into the procedure,
will probably be required before an effi-
cient fractionation can be consistently
achieved and before the overall tech-
nique can be considered for use in an
operational monitoring network.
Fractionation of Waste Site
Material
The hazardous waste material used
during the evaluation was collected
from two different waste sites. While
known toxic and carcinogenic com-
pounds were present in the waste mate-
rial from both sites, these particular
sites were selected (one in California
and one in Oregon) because of the di-
versity of chemical compounds known
to be present at each location. Material
from the different locations was desig-
nated as collection A and collection B,
respectively.
Tables 1 and 2 show the recovery of
selected compounds that were identi-
fied in the initial waste samples. Com-
puter programs (i.e., computer assisted
GC/MS) confirmed the identification of
many additional waste fraction and
waste subfraction compounds, but
these selected organics are presented
mainly for illustrative purposes. Con-
centrations are given for the original
sample material, the three organic sub-
fractions, and for the total inorganics
fraction. Approximate detection limits
provided with each table have been
based on the recovery of internal stand-
ards and have attempted to include
some estimate of potential interference
caused by the presence of many addi-
tional compounds. The selected com-
pound results are, in most cases, ex-
trapolated values based on the original
2 liter sample volume, i.e., corrections
made for volume adjustments and sub-
sampling that occurred during the frac-
tionation process. Obviously, the organ-
ics present in the inorganic fraction
represent a deficiency in the fractiona-
tion procedure. Tables 3 and 4 present
the respective inorganic results ac-
quired using inductively coupled argon
plasma spectroscopy (ICAP).
Collection A contained fairly high
concentrations of polar compounds,
and, when the current fractionation pro-
cedure was followed, the overall recov-
ery of these polar organics was not as
efficient as the observed recovery of the
base/neutrals. In addition, those polar
compounds which were recovered from
the XAD-4 and XAD-8 resins were fre-
quently eluted with the aqueous phase
into the total inorganic fraction (Fig-
ure 1). This failure to achieve the in-
tended partitioning of component com-
pounds would have caused obvious
data interpretation problems had these
collection A fractionation samples been
biologically tested. A bioassay result for
the total inorganic sample would actu-
ally have been a test response, not only
to the total inorganics but also to many
organic compounds. Correspondingly,
a bioassay test result for the total or-
ganic fraction, or for any of the organic
subfractions, would have been a test re-
sponse to samples that did not contain
many of the respective organics that
were, in fact, present in the original ma-
terial. Polar organics were also present
in the collection B material, but they ap-
parently did not overload the binding
capacity of the resins, and consequently
the compounds were eluted, for the
most part, into the total organic fraction.
Conclusions
The chemical fractionation/aquatic
bioassay procedure is designed to test
samples of leachate, surface water, or
liquid waste material such as might be
encountered when monitoring at haz-
ardous waste sites. The procedure has
been successfully used to monitor in-
dustrial pollutants, and the current
demonstration was conducted to test
the procedure using a more complex
waste sample. The demonstration indi-
cated some areas in which the proce-
dure seemed to be fairly effective, but it
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Waste or
Lea chat e
Sample
Sample ttt
Original
Whole Waste
Filter
(0.45 fjmj
XAD-4
Resin
- Acetone
Acetone Eluate
Aqueous Eluate 1
Aqueous Eluate
Total
Inorganics
Sample #7
Total Inorganics
Dowex
50W-X8
Resin
Anions
XAD-8
Resin
• Diethyl-
ether
Diethylether
Eluate
Sample #5
Anions
Total
Organics
Sample #2
Total Organics
pH>J2
Organic Phase
Extract with
Methylene
Chloride
Aqueous
Phase
1 pH<1
Sa
Base
+
mp/e #3
/Neutrals
±
Organic
Extract with
Methylene
Chloride
Phase
Aque
ousP
Organic
Acids
1
Evaporate
to Dryness
Sample #4
Acids
Organic
Residuals
Organic
Fractions
Recombined
Sample #5
Organic
Residuals
Sample #6
Recombination
Figure 1. Chemical fractionation phase of the chemical fractionation/aquatic bioassay procedure. A total of nine separate samples are ultimately
provided for biological testing. The overall procedure was initially developed by G. E. Walsh and Ft. L GarnasattheEPA laboratory in Gulf
Breeze. Florida. t
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Table 1. Recovery and Partitioning of Selected Organic Compounds Following Chemical Fractionation of Collection A Waste Site Material
Whole Waste Base/Neutrals Organic Acids
Compound
benzaldehyde
benzole acid
pentachlorophenol
9, W-anthracenedione
phenanthrene
anthracene
carbazole
biphenylene
fluoranthene
pyrene
p-phenylcarbanilic
acid
4-hydroxybenzene acetic
acid
1,2-benzenedicarboxylic
acid
Concentration '
(M/l)
2,000
8,200
20,000
16,000
ND
P
P
ND
1,600
800
28,000
2,900
360
Estimated
D.L2
(v.g/1)
1000
1000
1000
1000
3000
3000
3000
1000
1000
1000
1000
100
100
Concentration '
(w/i)
740
ND
210
4,400
1,900
2,500
2,100
1,200
1,400
950
ND
ND
ND
(36)
f D
(26)
(94)
(119)
Estimated
D.L2
(w/D
50
50
200
200
200
200
200
50
50
50
50
50
50
Concentration '
<\Lg/l)
170
70
1,400
220
140
180
50
70
110
70
ND
ND
ND
<9)
(1)
(7)
(1)
(7)
(9)
Estimated
D.L2
(M/l)
20
100
20
20
20
20
20
20
20
20
20
20
20
Organic Residuals
Total Inorganics
Compound
benzaldehyde
benzole acid
pentachlorophenol
9, 10-anthracenedione
phenanthrene
anthracene
carbazole
biphenylene
fluoranthene
pyrene
p-phenylcarbanilic acid
4-hydroxybenzene acetic
acid
1, 2-benzenedicarboxylic
acid
Concentration1
(W/D
350
700
ND
2,900
NA
NA
NA
NA
NA
NA
2,700
280
420
(18)
(9)
(18)
(10)
(10)
(117)
Estimated D.L.2
(M/D
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Concentration '
720
2,900
7,200
5,700
ND
ND
ND
ND
ND
ND
10,000
1,000
130
(34)
(36)
(34)
(34)
(36)
(34)
(36)
Estimated D.L2
(v.g/1)
10
10
10
10
10
10
10
10
10
10
10
10
10
Total Recovery
97
54
42
79
707
728
46
44
153
i - Calculated concentrations, given as n.g/1, are based on a 2-liter total volume. The original extract concentrations have been multiplied by factors that correct for
volume adjustments made during the fractionation. The percentage of compound recovered in a particular fraction is shown in parentheses.
2 - Estimated detection limit: Estimate derived from GC/MS analysis of reference samples and from the respective volume adjustments that occurred during the
extraction process.
ND - Not detected.
P - Present, but quantitative data not available due either to the presence of interfering compounds or to very low concentrations of the respective compound.
NA - Data not accessible.
Table 2. Recovery and Partitioning of Selected Organic Compounds Following Chemical Fractionation of Collection B Waste Site Material
Whole Waste
Base/Neutrals
Organic Acids
Compound
pyridine
2, 6-dimethylpyridine
phenol
quinoline
benzaldehyde
2-methyl-2-hexanol
methyl pyridine
1,2,3,4-tetrahydro
quinoline
Concentration '
(M/l)
610
530
550
350
P
ND
660
630
Estimated
D.L.2
(M/l)
50
50
20
20
1,000
700
50
50
Concentration '
(W/l)
410 (67)
74 (14)
ND
172 (49)
ND
ND
320 (48)
172 (27)
Estimated
D.L2
(M/l)
100
100
100
100
100
100
100
100
Concentration '
(v.g/\)
ND
ND
720 (131)
ND
1100
280
ND
ND
Estimated
D.L.2
(v-g/D
100
100
100
100
100
100
100
100
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Table 2. (Continued)
Organic Residuals
Compound
pyridine
2,6-dimethylpyridine
phenol
quinoline
benzaldehyde
2-methyl-2-hexanol
methyl pyridine
1,2,3,4-tetrahydro
quinoline
Concentration '
tvefl
P
NO
P
ND
ND
ND
ND
ND
Estimated D.L.2
(v.g/1)
10
10
100
10
10
100
10
10
Total Inorganics
Concentration '
(W/l)
300 (49)
45 (8)
ND
ND
ND
110
370 (S6)
ND
Estimated D.L2
(v-g/l)
200
200
200
200
200
200
200
200
Total Recovery
(%)
116
22
131
49
104
27
i - Calculated concentrations, given as (ig/l. are based on a 2-liter total volume. The original extract concentrations have been multiplied by factors that correct for
volume adjustments made during the fractionation. The percentage of compound recovered in a particular fraction is shown in parentheses.
2 - Estimated detection limit: Estimate derived from GC/MS analysis of reference samples and from the respective volume adjustments that occurred during the
extraction process.
ND - Not detected.
P - Present, but quantitative data not available due either to the presence of interfering compounds or to very low concentrations of the respective compound.
Table 3. Inorganic Concentrations for Original Collection A Waste Material, Total Inorganics Fraction, Cation Subtraction, and Anion Subtrac-
tion (All concentrations given as mg/l.)
Aluminum
Antimony
Arsenic
Barium
Beryllium
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Molybdenum
Nickel
Selenium
Silver
Strontium
Thallium
Titanium
Vanadium
Zinc
Fluoride
Chloride
Nitrate
Phosphate
Sulfate
Nitrite
Cyanide
Original
Waste Material
(sample 1)
420
<15
<15
<7
<1.5
14
200
440
130
710
470
3
690
38
21
130
<1S
<1S
<1.5
20
<1.5
<7
48
710
14,000
31,000
<200
77,000
41,000
800
Total Inorganics
Fraction
%
(sample 7) Recovery
130
<6
<6
<3
<0.3
4.7
52
140
41
190
160
<1.2
200
11
7.8
50
<6
<6
<0.6
6.6
<0.6
<3
17
360
4,500
11,000
<200
16,000
13,000
320
80
—
~
—
-
87
68
83
82
70
89
—
75
75
97
100
..
_
—
86
.„
—
92
132
84
92
-
54
82
104
Cation
Subtraction
(sample 8)
140
<1
<1
9.0
-------
Table 4. Inorganic Concentrations for Original Collection B Waste Material, Total Inorganics Fraction, Cation Subtraction, and Anion Subtrac-
tion (All concentrations given as mg/l.)
Aluminum
Antimony
Arsenic
Barium
Beryllium
Cadmium
Chromium
Cobalt
Copper
Original
Waste Material
(sample 1)
110
<2
<2
<1
<0.1
<0.5
0.4
0.4
<0.5
Total Inorganics
Fraction
%
(sample 7) Recovery
160
<10
<10
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Viorica Lopez-Avila. Ray Kaminsky. Ursula Spanagel, and John Taylor are with
Acurex Corporation, Mountain View. CA 94039; William McKenzie and
Theodore Olsson are with Bioassay Systems Corporation, Woburn. MA 01801;
and the EPA author W. W. Sutton (also the EPA Project Officer, see below) is
with the Environmental Monitoring Systems Laboratory, Las Vegas, NV89114.
The complete report, entitled "Application of Chemical Fractionation/Aquatic
Bioassay Procedure to Hazardous Waste Site Monitoring," (Order No. PB 86-
109 493/AS; Cost: $16.95, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
P.O. Box 15027
Las Vegas, NV 89114
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S4-85/059
0000329 PS
U $ CNVIR PROTECTION Af*6NCt
CHICAGO
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