United States
                    Environmental Protection
                    Agency
Environmental Research
Laboratory
Duluth MN 55804
                    Research and Development
EPA-600/S3-84-009  Feb. 1984
&EPA          Project  Summary
                    Aquatic  Toxicity Tests to
                    Characterize  the  Hazard  of
                    Volatile  Organic  Chemicals  in
                    Water: AToxicity Data Summary
                    N. Ahmad, D. Benoit, L. Brooke, D. Call, A. Carlson, D. DeFoe, J. Huot, A.
                    Moriarity, J. Richter, P. Shubat, G. Veith, and C. Walbridge
                      This summary  presents  acute and
                    chronic toxicity test data and bioconcen-
                    tration factors compiled over a two-
                    year period on fish and invertebrates
                    exposed to several representative chem-
                    icals from five chemical classes (chlor-
                    inated ethanes, chlorinated benzenes,
                    chlorinated ethylenes, chlorinated di-
                    enes, and chlorinated propanes).
                      The fathead minnows and Daphnia
                    were quite similar in their sensitivities
                    (acute and chronic) to each chemical
                    class, while the  rainbow trout  were
                    considerably more sensitive to all class-
                    es during acute tests, except for the
                    chlorinated diene exposures, where
                    they were more resistant. The ranking
                    of acute and chronic sensitivity was
                    generally the same  for each chemical
                    within each class of chemicals for all
                    three species tested.
                      Both the acute and chronic toxicity of
                    all chemicals within a class increased as
                    the number of chlorines in the chemical
                    structure increased.
                      Bioconcentration factors for fathead
                    minnows were determined  for four of
                    the chemical classes tested. Hexachlo-
                    robenzene was bioconcentrated the
                    most (23,000x), and tetrachloroethane
                    was bioconcentrated the  least (8x).
                    Again, as with the toxicity experiments,
                    the greater the number of chlorines on
                    the molecule the greater the bioconcen-
                    tration within each class of chemicals.

                      This Project Summary was developed
                    by EPA's Environmental Research Labo-
ratory. Duluth, MN. to announce key
findings of the research project that is
fully documented in a separate report of
the same title (see Project Report order-
ing information at back).

Introduction
  The objectives of this study were to
evaluate the use of sensitive aquatic
toxicity tests in determining the relative
hazard of  volatile organic compounds;
and to compare the sensitivities of three
aquatic species to  several homologous
series of organic chemicals.  Investiga-
tions were divided into two major areas.
First, preliminary studies were conducted
to determine similarities and differences
in metabolism of selected xenobiotics
between aquatic  and mammalian organ-
isms. The second phase, reported here,
represents the largest effort, which was
to develop methods for testing volatile
chemicals  and to determine the differ-
ences in toxicity between daphnids (Daph-
nia magna) and fish to selected volatile
chemicals.
  Both acute and chronic toxicitv tests
were completed  with fathead minnows
(Pimephales promelas) and daphnids.
Only acute tests were performed using
rainbow trout (Salmo gairdneri).  Test
chemicals were selected from the follow-
ing classes of compounds: chlorinated
ethanes; chlorinated benzenes; chlori-
nated ethylenes;  chlorinated dienes and
chlorinated propanes. Bioconcentration
factors for fathead minnows exposed to

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10  chemicals  from the above  classes
were also determined.
  The full report represents an overview
of the results from these investigations.
Most data have been or are scheduled to
be published in scientific journals.

Methods and Procedures
Bioassay-Chemical
  Routine toxicity test met hods and chem-
ical procedures followed closely those
described by Mount and Brungs (1967),
APHA(1975),USEPA(1975), Phippsetal
(1981), and Benoit et al (1982)
  All tests,  except for the static Daphnia
exposures, were performed using propor-
tional diluters. Lake Superior water was
used for all tests. Nominal water temper-
atures were 12, 20, and 25° C for rainbow
trout, Daphnia, and fathead minnows,
respectively Acute tests were for 96 hrs
forfatheadsand rainbowtroutand48 hrs
for Daphnia. Chronic fish (embryo-larval)
tests were  conducted for 32 days, and
chronic Daphnia  tests were of 28-day
duration
  Chemical characteristics of the control
and experimental waters were as follows:
dissolved oxygen  7.0-9.6 mg/l; pH 6.7-
7.6, total hardness (as CaCO3) 43-57
mg/l, and alkalinity 35-53 mg/l  Several
statistical procedures (Spearman-Karber,
ANOV, Probit analyses) were used to test
the significance of the results.
  Chemicals selected  for testing ranged
in purity from 95-99  percent  Solvents
used  in the extraction process  were of
glass-distilled, gas-chromatography
grade. The effectiveness of the extraction
procedure was examined by determining
the percent recovery of a known amount
of each  chemical in  the  experimental
water. All  toxicant concentrations in
water and tissues were analyzed by gas
chromatography  Further description of
these analytical procedures, according to
chemical, appear in the full report.

Development of Early Life
Stage,  Mini-Diluter Apparatus
  In order to successfully and safely test
volatile chemicals,  it  was necessary to
develop  specialized exposure  systems
and methods for monitoring air and water
leaving these systems.  Interest in develop-
ing fish early life-stage (ELS)toxicity tests
led to the design of a compact continuous
flow mini-diluter exposure system which
accurately delivered as little as 3 liters of
test water  per hour  to  each of five
concentrations plus a control This system
can be used to test the effects of either
single chemicals or treated  complex
effluents on young fish in the laboratory
or in the field. The small ELS test appa-
ratus takes  less space  and  requires
smaller volumes of test water, which is a
critical factor when effluents are shipped
to the laboratory or on-site toxicity tests
are conducted. Smaller volumes of test
water also reduce filtration costs for the
removal  of  hazardous test  chemicals
before discharging waste water to the
sewer.
  The ELS test system has been tested
and evaluated in the  laboratory and on-
site  in a mobile  trailer (Benoit et al.,
1982). This apparatus has been used  to
conduct  fathead  minnow (Pimephales
promelas) ELS exposures to various toxi-
cants including volatile  organic  com-
pounds, metals, pesticides, and treated
complex effluents from metal plating, oil
refinery,  and  sewage treatment plants
The system has also been successfully
used for testing macromvertebrates. All
embryo-larval fish tests described herein
were conducted in this mini-diluter sys-
tem.

Summary and Conclusions
  It has long been known that aquatic
animals are extremely sensitive to chemi-
cals at very low /ug/L to mg/L concentra-
tions in the aqueous environment. This
knowledge led us to the position  of
evaluating both acute and chronictoxicity
tests with several sensitive aquatic spe-
cies in an effort to deter mi net he range of
sensitivities and the possible application
of the data screening out chemicals,
which after short inexpensive tests with
selected aquatic species are shown to be
extremely toxic, and/or highly bioaccumu-
lable
  Phase  one  of these studies  was de-
signedto provide preliminary information
on the metabolic capabilities of rainbow
trout and daphnids These studies, coupled
with earlier studies  on  mixed  function
oxidase activity in mammals and  other
animals, indicated the metabolic systems
to be similar qualitatively, therefore, the
mechanisms leading to toxicity  and neo-
plasia, for example, are presumed  to be
similar in all organisms. Phase two was to
determine the acute and chronic toxicity
of five  classes of chlorinated organic
compounds to selected fish and inverte-
brate animals In addition, the bioconcen-
tration potential of these chemicals was
measured to  determine possible  food-
chain problems involving man.
  Daphnia  showed generally the  same
order of acute and chronic sensitivity  as
the fish for  all classes of  chemicals
tested. A comparison of species  acute
sensitivities (Table 1) indicated that Daph-
nia were slightly more resistant in mosl
cases than the fathead minnow, whereas
the rainbow trout was considerably more
sensitive than either the fathead minnow
or Daphnia to all  compounds  excep!
hexachlorobutadiene
  One of the more interesting findings ol
the acute studies was that the toxicity ol
the ethanes, benzenes, and ethylenes
increased as the number of chlorines on
the molecule increased. This was true for
both fathead minnows and Daphnia (Table
1).
  These data indicate that either fathead
minnows or Daphnia would  provide es-
sentially the same acute values for these
particular chemicals. These chemicals
are not considered to  be very toxic tc
aquatic species, since their 96-hr LC50s
are one to two orders of magnitude above
those environmental chemicals consid-
ered as extremely toxic.

  Fifteen chronic toxicity tests with fish
were also conducted on chemicals in the
five chemical classes. The order of sensi-
tivity for all chemicals tested chronically
(Table 2) was the same as that established
for acute toxicity  (Table 1).  Six chronic
values were also determined for Daphnia
and  in most  cases the sensitivity  was
similar except for the  ethanes,  where
there seemed to be considerable variation
between the fathead and Daphnia results.
Again, chronic toxicity increased consid-
erably for both species as the number of
chlorines on the  molecule increased
(Table 2).
  The  bioconcentration potentials of
these chemicals  were  determined by
establishing a bioconcentration factor
(BCF) Cp.sh/Cwater) for fathead  minnows
exposed  for 32 days to each  chemical
during an early  life-stage toxicity  test.
These BCFs were then compared to BCF
values for other species of fish found in
the literature (Table 3). In the  two classes
where comparisons were possible (chlori-
nated benzenes and ethanes), bioconcen-
tration as well as toxicity increased as the
number  of chlorines on the  molecule
increased. The literature values for other
fathead minnow studies as well as studies
with bluegill, sunfish (Lepomis macro-
chirus),  and guppies (Poecilia reticulata]
(Table 3) all agree very closely with the
BCFs generated here. This indicates that
age,  size, and species of fish have  little
effect on the BCF generated over a 32-day
period of water exposures. Based on BCF
values, hexachlorobenzene, hexachloro-
butadiene,  and 1,2,3,4-tetrachloroben-
zene are the chemicals from the groups

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Table  1.    Summary of Acute Toxicity Data for Fathead Minnows, Rainbow Trout, and Daphnia
        Compound
Fathead Minnow
  96-hr LCSO
    fmg/IJ
Rainbow Trout
 96-hr LCSO
   (mg/lj
 Daphnia
48-hr LCSO
  (mg/l)
Chlorinated Ethanes
  Hexach/oroethane
  Pentachloroethane
  1,1,2,2-Tetrachloroethane
  1,1,2-Tnchloroethane
  1,2-Dichloroethane
Chlorinated Benzenes
  Hexachlorobenzene
  Pentachlorobenzene
  1,2,3,4- Tetrachlorobenzene
  1,2,4 • Tnchlorobenzene
  1,3-Dichlorobenzene
  1,4-Dichlorobenzene
Chlorinated Ethylenes
  Tetrachloroethylene
  1,1,2-Tnchloroethylene
Chlorinated Propanes
  1,2-Dichloropropane
  1,3-Dichloropropane
Chlorinated Butadienes
  Hexachlorobutadiene
      1.53
      730
     2030
     81 70
    7 77 SO
      1 07
      •276
      779
      4 re

      1350
     44 10

    139 30
    131 10

      0.10
                     084
    1.52
    1.61
    1.12

    499
                     290
                     732
                    62.10
                   186.0
                   268.0
    2.09
    7.43
   18 10
                     032
"Not tested
"Not toxic at water saturation
Table 2.    Summary of Fathead Minnow and Daphnia Chronic Toxicity Data
        Compound
                                      Fathead Minnow
                                     32-day (ELS) MA TC
                                 Daphnia
                             28-day" Chronic
Chlorinated Ethanes
  Hexach/oroethane
  Pentachloroethane
  1,1,2,2-Tetrachloroethane
  1,1,2-Trichloroethane
  1,2 -Dichloroethane
Chlorinated Benzenes
  Hexachlorobenzene
  Pentachlorobenzene
  1,2,3,4- Tetrachlorobenzene
  1,2,4-Tnchlorobenzene
  1,3-Dichlorobenzene
  1,4-Dichlorobenzene
Chlorinated Ethylenes
  Tetrachloroethylene
  1,1,2-Tnchloroethylene
Chlorinated Propanes
  1,2-Dichloropropane
  1,3-Dichloropropane
Chlorinated Butadienes
  Hexachlorobutadiene
           69-207
          900-1,400
        1,40O-4,OOO
        6,000-14,800
       29,000-59,000

            4.76"
           549"
          245-412
          499-1,008
        1.000-2,267
          565-1,040

          500-1,400
        6,000-11,000
        8,000-16.000

          65-13.0
              6,850-14,4OO
             13,200-26,000
             10,600-20,700
               363-694
               689-1,450
               505-1.110
"Effect—no effect concentrations
"Water saturation—no effects noted
tested that pose the greatest bioconcen-
tration threat to the environment.
  The acute toxicity tests run with both
fish and invertebrates established a rela-
tive order  of toxicity for  the individual
chemicals that was nearly identical to the
order seen in  the  more sensitive
chronic exposures. Therefore, the short
96-hr LCSO fish exposures or the 48-hr
Daphnia exposures could  be used to
establish a ranking of chemicals found in
water that will  permit  the researcher to
initially concentrate the expensive chronic
testing on the more toxic materials
  The Daphnia acute test may be better
than a fish acute test, since it takes only
48-hrs and does  not  require the more
difficult flow-thro ugh system required for
a fish 96-hour LC50 estimate, yet it gives
the same  relative order of chemical
sensitivity for these classes of chemicals
(Tables 1 and 2).
  Early life-stage toxicity tests with fish
(32-days) and/or 28-day Daphnia chron-
ics would provide a more sensitive test
and the fish exposures would also allow
the determination of a BCF, which would
provide further information on the envi-
ronmental  impact of a chemical.
  The usefulness of aquatic tests as an
early warning system for toxic chemicals
in these classes may be somewhat limit-
ed, because of their low toxicity and low
ambient water concentrations. However,
this approach for  more toxic chemicals
has considerable  promise as an early
warning system for higher animals includ-
ing man.


References
American Public Health Association, A-
  merican Water Works Association, and
  Water  Pollution Control  Federation.
  1975. Standard methods for the exami-
  nation of water  and  wastewater  14th
  ed.  American Public Health Associa-
  tion, Washington, DC 20036.
Benoit,  D.  A., V. R  Mattson, and  D L.
  Olson  1982. A continuous-flow mini-
  diluter system for toxicity testing. Water
  Res 16-457-464.
Mount, D  I. and W. A. Brungs. 1967. A
  simplified  dosing apparatus  for  fish
  toxicology studies. Water Res., Vol 1,
  pp  21-29.
Phipps, G. L., G. W. Holcombe, and J. T.
  Fiandt. 1981. Acute toxicity of phenol
  and substituted phenols to the fathead
  minnow. Bull. Environ. Contam. Toxicol.
  26:585-593.
U.S.  Environmental Protection Agency,
  Committee on  Methods  for  Toxicity
  Tests with Aquatic Organisms. 1975.

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  Table 3.    A Comparison of Bioconcentration Factors for Chemicals Tested in Present Study in
             Fathead Minnows vs. Other Species of Fish in Other Studies
          Chemicals
   Present Study
Fathead Minnows °
  BCF    Log BCF
      Literature Values
Fathead
Minnow*   Bluegilf   Guppyc
Chlorinated Ethanes
Hexach/oroethane
Pentachloroethane
1 ', 1 ,2,2-Tetrachloroethane
Chlorinated Ethylenes
Tetrachloroethylene
Chlorinated Butadienes
Hexachloro- 1,3-butadiene
Chlorinated Benzenes
Hexachlorobenzene
1 .2,3,4- Tetrachlorobenzene
1 ,2,4- Trichlorobenzene
1 , 3-Dichlorobenzene
1 ,4-Dichlorobenzene

703.4
60.4
8.1

61.5

6,988.6

23,391 5
2,567.3
398.5
97.9
112.4

285
1.78
0.91

1.79

3.84

4.37
3.41
260
1.99
205

138
68
8

49

-

21,878
1,820
1,698
66
60


-
-

-

-

14,454
3.631
646

91
  ^32-day exposure ELS toxicity test.
  ^30-day old fish exposed for 30 days.
  1Adult fish exposed for 30 days.
    Standard practice for conducting acute
    toxicity tests with fishes and macro-
    invertebrates, and  amphibians. EPA-
    660/3-75-009. Duluth, MN. 67 p.
           N. Ahmad, J. Huot, and A.  Moriarity are with  the  University of Wisconsin,
             Superior,  Wl 54880; the EPA authors D.  Benoit, L. Brooke. D.  Call, A.
             Carlson, D. Defoe, J. Richter. P. Shubat, G. Ve/th, and C. Walbridge are with
             the Environmental Research Laboratory, Duluth, MN 55804.
           J. M. McKim and R. A. Drummond are the Co-Project Coordinators (see below).
           The complete report, entitled "Aquatic Toxicity Tests to Characterize the Hazard
             of Volatile Organic Chemicals in Water: A Toxicity Data Summary," (Order No.
             PB 84-141 506; Cost: $13.00,  subject to change) will be available only from:
                   National Technical Information Service
                   5285 Port Royal Road
                   Springfield, VA 22161
                   Telephone: 703-487-4650
           The Co-Project Coordinators can be contacted at:
                   Environmental Research Laboratory
                   U.S. Environmental Protection Agency
                   6201 Congdon Blvd.
                   Duluth, MN 558O4
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