United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA-600/S3-81-043 Aug 1981
Project Summary
Reduction of Toxicity to
Aquatic Organisms by
Industrial Wastewater
Treatment
George A. Gary and Michael E. Barrows
The specific goal of this research
was to conduct 24-hour static acute
bioassays with "untreated" influent
and "treated" effluent using fathead
minnows (Pimephales promelas) and
water flea (Daphnia magna) to biolog-
ically evaluate the effectiveness of
industrial wastewater facilities in
reducing effluent toxicity to aquatic
organisms. Of primary interest to the
EPA was an evaluation of the capacity
of the wastewater treatment facilities
of the pesticide industry for reducing
toxicity. To accomplish the stated
goal, on-site 24-hour static acute
toxicity tests were performed during
ten consecutive workdays at seven
industrial sites. Five of the test sites
are defined as pesticide manufacturers,
while the remaining sites consisted of
an organo-chemical manufacturer and
a bleached-kraft paper mill. The effec-
tiveness of the treatment plants was
determined by performing static acute
toxicity tests with the fathead minnow
(Pimephales promelas) and the water
flea (Daphnia magna) on samples of
the wastewater collected before and
after treatment. Results of the studies
are expressed in terms of both median
lethal concentrations (LCBO's) as
percent effluent and lethal units.
The data from these studies indicate
that the wastewater treatment plants
provided an average efficiency of 98%
in reducing the toxicity of "untreated"
wastewaters. Neither species tested
proved to be a more sensitive indicator
of toxicity, though a larger data base is
required to make a valid appraisal.
Of interest was the observation that
while some wastewater treatment
facilities provide good efficiency
(98+%) in reducing toxicity, the result-
ing effluent still represented a relatively
high number of lethal units. This was a
result of the fact that the "untreated"
influent entering the waste treatment
system contained a very high level of
lethal units and a subsequent 98%
reduction of that level still resulted in a
toxic wastewater.
This Project Summary was devel-
oped by EPA's Environmental Re-
search Laboratory. 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 ordering information at
back).
Introduction
In 1977, the United States Environ-
mental Protection Agency was charged
by Congress under the Federal Water
Pollution Control Act Amendments (PL-
95-217) to develop a program which
would protect aquatic life by eliminating
the discharge into the nation's water-
ways of toxic pollutants in toxic amounts.
EG&G, Bionomics' personnel performed
on-site toxicity testing at seven indus-
trial wastewater treatment facilities
throughout the United States. The data
-------
generated from these studies will aid
EPA in developing effluent discharge
guidelines related to the toxicity to
aquatic organisms of industrial waste-
water streams.
The specific goal of this research was
to conduct 24-hour static acute
bioassays with untreated influent and
treated effluent using fathead minnows
(Pimephales promelas) and water flea
(Daphnia magna) to biologically
evaluate the effectiveness of industrial
wastewater facilities in reducing
effluent toxicity to aquatic organisms.
To accomplish this task, it was
necessary to evaluate the toxicity of the
wastewaters before and after biological
and/or chemical treatment procedures.
Results of the acute toxicity studies are
expressed in both the conventional
format as median lethal concentrations
(LCSO's) and as lethal units, a concept
which is similar to that of toxic units as
described by Esvelt, Kaufman, and
Selleck (1971). The lethal unit concept,
as it applies to effluents, is the
concentration of the effluent divided by
the 24-hour LC50. This unitless
expression of toxicity may be ideally
suited for use with wastewater
discharges of diversified effluents and
can be incorporated into the regulatory
process of controlling and limiting toxic
discharges.
Of primary interest to EPA was an
evaluation of the capacity of the
wastewater treatment facilities of the
pesticide industry for reducing toxicity.
The Agency's rationale in selecting this
industry was based on the premise that
discharges from pesticide manufacturers
might be expected to be relatively toxic,
and perhaps not readily degraded even
during biological treatment. The
industries which participated in this
study were Monsanto Company,
Muscatme, Iowa; Mobay Chemical
Company, Kansas City, Missouri;
Monsanto Company, Luling, Louisiana;
and Diamond Shamrock Corporation,
Green Bayou, Texas. Most chemical
manufacturing complexes are not
limited to the production of pesticides
alone. These sites were selected
because they had segregated pesticide
wastewater treatment facilities which
do not (for the most part) receive
wastewater from other manufacturing
processes, and because the majority of
the treatment systems were candidates
for best available technology economi-
cally achievable (BAT) systems or
portions thereof. In addition to the above
four pesticide manufacturers, studies
were conducted with a complex organo-
chemical effluent at Union Carbide
Corporation, South Charleston, West
Virginia; a complex organo-pesticide
effluent at E.I. duPont Nemours &
Company, Inc., LaPorte, Texas; and a
bleached-kraft paper mill effluent from
International Paper Company, George-
town, South Carolina.
Technical Approach
On-site testing was carried out at
each site in one of EG&G, Bionomics'
Mobile Aquatic Toxicology Laboratories.
Unless otherwise stated, procedures
used in conducting the on-site tests
followed "Methods for Acute Toxicity
Tests with Fish, Macroinvertebrates,
and Amphibians" (EPA, 1975). All raw
data generated from these studies are
permanently stored in the archives at
EG&G, Bionomics, Wareham, Massa-
chusetts.
The majority of the wastewater treat-
ment systems evaluated consisted of
the following (generalized) treatment
steps: coarse screening of particulates,
pH neutralization, activated sludge
digestion, and clarification. The specific
design of the wastewater treatment
plants varied from one site to the next
depending on the nature and composition
of the wastewater being treated. Waste-
water samples, which were generally
representative of the major contributory
pesticide wastewater stream entering
the treatment plant (untreated influent)
and the final discharge stream from the
wastewater treatment plant (treated
effluent), were collected at each site.
Depending on the site, one or two
untreated influent wastewater streams
were tested. In certain instances, direct
access to an incoming wastewater
stream was not possible until after it
had already entered some portion of the
treatment system (e.g., grit basin or pH
neutralization basin). At these sites the
influent samples were obtained imme-
diately prior to the next step in the
treatment process. The complexity of
some wastewater treatment plants
made it impossible to test influent
samples which were wholly representa-
tive of the total pesticide wastewater
load entering the treatment system.
This was a result of multiple wastewater
streams entering the treatment plant at
different stages in the treatment process.
Treated effluent samples were ob-
tained at sampling points located imme-
diately after the last step in the treatment
process. Since many of the sites hav
multiple wastewater treatment systerr
which are utilized for treating othe
manufacturing processes, the treate
effluent tested in these studies rep re
sented only the wastewater discharge
directly from the pesticide wastewate
treatment system. These samples wer
collected prior to dilution with othe
process wastewater streams or non
contact cooling waters and, therefore
are not representative of the company'
total discharge entering the receivin
system.
Twenty-four hour static acute toxicit
tests were performed daily on sample
of untreated influent and treated effluer
at each of the study sites (Mobay Chemi
cal, only treated effluent) over te
consecutive test days. Fathead minnov
tests were conducted on wastewate
samples collected at two samplini
periods each day while daphnid test
were performed on alternating day.
beginning with test day 1. At four of tni
sites, Salmonella Bacterial Mutagenesh
Assays (Ames Tests) were performed 01
samples of the treated effluent.
Grab samples of each wastewate
(influent and effluent) were obtainec
twice a day (9:00a.m.73:00p.m.)forter
consecutive days to coincide with th<
test regimentation. A sample of eacf
wastewater was taken prior to the firs
day of testing in order that preliminary
range-finding tests could be conductec
with each test species. At certain sites
personnel from the host company col-
lected duplicate or split samples of the
wastewater with the laboratory person-
nel from EG&G, Bionomics.
A characterization of the chemica
and physical parameters of the dilutior
water and treated and untreated effluent
wastewaters was performed daily at
each site. On each test day, samples ol
reconstituted dilution water were anal-
yzed for total hardness, total alkalinity,
specific conductance, pH, temperature,
and total residual chlorine. Similarly,
aliquots of each wastewater sample
collected during each ten-day study
were analyzed for dissolved oxygen
concentration (DO), pH, specific con-
ductance, temperature, and total resid-
ual chlorine. These measurements
were made on samples of the waste-
waters brought back to the mobile
laboratory, but prior to temperature
adjustment.
During each test, the pH, dissolved
oxygen concentration, and temperature)!
were measured in the control, high!
-------
middle, and low test concentrations
prior to the addition of the test organisms
and at the termination of the test.
Specific conductance was monitored
similarly, but only at the initiation of
testing.
Mortality data derived from each
definitive test were used to calculate a
median lethal concentration (LC50)and
its 95% confidence limit utilizing the
moving average angle method (Harris,
1959). The data are presented as both
LC50 values (percent wastewater) and
lethal units (100/LC50). The LC50 is the
calculated nominal concentration of the
wastewater in diluent water which
produces mortality of 50% of the test
animal population at the stated time of
exposure; i.e., 24-hour LC50. In those
instances where LC50 values could not
be calculated due to <50% mortality in
100% wastewater, the lethal units have
been derived through graphical inter-
polation of the data on log x probit paper
and provide the most probable toxicity
concentration (Esvelt, et al., 1971).
Results and Discussion
In the present study, the results of
toxicity studies indicate a higher level of
lethal units present in the wastewaters
of the pesticide formulation, both before
and after treatment as compared to the
wastewaters of pulp and paper mill and
organo-chemical plants (Table 1). A
sufficient data base is not available to
determine whether these observations
are valid overall for the industries of
concern. The average efficiency of the
wastewater treatment plants in reducing
the toxicity of the "untreated" waste-
water was 98% (range, 92-100%). Ex-
cluded in the average were Mobay
Chemical Company and E.I. duPont.
Mobay was excluded since influent
tests were not performed, and conse-
quently the percent efficiency of the
treatment system could not be deter-
mined. E.I. duPont was excluded from
the calculation since a true representa-
tion of the toxicity of the entire influent
wastewater load could not be deter-
mined.
The results of ten consecutive days of
biological testing at each site indicate
that the toxicity of the treated effluent
samples remained relatively constant
from day to day. The average number of
lethal units present in the treated
effluents, as determined from tests with
fathead minnows, was 2.1 with a range
of 0-6 lethal units. Similarly, Daphnia
magna tests provide a mean of 6.5 lethal
units with a range of 0-34. As would be
expected, the toxicity of the untreated
influent samples was extremely variable
on a day-to-day basis. From the fathead
minnow studies, the untreated influent
contained a mean of 93 lethal units
(range, 4.6-373), while Daphnia magna
acute tests produced a mean of 261
lethal units (range, 1.7-782).
The results of the Ames Test per-
formed on the treated effluenfwater
samples, collected on test days 1,3,6,7,
and 9 at Monsanto (Luling, La.), indicate
a weak, but consistent, increase in the
reversion index of tester strain TA1535.
The dose-related increase was present
both with and without metabolic activa-
tion, but was most pronounced in the
presence of rat liver-microsomes.
A confirmation study of the His*
phenotype of revertant colonies of
strain TA1535 at the highest dose level
indicated that 91 % of the colonies were
true revertants. This result supports the
conclusion that the observed increase
in reversion index of TA1535 is real and
not an artifact of the experiment. The
Ames Test showed no significant in-
crease in the reversion index of tester
strains, with or without metabolic acti-
vation, in treated effluent samples
collected at Monsanto, Muscatine,
Iowa; Diamond Shamrock, Green Bayou,
Texas; or Mobay Chemical, Kansas City,
Missouri.
Toxicity of the treated effluent is
relative to the toxicity of the untreated
influent. For example, if an influent
contained 300 lethal units and under-
went biological and/or chemical treat-
ment which has an efficiency of 99%,
Tahiti.
*Mean Lethal Units for Fathead Minnows and Daphnia magna Exposed to Untreated Influent and Treated Effluent from
Industrial Wastewater Treatment System.
Fathead Minnow
Daphnia magna
Industry
Union Carbide
(South Charleston)
Monsanto
(Muscatine)
Mobay Chemical
(Kansas City)
Monsanto
(Luling)
Diamond Shamrock
(Green Bayou)
E.I. duPont tt1
(La Porte) #2
International Paper
(Georgetown)
* Lethal units - 100
24-hr LC50/EC50 (percent
**Unable to make valid comparison
Influent
10.0
47.0
—
74.0
360.0
4.5
140.0
2.8
waste)
Effluent
0
1.5
—
6.0
1.2
2.4
0
% Toxicity
Reduced
100.0
97.0
—
92.0
99.7
#*
100.0
Influent
12.0
41.0
3.8
88.0
130.0
4.8
770.0
1.7
Effluent
0
1.3
4.4
3.1
2.6
3.5
0
% Toxicity
Reduced
100.0
97.0
—
96.0
98.0
_**
100.0
-------
then the treated effluent would still
contain 3 lethal units. This would be
equal to an LC50 value of 33% effluent.
Conversely, if an influent having mod-
erate toxicity is subjected to the same
treatment efficiency, the resulting ef-
fluent will probably be non-toxic or of a
very low toxicity. The significance of this
observation must be viewed in terms of
what additional engineering steps have
been applied to the effluent between the
time the wastewater leaves the treat-
ment system and enters the receiving
water. Dilution with other treated
process streams and non-contact cool-
ing water must be taken into considera-
tion in both designing wastewater
treatment systems and regulating their
discharge.
The Mass Emission Rate, as proposed
by Esvelt, et al. (1971), provides a useful
method of incorporating lethal units and
flow rates in assessing the impact of a
wastewater stream on the total ecology
of the receiving water. To determine the
mass emission rate for a stream, one
must first determine the relative toxicity
(RT) of the wastestream which is defined
as the product of the toxicity concentra-
tion (expressed as lethal units) and the
flow rate.
RT=Tc x Flow Rate
The mass emission rate then for a
receiving water (Tc2) is defined as the
relative toxicity divided by the combined
waste and dilution water flow.
Tc2= RT
wastewater
flow rate
dilution water
flow rate
At present, the mass emission rate for
a receiving water is a somewhat nebu-
lous value requiring further regulatory
definition. If a program for water quality
management is to be developed, which
considers the toxic properties of wastes,
then additional information must be
obtained on the efficiency of the waste-
water treatment systems. The utilization
of a study program similar to that carried
out under the present contract is one
method by which the minimum efficiency
of a wastewater treatment plant, under
normal operating conditions, could be
determined. If the toxicity of an effluent
could be correlated with one or more
chemical parameters which can be
monitored continuously, then a mech-
anism would evolve by which regulators
and plant operators could monitor the
discharge of toxic wastes on a real time
basis before they had a chance to cause
damage to the environment.
In this study, neither the fathead
minnow nor the water flea proved to be
routinely more sensitive than the other
in tests with the "untreated" or "treated"
wastewaters. Since an adequate data
base has not been developed for these
species to the vast majority of industrial
effluents, it is suggested that both the
fathead minnow and daphnids be tested.
On the other hand, should an on-going
biomonitoring program be instituted for
an industrial discharge in which both
species provide similar results, then
future testing might be limited to only
one species. Daphnia magna are attrac-
tive for this purpose from the standpoint
that their use can greatly reduce the
economic burden to an industry for such
a program. Daphnids are relatively easy
to culture, require no capital investment,
and can be used by plant personnel to
generate required data. It may also be
assumed that with such a biomonitoring
tool available to them, industry will take
it upon themselves to incorporate its
use on a regular basis in monitoring the
efficiency of their wastewater treatment
systems.
Finally, the use of the lethal unit
concept should be applied to all effluent
toxicity studies. LC50 values as percent
effluent do not provide an accurate
assessment of the impact of the waste-
water. This is especially true for highly
toxic wastes. The mean of a number of
LC50 tests may not equal the mean from
the same tests expressed as lethal
units. A highly toxic effluent when
converted to toxic units will skew the
mean upwards and as a result will more
accurately represent the toxic load
entering the environment.
For those effluents resulting in less
than 50% mortality in 100% effluent,
the most probable toxicity concentration
(expressed as lethal units) is equally
important in determining mass emission
rates.
References
Esvelt, L. A., W. J. Kaufman and R. E
Seleck. October, 1971. A study c
toxicity and biostimulation in Sa
Francisco Bay - Delta Waters. Vol. IV
Sanitary Engineering Research Lab
oratory. Univ. California, Berkeley.
Harris, £. K. 1959. Confidence limits fc
the LC50 using the moving average
angle method. Biometrics, Vol. 4; #3
pp. 157-164.
L). S. EPA. 1975. Methods for acuti
toxicity tests with fish, macroinverte
brates and amphibians. Ecologica
Research Series (EPA-660/3-75-009;
61 pp.
-------
George A. Gary and Michael E. Barrows are with EG&G, Bionomics, 790 Main
Street, Wareham, MA 02571.
William Horning, II. is the EPA Project Officer (see below).
The complete report, entitled "Reduction of Toxicity to Aquatic Organisms by
Industrial Wastewater Treatment," (Order No. PB 81-222 366; Cost: $8 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 EPA Project Officer can be contacted at:
Newtown Fish Toxicology Station
U.S. Environmental Protection Agency
3411 Church Street
Cincinnati, OH 45244
•ft U S GOVERNMENT PRINTING OFFICE, 1981 — 757-012/7339
-------
Postage and
United States Center for Environmental Research Fees Paid
Environmental Protection Information Environmental
Agency Cincinnati OH 45268 Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
PS on 0
u s FhviR PKOTECTIOM
RtGjnw 5 LfBSARY
?30 S OEAW80RN STREET
CHlCAbO IL 6060a
------- |