v>EPA
United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/D-80-005 Dec. 1980
INDUSTRIAL
ENVIRONMENTAL
RESEARCH BRIEF
Use of Aquatic Oligochaete, Lumbriculus variegatus, for
Effluent Biomonitoring
C Evan Hornig, Department of Biological Sciences,
University of Las Vegas, Las Vegas, NV 89154
Introduction
In a recent document for effluent bioassay methods,
the U.S. Environmental Protection Agency (EPA)
notes the importance of toxicity testing as it relates to
the prevention of toxic discharges to the
environment
"The Declaration of Goals and Policy of the Federal
Water Pollution Control Act Amendments of 1972,
Section 101 (a) (3), states that 'it is the national goal
that the discharge of toxic pollutants in toxic amounts
be prohibited.' Current Agency programs for the
protection of aquatic life in receiving waters are
based, in,part, on effluent limitations for individual
chemicals. However, toxicity data are available for
only a limited number of compounds. The effluent
limitations, therefore, may not provide adequate
protection where the toxicity of the components in the
effluent is not known, where there are synergistic
effects between toxic substances m complex
effluents, and/or where a complete chemical
characterization of the effluent has not been carried
out. Since it is not economicallyfeasibletodetermine
the toxicity of each of the thousands of potentially
toxic substances in complex effluents or to conduct
an exhaustive chemical analysis of the effluent, the
most direct and cost-effective approach to the
measurement of the toxicity of effluents is to conduct
a bioassay with aquatic organisms representative of
indigenous populations For this reason, the use of
effluent bioassays to identify and control toxic
discharges is rapidly increasing within the Agency
and state NDPES programs "
A variety of biological approachestothedetection and
assessment of effluent toxicity have been developed
by various researchers. Monitoring objectives and,
practical limitations will determine the best available
approach One application of biological techniques is
the use of organisms as "early warning"
mechanisms to detect changes in the toxicity of
effluents and receiving waters While continuous on-
site monitoring is ideal in situations where rapid
detection of a potentially dangerous alteration in
water quality is critical, approaches tothe monitoring
of behavior and physiology in test fish require
complex equipment and techniques, and their wide-
spread use is impractical
This report focuses on a simple, inexpensive short-
term acute toxicity test which can be used in the
detection of gross changes in effluent and receiving
water toxicity. The approach is designed as an initial
screening technique for detecting toxicity of cooling-
water effluents. A "positive" toxicity test would
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identify locations where more intensive biological
and chemical analyses should be concentrated.
Although this simple approach to biological water
quality monitoring is not a substitute for more
rigorous testing, its widespread use will improve the
timely detection of toxic substances in the aquatic
environment. This report describes the use of
Lumbriculus variegatus, an aquatic earthworm, as a
test organism for short-term acute toxicity tests. This
organism's hardiness, sensitivity to fluctuating
effluent quality, and response to a commonly used
biocide (sodium pentachlorophenate) are described.
Conclusions and Recommendations
Oligochaetes may prove to be cost-effectively used to
detect changes in gross toxicity of effluents and
receiving waters because of their uncomplicated
biology and life-cycle.
The bioassay tested requires only holding containers,
temperature control, and the test organisms,
Lumbriculus variegatus (Oligochaeta:Lumbricuhdae).
While not recommended as a substitute for more
sophisticated biological techniques, bioassay testing
promises to be a means of increased monitoring
where the number of cooling-water biomonitoring
stations are limited, particularly in the western
United States, where recent emphasis on energy
development is expected to result m the rapid
increase of cooling towers and other sources of
industrial pollution
Short-term static effluent tests for acute toxicity
showed that L. variegatus responded differentially to
industrial effluents collected at different dates from
the same outlet, indicating their usefulness for
detecting changes in the gross toxicity of complex
effluents
L. variegatus also showed sensitivity to a specific
pollutant, sodium pentachlorophenate (Na-PCP),
which is commonly used as a fungicide in cooling
towers. Definitive tests with Na-PCP resulted in a 96-
hour LCso of 0.57 ppm and a 48-hour EC5o for
inactivity of 0 66 ppm.
Observations of effects of Na-PCP on/., variegatus at
shorter and longer exposure times is needed to
determine the minimum length of time required to
detect high levels (> 1 ppm) of a Na-PCPand whether
a threshold of LCso exists.
Additional work is recommended to determine
response of L. variegatus to other important cooling-
water toxicants and to a variety of cooling-water
effluents, ideally with the chemical constituents of
the effluents identified. Attempts can then be made to
relate results from effluent tests to changes in toxicity
predicted from chemical composition. It would also be
useful to compare toxicant and effluent responses of
this organism to those organisms indigenous to the
receiving waters.
The potential of other ohgochaetes or other hardy
organisms for simple, initial-stage monitoring should
be investigated.
Materials and Methods
Test Organisms
Most of the studies on the effects of pollutants on
oligochaetes looked at sewage or other organic efflu-
ents. A number of investigations are described here.
Laboratory studies on the effects of specific heavy ion
and biocide toxicants have been performed on a few
species of oligochaetes with varied results,
depending on the test species and toxicant. One study
by Whitten and Goodnight found DDT nontoxic to
worms at 100 mg/l, while marking fixed the LCso of
Tubifex tubifex to the pesticide, 2-(digeranylammo)-
ethanol, at 0.054 ppm. Whitten established the sensi-
tivity of tubificids to heavy metals from 24-hour LC50
of 49 0 ppm of lead and LC5o 46.0 ppm of zinc to a level
well below the 1 ppm of cadmium, copper, and
mercury reported by Brkovic and Popovic.
The sensitivity of many species of oligochaetes to
industrial toxicants is indicated by field studies of the
River Irwell m England and Kanawha River, West
Virginia made by Eyers and Maciorawski, respec-
tively. At some locations on the River Irwell,
oligochaete diversity was lower than could be
explained by organic pollution alone; the investiga-
tors speculated that toxins undetected by routine
chemical analysis may have had adverse effects on
the oligochaete community. Greatly reduced
populations of all oligochaete species were found m
the Kanawha River around areas of the highest
industrial activity, with many of the speciesgenerally
restricted to the cleaner water stations Lumbri-
culidae, although uncommon, was largely restricted
to the less polluted reaches. These studies indicate
aquatic oligochaetes are generally sensitive to a
variety of toxic substances; it should be beneficial to
investigate their applicability m simple bioassays for
screening of cooling water toxicity
L. variegatus (Oligochaeta Lumbriculidae) was
chosen for these investigations because it is readily
available and m uniform supply. These organ isms are
raised under constant conditions and are highly
sensitive to changes m their environment. For
example, failure to change their holding water will
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result in death of the organisms within two days
(probably due to self-pollution caused by high
organism density) Daily changing of their holding
water (with dechlormated tap water) insures their
health and activity not only in the laboratory, but also
in their function as test organisms
Test Media
Effluent samples were collected from a small outlet
ditch at Basic Management, Inc., of Henderson,
Nevada, an industrial complex. The effluents were
left at 100% concentration for all effluent tests.
Dechlorinated tap water was used as the control
medium for the effluent tests.
The toxicant tested was sodium pentachlorophenate
(Na-PCP). The Na-PCP was supplied by Dow Chemical
Company of Midland, Michigan, as Dowicide™ G-St
Beads (EPA Registration No. 464-380). The
ingredients of this product were listed by the manu-
facturer as 79% sodium pentachlorophenate, 11%
sodium salts of other chlorophenols, and 10% inert
ingredients.
Na-PCP was chosen as the test toxicant because of its
widespread and common usage. Na-PCP and
pentachlorophenol (PCP) collectively are the second
most heavily-used pesticides in the country. They are
chiefly used for wood preservation and treatment, but
also have many other uses and are registered by the
U.S. Environmental Protection Agency as insecti-
cides, fungicides, and algicides. Phenols and
chlorinated or phenylated phenols represent a major
class of cooling tower biocides used for slime control.
Of this group, Na-PCP is probablythe most frequently
used. Because PCP and Na-PCP can become
important and persistent environmental pollutants,
they pose a potential health hazard. Also, they have
been used extensively for toxicology testing.
The control and toxicant dilution water for the
toxicant range-finding tests was dechlorinated tap
water. The control and toxicant dilution water for the
toxicant definitive tests was reconstituted soft water
as recommended by EPA. This water was prepared by
adding 48 mg/l NaHCO3, 30 mg/l CaSO4-2H20, 30
mg/l MgSCU, and 2 mg/l KCI to triple-distilled water
(less than one micromho/cm). This water has the
following properties: pH =7.2-7.6, hardness = 40-48
mg/l CaCOs, and alkalinity = 30-35 mg/l CaC03.
Equipment and Test Conditions
A Freas-81 5 low-temperature incubator kept at 17°C
± 2°C was used for all experiments. After the
organisms were obtained, they were held in liter
flasks filled with dechlorinated tap water which was
changed daily. All effluent and toxicant tests were
static tests The test containers consisted of 3-cm
high, wide-mouth jars. For most tests these jars were
filled with 50 ml of the appropriate medium, five
organisms were placed in each filled jar. Maintaining
a low number of test organisms per container elimi-
nated self-contamination associated with over-
crowding and facilitated counting. No aeration or
other precautions were used to maintain the
organisms during the experiments.
Experimental Design
Effluent Study
This test was designed to assess the ability of the
organisms to respond to unknown and complex
industrial effluents and to changes in the toxicity of
those effluents. Effluent samples were collected
on five different dates in February and March 1979. In
the earliesttest, ten organisms were placed in each of
ten flasks filled with 100 ml of effluent. In all
subsequent tests, five organisms and 50 ml of
effluent were used with each experimental test set
After 96 hours, the worms were removed from the
incubator and inspected for mortality.
Toxicant Studies
Range Tests—Range tests were used to determine
the Na-PCP concentration levels to be employed in
the definitive toxicant tests. The first test utilized Na-
PCP concentration levels to be employed in the
definitive toxicant tests. The first test utilized Na-PCP
concentrations of 0.5, 1, 2, and 4 ppm, and mortality
was recorded at 24, 48, and 120 hours. The second
test was conducted using Na-PCP concentrations of
0.125, 0.25, 0.5, and 1 ppm, with mortality recorded
at 24, 48, 72, 96, and 168 hours. In each test, five
organisms in five separate jars containing 50 ml of
the appropriate medium were utilized for each
experimental concentration and a control.
Definitive Tests—Definitive toxicant tests were
carried out using nine concentrations of the test
toxicant, ranging from 0.2 ppm to 1.0 ppm Na-PCP at
intervals of 0.1 ppm. For each toxicant concentration
and control, five organ isms were placed in each of the
five test jars filled with 50 ml of the appropriate
medium. At 24, 48, 72, 96, 120, 144, and 168 hours
the organisms were removed from the incubator and
both mortality and inactivity were recorded.
Data Analysis
Graphical Determination of LCso and LE50 Levels—
ASTM graphical models were used for both toxicant
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range-finding and definitive tests. The toxicant
concentration was plotted on log scale against the
probit scale for the percent of organisms affected at a
given number of hours after the start of the experi-
ment. A straight line was drawn to the plotted points,
and the concentration corresponding to 50% mortal-
ity or inactivity was read from t he graph as the LC so or
EC50.
Probit Analysis of LC5o and ECso Levels—Probit
analysis was applied to results which met EPA
criteria for definitive toxicant tests. These criteria are
as follows (1) the concentration of toxicant in each
treatment must be at least 60% of the next higher
concentration, (2) one treatment other than the
control must affect less than 65% of the exposed
organism; and (3) graded responses must result for a
minimum of five levels of the toxicant (a minimum of
three partial kills or effects). Calculations were
completed with the aid of a programmable handheld
calculator
The probit analysis utilized was the maximum
likelihood estimation described by Finney in 1971.
The iteration was repeated until at no more than one
concentration did the corresponding estimated probit
value change by more than 0 1 and no concentration
had a probit value shift more than 0.2. The chi-square
value for precision of fit describes how well the data
conform to the probit model; a smaller value indicates
a better fit
Results from the probit analyses were used to plot
percentage mortality against Na-PCP concentration
for the exposure times of 48, 96, and 168 hours
These regression lines show the rate of increase in
mortality as a function of the increase in Na-PCP
concentration The LCsoS were plotted against their
exposure times (toxicity curve) to determine a
possible threshhold LCso. The threshold LC5o
indicates the level of toxicity at which acute lethality
has stopped
Results and Discussion
Suitability of Lumbriculus variegatus as a Test
Organism
Collection and preparation of L variegatus for
bioassay testing can be cost effective for the following
reasons (1) aquatic oligochaetes exhibit relatively
few complex biological and life-cycle characteristics;
(2) because they are hermaphroditic and do not molt,
they do not have to be sorted acordmg to sex or
molting stage; and, (3) since these organisms are
commercially raised and shipped, they can be
obtained from a uniform source. Variation factors that
could occur because of sex, growth, and other
complex biological characteristics are, thus, avoided
by using this particular organism.
Handling, holding, and conditioning of test organisms
can also require complex and expensive procedures
to keep organisms healthy and to assure consistent
results. A continuous flow of prepared water and
proper feeding are required for many organisms.
Survival rates exhibited by control organisms utilized
in the tests described here (Tables 1 -5) indicated that
minimal preparation and maintenance procedures
were adequate to assure that specimens remained
healthy.
Sensitivity of Lumbriculus variegatus to Effluent
Toxicity
The test organisms exhibited various responses to
effluent samples collected on different dates (Table
1) The industrial outlet ditch from which the effluent
samples were taken is shown to be subject to large
variations in water quality, such fluctuations in water
quality probably account for the observed variation in
test organism response. While chemical analyses of
the effluent are not available, the bioassay results
suggest that L. variegatus is sensitive to fluctuations
in water quality. The organism should be tested
further to determine its usefulness as a biological
screening agent for changes in effluent toxicity. For
of 3 and 5500 ppm chlorides.
TABLE 1 MORTALITY OPLumbr/cu/usvar/egatus£J96\-\O\JRS
TO 100% EFFLUEIMTCOLLECTEDATVARIOUS DATES
FROM THE OUTLET STREAM OF THE BMI INDUS-
TRIAL COMPLEX, HENDERSON, NEVADA
Date of
Collection
2/28/79
3/1/79
3/5/79
3/16/79
3/21/79
No of Test
Organisms
100
100
50
50
50
50
50
50
50
50
Test
Water
Effluent
Control
Effluent
Control
Effluent
Control
Effluent
Control
Effluent
Control
Percent
Mortality
97
0
2
2
2
0
0
0
28
0
Effects of Na-PCP on Lumbriculus variegatus
One-hundred-percent mortality occurred within 24
hours at 2.0 ppm Na-PCP and within 48 hours at 1.0
ppm Na-PCP during the range-finding tests (Tables 2-
3). The 24-hour LC50 was estimated to be slightly
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TABLE 2 MORTALITY OF Lumbnculus variegatus TO SODIUM PENTACHLOROPHENATE (RANGE-FINDING TEST 05-40 ppm)
No of Test
Organisms
25
25
25
25
25
LCso, ppm, estimated
Concentration
NaPCP (ppm)
40
20
1 0
05
00
from graph
24 hr
100
100
12
8
0
1 16
Percent Mortality
at 48 hr
100
100
92
28
0
060
120 hr
100
100
100
100
0
TABLE 3 MORTALITY OF Lumbnculus variegatus TO SODIUM PENTACHLOROPHENATE (RANGE-FINDING TEST 0 125-1 Oppm)
No of Test
Organisms
25
25
25
25
25
LCso, ppm, estimated
Concentration
of NaPCP (ppm)
1 0
05
025
0125
00
from graph
24 hr
36
0
0
0
0
1 04
48 hr
100
44
0
0
0
052
Percent
72 hr
100
76
4
0
0
044
Mortality at
96 hr
100
92
8
0
0
040
168 hr
100
100
8
0
0
0 36
above 1 ppm, while the 48-hour LC5o was estimated
to be slightly higher than 0.5 ppm. Only 2 of 25 worms
had died at the 0.25 ppm concentration level afterthe
full 7-day test period.
Results of the definitive test for mortality at the 1.0
and 0.3 ppm levels were similar to those of the range-
finding test results (Table 4). However, there was a
considerable difference between the results of the
two tests for the 0.5 ppm concentration after 48
hours. This discrepancy may be due to differences m
the dilution water used in the two tests.
The largest decrease in the estimated LC50 as
generated from definitive test data occurred between
24 and 48 hours from initiation. The rate of decrease
in estimated LC5o levels beyond 72 hours was low,
and indicates that where Na-PCP concentration is
sufficient to cause- substantial acute mortality, the
effects are largely apparent within 2 to 3 days. This
finding may be important for the use of L variegatus
in biological screening, where the necessity for a
longer test period would both increase cost and delay
results.
None of the chi-square values for precision of fit to the
probit line for the LC5o definitive test data showed
statistical significance at the 0.05 probability level,
that is, the probit model appears appropriate to these
data. However, the confidence interval tends to
narrow and the chi-square value decreases with
increasing time of exposure, suggesting greater
predictability of test organism response to Na-PCP
after longer exposure periods.
Figure 1 shows that the slope of the probit regression
line increases with increase in exposure time That is,
as exposure time lengthens, increase m toxicant
concentration will result in greater increases in
mortality. A threshold LCso is not apparent from the
toxicity curve (Figure 2)
In 1968 Whitley, using dilution water with a pH of
7.5, found 100% mortality after 24 hours at 0.5 ppm
concentration of Na-PCP for Tubifex and Limnodnlus
as opposed to the 4% mortality for Lumbnculus
variegatus reported here (with dilution of pH 7.2 -
7.6). Our results more closely approximate those
obtained by Whitley with dilution water of pH 8.5 (24-
hour mortality of 11 %).
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TABLE 4 MORTALITY OF Lumbncu/us vanegatus TO SODIUM PENTHAHLOROPHENATE
No of Test
Organisms
25
25
25
25
25
25
25
25
25
25
Concentration
of NaPCP (ppm)
1 0
09
08
07
06
05
04
03
02
00
LCso, ppm, estimated from graph
LCso, estimated
95% confidence
by probit analysis1
limits of LCso
X2 value for goodness of fit to probit line
degrees of freedom for X2
24
48
Exposure time
72 96
(hours)
120
144
168
Percent mortality
36
32
16
16
12
4
0
0
0
0
88
44
52
28
32
8
0
0
0
0
96
76
84
56
52
20
0
0
0
4
100
100
96
76
52
40
4
0
0
8
100
100
96
84
68
40
8
0
0
8
100
100
100
96
88
44
20
0
0
8
100
100
100
100
96
68
28
0
0
8
LCso Results
1 22
—
—
—
—
079
080
074
087
9.66
5
063
065
060
069
6.62
5
056
057
053
060
4.55
5
055
054
051
058
1.17
5
049
049
046
052
2.18
4
044
045
042
048
0.96
3
1 Probit analysis was applied only for those time intervals yielding definitive results A definitive result must show graded responses at a
minimum of five concentrations, including at least one response at greater than 65% and at least one at less than 35% (USEPA 1 975)
98-
3 4 5 678910
Concentration of Na-PCP (ppm) - log scale
4 567
LCso (ppm) - log scale
9 1 0
1 2
Figure 1. Probit regression lines showing relation
of Lumbriculus variegatus mortality to Figure 2. Toxicity curve showing change in LCso as
concentration of Na-PCP (from Table 4). test proceeded (from Table 4)
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ECso levels for inactivity, estimated from the definitive
test data, diverged most from the LCso levels at
shorter exposures and higher toxicant concentration
levels (Tables 4-5). The lower EC5o levels underthese
conditions (in comparison to the LC5o levels) suggest
that the use of sublethal criteria may be most effec-
tive in increasing test sensitivity where quick results
are important and where relatively high
concentrations are involved Allchi-square valuesfor
precision of fit for the ECso data fell within acceptable
levels, except for the 24-hour exposure time (border-
line at the 0 05 probability level).
In general, differences among investigators in deter-
minations of mortality and inactivity were minimal,
although there were some discrepancies at higher
concentration levels for the shorter (48 hour) test
period (Tables 6-7) A thorough familiarization with
response criteria is recommended for improving the
reproducibility of results.
TABLE 5 INACTIVITY RESPONSE OF Lumbriculus vanegatus TO SODIUM PENTACHLOROPHENATE
No of Test
Organisms
25
25
25
25
25
25
25
25
25
25
Concentration
of NaPCP (ppm)
1 0
09
08
07
06
05
04
03
02
00
EC5o, ppm, estimated from graph
EC5o, estimated
95% confidence
X2
by probit analysis1
limits of ECso
value for goodness of fit to probit line
Degrees of freedom from X2
24
48
Exposure time
72 96
(hours)
120
144
168
Percent inactive
80
44
16
24
20
12
8
0
0
0
100
92
88
48
36
12
0
0
0
0
100
88
88
88
60
28
8
0
0
4
100
100
96
76
56
40
12
0
0
8
100
100
96
92
76
40
12
0
0
8
100
100
100
100
88
64
20
0
0
8
100
100
100
100
100
68
32
0
0
8
ECso results
088
091
082
1 07
12 6
6
066
066
062
069
520
5
0 59
057
053
061
475
6
055
055
052
059
3 43
5
052
052
049
055
045
5
047
047
044
050
085
3
045
—
—
—
—
'Probit analysis was applied only for those time intervals yielding definitive results A definitive result must show graded responses at a
minimum of five concentrations, including at least one response greater than 65% and at least one less than 35% (USEPA 1975)
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TABLE 6 MORTALITY OF Lumbnculus variegatus TO SODIUM PENTACHLOROPHENATE AS RECORDED FOR THE SAME SETS OF
ORGANISMS BY TWO INVESTIGATORS
No of Test Concentration
Organisms of NaPCP (ppm)
25
25
25
25
25
25
25
25
25
25
1 0
09
08
07
0.6
05
04
03
02
00
LCso, ppm, estimated from graph
LCso, estimated
analysis
95% confidence
by probit
limits of LCso
48 hr
Investigator
I II
144 hr
Investigator
I II
168 hr
Investigator
I II
Percent Mortality
88 48
44 40
52 44
28 28
32 32
8 8
0 0
0 0
0 0
0 0
079 089
— —
— —
100
100
100
96
88
44
20
0
0
8
100
100
100
96
88
44
20
0
0
8
LCso Results
049
049
046
052
049
049
046
052
100
100
100
100
96
68
28
0
0
8
0 44
045
042
048
100
100
100
96
92
68
20
0
0
8
0.47
047
044
050
8
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TABLE 7 INACTIVITY RESPONSE OF Lumbriculus vanegatus TO SODIUM PENTACHLOROPHENATE AS RECORDED FOR THE SAME
SETS OF ORGANISMS BY TWO INVESTIGATORS
No of Test
Organisms
25
25
25
25
25
25
25
25
25
25
Concentration
of NaPCP(ppm)
1 0
09
08
07
06
05
04
03
02
00
ECso, ppm, estimated from graph
ECso, estimated
analysis
95% confidence
by probit
limits of ECso
1
48 hr
Investigator
I!
144 hr
Investigator
I
II
168 hr
Investigator
I II
Percent Inactive
100
92
88
48
36
12
0
0
0
0
76
56
68
40
36
16
4
4
0
0
100
100
100
100
88
64
20
0
0
8
100
100
100
96
88
64
20
0
0
8
1 00 1 00
100 100
100 100
100 100
1 00 1 00
68 68
32 20
0 0
0 0
8 8
EC5o Results
066
066
062
069
076
075
068
083
047
047
044
050
048
047
044
050
045 046
— —
— —
i US GOVERNMENT PRINTING OFFICE 1981 -757-064/OaOO
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