TABLE 2. CONCENTRATIONS OF TRACE METALS AND OTHER

IONS IN COAL LEACHATES

Parameter 0.004 w/w 0.016 w/w
leachate leachate

Conductivity (ymhos/cm) 15-17 28-36
pH 7.0-7.4 7.4-7.7
Reactive phosphorus - <1
Nitrate nitrogen - 75-166
Lead <0.5 <0.5
Zine 0.4-2.1 0.6-2.0
Manganese <0.2 <0.2-0.5
Copper 0.1-0.6 <0.2-0.6
Cadmium <0.02 0.05-0.08
Chromium 0.2 <0.5
Iron 1.7-2.5 -
Nickel - <2.0

*
All element concentrations are in ug/l.

11


60
=
240
<
all
=i
>
=
a.
°
ae
© 20
<=
1S)

Figure 17,

C:CONTROL
FL: FILTERED LEACHATE

NITZSCHIA

3 4
DAY

Effects of 20% v/v leachate on the growth of Nitzschia
palea in Lake Superior water in batch cultures. Each
treatment had 3 replicate flasks. Error bars indicate
one standard deviation. Leachate conductivity was 71
ymhos/cm; turbidity ee 14 NTU.


A

240
® CONTROL 20ug/I P
OCONTROL 200ng9/1P
200 | OLEACHATE 20 y9/IP 4
@ LEACHATE 200yg/1 P i
= —6
[160+ aw 4
EY —
<i
3120F 4
: 9
a
9 sok 4
°
ba
& —
40r TM~ — 4
4 —._|, ~
$—s e
oO 1 1 L Ll L L
120 B
100+ — 24
r VA ?
<
Ff 80
z 2 —_—
G 60 4
Zz
we
w
a
=)
= t
2 20h 4
0 L 1 L 1 1 1 1
0 1 2 3 4 5 6
DAY

Figure 13, Effects of 20% v/v leachate on the growth of Selenastrum
capricornutum in Lake Superior water in 50%/day replacement
cultures, Test 1, Each treatment had 4 replicate flasks,
Error bars indicate one standard deviation, No error bars
are shown where the bar is smaller than the symbol. Leachate
conductivity was 76 wmhos/cm. A- Chlorophyll a; B- Photo-
synthetic capacity.

28


dominant species was not very different in distillate and control streams,
and most species appeared to be inhibited to an approximately equal degree.
An unexpected result of the cell count data was the finding that distillate
streams supported as many or more diatom species than control streams
(Table 9). However, many of the extremely rare species may have been rep-
resented only by dead frustules in both distillate and control streams.
Hence, their increased presence in distillate streams was likely due to an
increased probability of their being counted when total frustule counts were
radically reduced.

TABLE 8. RATIOS OF ASH-FREE DRY WEIGHT TO CELLULAR ORGANIC CARBON
IN STREAMS TREATED WITH 15% COAL DISTILLATE AND 15%
DISTILLED WATER

Mean for Control Mean for Distillate
Day Streams Streams
10 35.5 37.4
14 9.9 49.6
15 9.3 51.5
17 5.8 22.2

37


CEMENT BLOCK

Figure 1. Field bioassay enclosure system.


10.

11.

REFERENCES

American Public Health Association. 1975. Standard methods for the
examination of water and wastewater. 14th ed. APHA, New York.
1193 pp.

American Society of Testing and Materials. 1976. Annual book of ASTM
standards. Part 31, Sec. D, 5-12. ASTM, Philadelphia. pp. 274-5.

Atkinson, L.P., W.M. Dunstan, and J.G. Natoli. 1977. The analysis and
control of volatile hydrocarbon concentrations (e.g., benzene) during
oil bioassays. Water, Air, and Soil Pollution 8: 235-242.

Carlson, R.M., A.R. Oyler, E.H. Gerhart, R. Caple, K.J. Welch, H.L.
Kopperman, D. Bodenner, and D. Swanson. 1978. Environmental impli-
cations of polynuclear aromatic hydrocarbons liberated from northern
Great Plains coal. Final report. Environmental Research Laboratory,
U.S. Environmental Protection Agency, Duluth, Minn. 169 pp.

Chau, Y.K. and K. Lum-Shu-Chan. 1974. Determination of labile and
strongly bound metals in lake water. Water Research 8: 383-388.

Coward, N.A., J.W. Horton, R.G. Koch, and R.D. Morden. 1978. Static
coal storage--biological and chemical effects on the aquatic environ-
ment. Final report. Environmental Protection Agency, Duluth, Minn.
169. pp.

Dunstan, W.M., L.P. Atkinson, and J. Natoli. 1975. Stimulation and
inhibition of phytoplankton growth by low molecular weight hydrocarbons.
Marine Biology 31: 305-310.

Ellis, B.E. 1977. Degradation of phenolic compounds by fresh-water
algae. Plant Science Letters 8: 213-216.

Environmental Protection Agency. 1971. Algal assay procedure bottle
test. National Eutrophication Research Program, U.S. EPA. 82 pp.

Gerhart, D.Z., S.M. Anderson, and J. Richter. 1977, Toxicity bio-
assays with periphyton communities: design of experimental streams.
Water Research 11: 567-570.

Gerhart, D.Z. and T.E. Davis. 1978. Effect of heavy metals and sulfur

dioxide on phytoplankton. Final report. Minnesota Regional Copper-
Nickel Study, Minneapolis. 44 pp.

65


A-TEST 1, SELENASTRUM

NEOPRENE FOAM
25 DAY 3 I 4 100
20 ol al I a= 2°
ql \ ee at
steal Vi fal it 1h 460
\\ NY VN
\
og el te ee
oe Ve) he ent
\ \ \
i \ i Ney ou
\\ n NY N NN NH
a) ay \\ iN \\ 7? 3
3 YP PRU Pu] 2
= ° ul My NN NN Lilo 5
2 Zz
= DAY 4 I a
@ 125+ 100 ¥
(3) Fo
2
° I a
z w
5 100 480
NN al 3
VN AY =]
7 i i =
vs Pe aly
a iy Vy \\
rf tin ee et
50> Vt Ny NN \\ 440
Nog ool ft i it
\ \ \
att N \\ \ M20
NN NN iN iN ‘
NN NN yy wn uN
0 iN NN nN i! Nd,
CONTROL UNBUBBLED BUBBLED CONTROL UNBUBBLED
(0.168) (0.059) (0.168)

Figure 27A, Effects of 20% v/v unbubbled and bubbled distillate on the
growth of Selenastrum capricornutum in Lake Superior water
in batch cultures. Each treatment had 3 replicate flasks.
Error bars indicate one standard deviation. Numbers in
parentheses indicate absorbance values of distillate in
l-cm cells at 254 nm. Test 1 employed both neoprene and
foam stoppered flasks.

47


(ey

CONTROLS D: DISTILLATES
DAY 3 DAY 6

2% 5% 10% 2% 5% 10%

CELLS/ MLx 10°

1+ [Slmrc18 1912

Figure 25.

2% 5% 10% 2% 5% 10%

Effects of 2%, 5%, and 10% v/v distillate on the. growth
of Selenastrum capricornutum in synthetic nutrient
medium in 50%/day replacement cultures, Each treatment
had 5 replicate flasks. Error bars indicate one
standard deviation. Fresh distillate was used in Test
1. Distillate used in Test 2 had been stored under
refrigeration for one week.

45


ANABAENA

300 NEOPRENE FOAM 150
DAY 4
250 4125
nl
200° a| i n TY 4 100
NN Ni HH wi"
\
NN \
- \\ i \\ \
eo TP HH YP 7 fs
ny it TM WN WN =
ie ie aie)
100 > NN \\ WN ny WY 450 Nt
| it \\ i i ee
N nN \
m soh \\ iN Mt NN Ny dos 2
S \\ i nh \\ \ =
2 \ ut Nh NN 5
<9 nn nh WN NN ij, 7
3 DAY 5 8
x= wu
z dias
Oo 250 R25 av
5 o
2 6
= 3
2 200 400 =
I | *
a MN
150+ al ut dos
Nh Nh
scotm| ql i i so
in in = NN ] NN
Ny ut Ny NN NH
\ \
ore i i Hoge it as
Ny uw ul AN nN
° Ny N NH Ht No,
CONTROL UNBUBBLED BUBBLED CONTROL UNBUBBLED
(0.157) (0.113) (0.157)

Figure 29. Effects of 20% v/v unbubbled and bubbled distillate on the
growth of Anabaena flos-aquae in Lake Superior water in
batch cultures. Each treatment had 3 replicate flasks.
Error bars indicate one standard deviation. Numbers in
parentheses indicate absorbance values of distillates in
l-cm cells at 254 nm. Tests were conducted in both
neoprene and foam stoppered flasks.

51


A second field bioassay was conducted to verify these results and to
determine whether higher chlorophyll levels in distillate carboys were a
result of toxic effects of distillate on zooplankters. To test this possi-
bility, zooplankters were experimentally removed from one set of carboys
(Table 10) by filtering lake water through an 80-» plankton net. The net
was fitted over the sampling tube prior to pumping lake water into the car-
boys both at the time of the initial filling and during the daily replace-
ment procedure. Coal distillate used in this experiment had a pH of 4.8
and a conductivity of 25 ymhos/cm. Chlorophyll a analyses indicated con-
sistently lower pigment concentrations in control than in treatment car-
boys on days 5, 7, and 9 (Figure 31). That this effect was observed even
in filtered carboys (Figure 31B) suggests that reduced zooplankton grazing
in distillate carboys was not the causative factor. Bacterial counts
yielded results similar to those of the first experiment (Table 11), and
ATP analyses for unfiltered carboys showed higher biomass levels in dis-
tillate carboys (Figure 32). Distillate carboys exhibited somewhat lower
pH's (6.4-6.5) than did control carboys and the lake (7.0).

55


£y

o

‘o
=
ta
a4
=
~
an
S
wu 2
rw) Cc
0)
Figure 24,

C:CONTROLS DD: DISTILLATES
TEST 1 TEST 2

H
iw]

DAY

Effects of 20% v/v distillate on the growth of Selenastrum capricornutum
in synthetic nutrient medium in 50%/day replacement cultures. Each

treatment had 3 replicate flasks. Error bars indicate one standard
deviation.



TABLE 4. ABSORBANCE VALUES (1-CM CELLS) FOR COAL DISTILLATE
BEFORE AND AFTER VARIOUS PERIODS OF BUBBLING

Wavelength Bubbling Time (min)
(nm) 0 0.5 1.0 3.0 5.0 15.0
200 0.780 0.753 0.713 0.650 0.602 0.504
220 +679 +663 +633 -587 +540 +425
230 +354 +346 +333 -301 +285 +220
240 +297 291 +286 +273 +252 +210
254 496 487 +473 +455 +423 +342
546 002 004 » 003 +003 -002 ~006

TABLE 5. EFFECTS OF A 30-MIN BUBBLING PERIOD ON COAL
DISTILLATE pH AND CONDUCTIVITY

pH Conductivity (umhos/cm)
Sample Initial Final Initial Final
1 4.9 6.5 14.8 7.9
2 4.6 6.4 16.7 7.2

15


tissue analysis. Communities were allowed to develop in six streams for 26
days. Following this period, three streams received 12% v/v coal distillate
and three received 12% distilled water for 10 days. All streams were
enriched with 65 yg/l POy-P and 456 yg/1 NO3-N. On day 33 single substrate
plates were sampled from each stream, and on day 36 all remaining plates
(ten from each stream) were’ removed and combined into a single composite
sample for distillate streams (30 plates) and a single composite sample for
control streams (30 plates). After dilution to 2,000 ml, 100 ml was removed
from each sample for cell counts and other biomass measurements. The
remaining 1,900 ml was centrifuged twice at 15,000 rpm in a continuous-flow
centrifuge and the resulting pellets prepared for tissue analysis (see
Chemical Analyses).

Stream temperatures averaged 9-12°C during the experiment. No differ -
ences in pH or conductivity were observed between treatment and control
streams. Chlorophyll a determinations and cell counts suggested somewhat
less growth in distillate than control streams (Table 6, Figure 18).
Nitzschia spp. were significantly more abundant (95% level) in control than
in distillate streams. Tissue analysis showed four peaks in the aliphatic
fraction present in the distillate-exposed periphyton which were absent in
control periphyton (Figure 19). Also, one of the n-alkanes was present in
much higher concentration in the exposed sample. In the aromatic fraction
there was a general correspondence of peaks in the distillate and control
algal samples, but the quantities of several compounds varied considerably.
There was no evident correspondence between dominant peaks in coal distil-
late and accumulated peaks in exposed periphyton cells. The two periphyton
samples were similar in lipid content: 20.5% for exposed algae and 20.3%
for control algae.

TABLE 6. CHLOROPHYLL A (ug/em?) IN STREAMS TREATED WITH 12%
COAL DISTILLATE AND 12% DISTILLED WATER FOR 10 DAYS

Day 33 Day 36
Treatment Mean Std. Dev. Mean*
12% Coal 1.6 0.2 2.2
Distillate
12% Distilled 2.7 0.7 3.1
Water

*
Means for duplicate determinations on composite samples.

33


Further study is necessary to determine the extent of the problem of
algal bioaccumulation of organics from coal. Our work is in agreement with
that of Thompson and Eglinton (1976) with crude oil in suggesting that
diatoms accumulate aliphatics from fossil fuels. However, algal uptake of
aromatics upon exposure to specific aromatic compounds has also been demons-
trated (Payer and Soeder, 1975; Soto et al., 1975b; Walsh et al., 1977), and
additional work might demonstrate the accumulation of aromatics from coal
volatilization products.

Several problems arise in using the results reported here to predict
the environmental effects of coal storage facilities. Our results are
reported in terms of volume percent additions of standard leachates and
distillates. Yet these "standard" test materials exhibited considerable
variation in physical and chemical characteristics, depending on variation in
coal samples from which they were prepared. Coal samples obtained from
different western mines differed in their products of leaching and volatil-
ization. In addition, the resemblance of our leachates and distillates to
materials actually entering the aquatic environment from coal piles is
unknown. Considering the very high coal/water ratios existing in coal
storage piles, it seems likely that concentrations of dissolved and
particulate materials in our laboratory-prepared leachates and distillates
were unrealistically low. There is a need for site-specific studies of
coal storage facilities which are designed to assess both the quality and
quantity of runoff.

64


FOREWORD

Our nation's fresh waters are vital for all animal and plant life, yet
our diverse uses of water -- for recreation, food, energy, transportation,
and industry -- can physically and chemically alter lakes, rivers, and streams.
Such alterations threaten terrestrial organisms, as well as the aquatic ones.
The Environmental Research Laboratory in Duluth, Minnesota, develops methods,
conducts laboratory and field studies, and extrapolates research findings
from both inhouse and extramural projects

-- to determine how physical and chemical pollution effects aquatic
life;

-- to assess the effects of ecosystems on pollutants;

-- to predict effects of pollutants on the ecosystems through use
of models; and

-- to measure the rate of uptake and bioaccumulation of pollutants
in aquatic organisms that are consumed by other animals, including
man.

A comprehensive program was designed in 1974 because of the "Energy
Crisis" of 1973 to study the adverse effects on the aquatic environment
being created by new energy sources and technologies. Data being reported
in this grant are a part of the comprehensive program and deal with the
possible effects of coal storage on periphyton.

Laboratory periphyton communities were generally stimulated by coal
leachates and inhibited by coal distillates. Whereas distillates and
leachates were toxic to algae in bottle tests. In contrast, in situ
experiments with natural phytoplankton and coal distillates indicated
stimulation.

These findings will be integrated with other studies to aid elected
officials in making environmentally sound decisions on future energy
developments.

J. David Yount, Ph.D.
Acting Director
Environmental Research Laboratory~Duluth

aii


22.

23.

24.

25.

26.

27.

28.

29,

30.

REFERENCES (CONT.)

Payer, H.D. and C.J. Soeder. 1975. Accumulation of polycyclic aromat-
ic hydrocarbons in cultivated microalgae. Naturwissenschaften 62:
536-537.

Prouse, N.J., D.C. Gordon Jr., and P.D. Keizer. 1976. Effects of low
concentrations of oil accommodated in sea water on the growth of uni-
algal marine phytoplankton cultures. Journal of the Fisheries Research
Board of Canada 33: 810-818.

Pulich, W.M., Jr., K. Winters, and C. Van Baalen. 1974. The effects
of a no. 2 fuel oil and two crude oils on the growth and photosynthesis
of microalgae. Marine Biology 28: 87-94.

Rudd, J.W.M. and R.D. Hamilton. 1973. Measurement of adenosine tri-
phosphate (ATP) in two Precambrian Shield lakes of Northwestern Ontario.
Journal of the Fisheries Research Board of Canada 30: 1537-1546.

Samuelsson, G., G. Oquist, and P. Halldal. 1978. The variable chloro-
phyll a fluorescence as a measure of photoshythetic capacity in algae.
Mitt. Internat. Verein. Limnol. 21: in press.

Schindler, D.B., B.F. Scott, and D.B. Carlisle. 1975. Effect of. crude
oil on populations of bacteria and algae in artificial ponds subject to
winter weather and ice formation. Verh. Internat. Verein. Limnol.
19: 2138-2144.

Slovacek, R.E. and P.J. Hannan. 1977. In vivo fluorescence determina-
tions of phytoplankton chlorophyll a. Limnology and Oceanography 22:
919-924.

Soto, C., J.A. Hellebust, T.C. Hutchinson, and T. Sawa. 1975. Effect
of naphthalene and aqueous crude oil extracts on the green flagellate
Chlamydomonas angulosa. I. Growth. Canadian Journal of Botany 53:
109-117.

Soto, C., J.A. Hellebust, and T.C. Hutchinson. 1975. Effect of naph-
thalene and aqueous crude oil extracts on the green flagellate
Chlamydomonas angulosa. II. Photosynthesis and the uptake and release
of naphthalene. Canadian Journal of Botany 53: 118-126.

67


12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

REFERENCES (CONT.)

Gordon, D.C., Jr. and N.J. Prouse. 1973. The effects of three oils on
marine phytoplankton photosynthesis. Marine Biology 22: 329-333.

Holm-Hansen, O. and C.R. Booth. 1966. The measurement of adenosine
triphosphate in the ocean and its ecological significance. Limnology
and Oceanography 11: 510-519.

Ishio, S., K. Kawabe, and T. Tomiyama. 1972. Algal cancer and its
causes. I. Carcinogenic potencies of water and suspended solids dis-
charged into the River Ohmuta. Bulletin of the Japanese Society of
Scientific Fisheries 38: 17-24.

Ishio, S., H. Nakagawa, and T. Tomiyama. 1972. Algal cancer and its
causes. II. Separation of carcinogenic compounds from sea bottom mud
polluted by wastes of the coal chemical industry. Bulletin of the
Japanese Society of Scientific Fisheries 38: 571-576.

Kauss, P., T.C. Hutchinson, C. Soto, J. Hellebust, and M. Griffiths.
1973. The toxicity of crude oil and its components to freshwater algae.
In: Proceedings of Joint Conference on Prevention and Control of Oil
Spills, March 13-15, Washington, D.C. pp. 703-714.

Kauss, P.B. and T.C. Hutchinson. 1975. Studies on the susceptibility
of Ankistrodesmus species to crude oil components. Verh. Internat.
Verein. Limnol. 19: 2155-2164.

Mattson, J.S., C.A. Smith, T.T. Jones, S.M. Gerchakov, and B.D. Epstein.
1974. Continuous monitoring of dissolved organic matter by UV-visible
photometry. Limnology and Oceanography 19: 530-535.

Mrkva, M. 1969. Investigation of organic pollution of surface waters
by ultraviolet spectrophotometry. Journal of the Water Pollution
Control Federation 41: 1923-1931.

Niemi, A. 1972. Effects of toxicants on brackish-water phytoplankton
assimilation. Commentat. Biol. 55: 1-19.

Parsons, T.R., W.K. Li, and R. Waters. 1976. Some preliminary obser-

vations on the enhancement of phytoplankton growth by low levels of
mineral hydrocarbons. Hydrobiologia 51: 85-89.

66


TECHNICAL REPORT DATA

(Please read Instructions on the reverse before completing)

1, REPORT NO. z
EPA-600/3-80-051

3. RECIPIENT'S ACCESSION NO.

4. TITLE ANO SUBTITLE
Algal Bioassays with Leachates and Distillates from
Western Coal

Is. REPORT DATE

Ma:
6, PERFORMING ORGANIZATION CODE

1980 issuing date

7. AUTHOR(S)
David Z. Gerhart, Joseph E. Richter, Sidney J. Curran,
and Thomas E. Robertson

8, PERFORMING ORGANIZATION REPORT NO.

9, PERFORMING ORGANIZATION NAME AND AODRESS
Department of Biology and Lake Superior Basin

70. PROGRAM ELEMENT NO.
1NE625

Studies Center
University of Minnesota-Duluth
Duluth, Minnesota 55812

Grant No.

TT. CONTRACT/GRANT NO.

R803932

12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Duluth, MN

13, TYPE OF REPORT AND PERIOD COVERED

Office of Research and Development

U.S. Environmental Protection Agency
Duluth, Minnesota 55804

14. SPONSORING AGENCY CODE

EPA/600/03

15. SUPPLEMENTARY NOTES

76. ABSTRACT

of materials derived from coal storage piles.
prepared in the laboratory from low-sulfur Montana coal.
bioassays were conducted:

composition changes in response to coal leachates.
inhibited growth,

distillates.

The objective of this research was to assess the effects on freshwater algae
Coal leachates and distillates were
Three types of algal

1) A laboratory stream facility was constructed which supported periphyton
communities of 50-80 species growing on artificial substrates.
communities generally showed stimulation of growth and some species

These

Coal distillates

Periphyton exposed to distillates accumulated aliphatic

hydrocarbons.

2) Short-term laboratory bottle tests with test species of algae generally
showed growth inhibition in response to leachates and distillates. When
distillates were bubbled to remove volatile organic compounds, growth
stimulation was observed.

3) Three in situ experiments in a small lake were conducted with coal

Increases in algal biomass and bacterial populations in
distillate-treated enclosures were observed in each of these tests.

fh? KEY WORDS AND DOCUMENT ANALYSIS

a. DESCRIPTORS b.IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Coal Energy Periphyton DCMU 06/A
Leaching Phytoplankton 06/C
Volatilization Diatoms 06/F
Algal bioassay Selenastrum 06/M
Field bioassay Anabaena 06/T
Laboratory streams Nitzschia 08/H
Lake Superior Chlorophyll

118. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) ‘21. NO. OF PAGES

UNCLASSIFIED 7
20. SECURITY CLASS (This page) 22. PRICE
RELEASE TO PUBLIC UNCLASSIFIED

PREVIOUS EDITION IS OBSOLETE

~ 69

EPA Form 2220-1 (Rev. 4-77)

‘US. GOVERNMENT PRINTING OFFICE: 1980-657-146/5691



9%

CELLS/ ML x 10°

NS

C:CONTROL CL: CENTRIFUGED LEACHATE

FL: FILTERED LEACHATE

6~= 7

TEST 1

wW

FL

TEST 2

al

|

TEST 3

Figure 12. Effects of 20% v/v leachate on the growth of Selenastrum capricornutum in
synthetic nutrient medium in batch cultures, day 6, Each treatment had
Error bars indicate one standard deviation.

6 replicate flasks.



and small but significant quantities of dissolved organic and particulate
phosphorus (Table 3). The pH of leachates was always between 7.0 and 8.0.

Direct GC analysis of a methylene chloride extraction of a 2-1 sample of
membrane filtered leachate showed that few non-polar organic compounds were
present and that concentrations of these compounds were extremely low (Figure
3). Bubbling leachates with filtered air was not effective in reducing the
concentrations of organic compounds. The UV absorption spectrum for fil-
tered leachates showed gradually decreasing absorbance values as the wave-
length was increased from 210 nm. There were no absorbance peaks in the UV
spectrum. It is likely that absorbances in the UV were due largely to the
presence of extremely fine particles (<0.45 diameter) in filtered
leachates. Similarly, most of the total organic carbon (TOC) content of fil-
tered leachates was probably due to these particulates rather than to the
presence of dissolved organic compounds. TOC values for membrane filtered
leachates averaged 19 mg/1, while a single measurement of a centrifuged (un-
filtered) leachate sample yielded a TOC value of 78 mg/l.

COAL DISTILLATES

All coal distillate was prepared by distilling 4 1 of double-distilled,
deionized water containing 1,000 g ground, unsieved coal and collecting the
condensate. Coal distillate prepared in this manner had a conductivity of
5-30 umhos/em, a pH of 4.5-5.5, and a total phosphorus content of approxi-
mately 1 ug/l. A single batch of distillate was found to have a phenol
concentration of 0.24 mg/l.

The organic carbon content of coal distillates varied considerably with
different coal samples. Chemical oxygen demand (COD) values as low as 15
and as high as 127 mg/l were recorded, while TOC ranged from 5-34 mg/1. The
UV absorption spectrum for distillates had a minimum at 236 nm and a maximum
at 254-256 nm. Below 236 nm, absorbance increased rapidly. Most of the
organic compounds present were highly volatile. When coal distillate was
bubbled with filtered air in 250-ml Erlenmeyer flasks, UV absorbances dec-
reased significantly, and this decrease was greater at lower wavelengths than
at 254 nm (Table 4). Swirling flasks of distillate on a mechanical shaker
and warming the samples on a hotplate were also observed to reduce UV absor-
bances. In addition, some loss of organic compounds occurred when distillate
was stored, even when tightly capped and refrigerated. Bubbling also affect-
ed distillate pH and conductivity (Table 5). The pH of distillate was near-
ly two units higher after bubbling for 30 min, while the conductivity
dropped to approximately half that of the unbubbled sample.

Fractionation and GC analysis of a 1-1 sample of coal distillate indi-
cated that the predominant organics were aliphatics, with lesser concentra-
tions of aromatic and polar compounds (Figure 4). Direct GC analysis of a
methylene chloride extraction of a 2-1 distillate sample showed the predomi-
nance of early-eluting (volatile) organic compounds. Most compounds appeared
on the chromatogram before the temperature reached 150°C during the GC tem-
perature program. GC analysis of bubbled distillate showed decreased concen-
trations of many non-polar organic compounds relative to unbubbled distil-

12


SECTION 5

EXPERIMENTAL PROCEDURES AND RESULTS

COAL LEACHATES

Laboratory Streams

Early experiments with extremely low concentrations of coal leachate
showed no effects on periphyton growth in the laboratory streams (Gerhart et
al., 1977). However, three experiments performed with 3.2% v/v leachate
suggested some stimulation of periphyton growth by leachates. These tests
were conducted at water temperatures ranging from 11-16°C and lasted from
18-24 days. The first was conducted in June, the second in July, and the
third in September 1976.

With few exceptions, chlorophyll a concentrations were higher in
leachate streams than in controls (Figure 6). When cell counts were per-
formed, these showed increases in diatom numbers in leachate streams (Figure
7). In the September experiment (Figure 7B) a species dominance shift was
observed in leachate streams from Achnanthes microcephala to Synedra spp.
This experiment also included a treatment of leachate plus 1 mg/1 Cu to
test the possibility that coal particles settling on the substrates in
leachate streams might interfere with chlorophyll determinations. No signi-
ficant interference was found (Figure 6C).

Visual differences between treatment and control streams were evident
in these experiments. Substrates in leachate streams had a definite dark
brown color relative to controls, due both to heavier algal growth and
sedimentation of coal particles.

Experiments conducted with higher concentrations of coal leachates also
indicated growth stimulation. One experiment was conducted with 17% v/v
leachate in December 1976 and January 1977 when stream water temperatures
were 8-10°C. The conductivity of the leachate used in this run averaged 239
wmhos/cm, and leachate streams had somewhat higher conductivities (avg. = 111
umhos/cm) than control streams (avg. = 95 umhos/cm). The leachate had a
suspended solids concentration of approximately 134 mg/1. Growth was
initially slow in all streams, and on the 23rd day of the experiment the
streams were enriched on a continuing basis with 5 ug/l POy-P. Growth was
tapid following this enrichment, with leachate streams showing higher values
for chlorophyll a and ash-free dry weight than controls (Figure 8). Changes
in species composition were also evident (Figure 9). Leachate streams
showed decreased importance of Nitzschia palea and Fragilaria spp.

18


F.1D. RESPONSE

TIME (MINUTES)

Figure 3. GC analysis of a 2-1 sample of membrane filtered leachate.

A- Membrane filtered distilled water blank; B- Membrane filtered
leachate.

14


particulates and were more inhibitory than filtered leachates. The conduc-
tivities of centrifuged and filtered leachates were similar. Algae exposed
to leachates exhibited abnormal cell structure when viewed under the light
microscope.

Two 50%/day replacement experiments with 20% v/v membrane filtered
leachate were conducted with Selenastrum growing in enriched Lake Superior
water. Phosphorus enrichment of the lake water was 5, 20, or 200 ug/l POy-P.
Light intensity in these and subsequent experiments was 3,700 lux. Fifty
percent of the culture volume was replaced daily, and chlorophyll a was
determined by in vivo fluorescence measurements after the addition of DCMU.
The percent increase in fluorescence after the addition of DCMU was used as
a measure of photosynthetic capacity. Leachates were prepared from two
different coal samples, originating in different strip mines. The conduc-
tivity of the leachate used in the first test was 76 wmhos/cm, while that in
the second test was 275 umhos/cm. In every case leachate cultures achieved
much lower chlorophyll levels than control cultures (Figure 13 and 14). Only
slightly less inhibition was observed in cultures receiving the higher P
additions. Percent increases in fluorescence after DCMU addition also indi-
cated a reduction of photosynthetic capacity in leachate cultures. This
trend was especially pronounced in the second test.

Two replacement tests were conducted simultaneously with v/v concen-
trations of 5, 10, and 20% filtered leachate. The test alga was Selenastrum
capricornutum in the first test and Nitzschia palea in the second. The
culture medium was filtered lake water enriched with 200 ug/1 PO,-P.
Leachate conductivity was 143 ymhos/cm; turbidity was 11-12 NTU. Growth
inhibition was observed at all concentrations with Selenastrum and at the 10
and 20% concentrations with Nitzschia (Figure 15). Differences in photo-
synthetic capacity between treatment and control flasks were generally not
significant in these tests.

Phosphorus enriched (200 ug/l PO,-P) lake water was also used as the
growth medium for four batch tests with 20% v/v filtered leachate. When
leachates having conductivities of 103-132 wmhos/cm and turbidities of 3-5
NIU were tested, no significant growth inhibition of Selenastrum or Nitzschia
was observed (Figure 16). Anabaena flos-aquae may have been inhibited
slightly by this treatment. However, a leachate having a conductivity of
71 ymhos/cm and a turbidity of 14 NTU did inhibit the growth of Nitzschia
(Figure 17). Significant differences in photosynthetic capacity between
treatment and control flasks were not observed in any of these tests.

COAL DISTILLATES

Laboratory Streams

Several stream bioassays were performed in which periphyton communities
were exposed to 10-20% v/v coal distillate for periods of 1-10 days. All of
these experiments suggested some inhibition of periphyton growth by distil-
lates, but the results were usually inconclusive. One such experiment was
conducted in February and March, 1977, primarily to obtain algal cells for

27


lipid analysis, and 5 ml were transferred to a silicic acid colum. Ali-
phatics, aromatics, and polar compounds were collected with 12-ml washes
using hexane, benzene, and methanol, respectively. The benzene fraction was
transferred to a Florisil column and eluted with 50 ml hexane to remove
lipids. The hexane and benzene fractions were concentrated in a Kuderna-
Danish evaporator, and 4 yl fractions were injected into a Varian 1700 gas
chromatograph (GC).

Coal distillate and leachate were also analyzed by GC. A 1-1 sample
was extracted for 2 h with 50 ml of 50:50 methylene chloride/hexane. The
solvent layer was removed, run through sodium sulfate, and concentrated in a
Kuderna-Danish evaporator. The 1-ml concentrate was then added to a silicic
acid column and eluted with hexane, benzene, and methylene chloride. GC
analysis was as described for algal tissue except that no Florisil cleanup
was used. Blanks were performed on the silicic acid column, and no conta-
mination was found. A simplified method was used to analyze 2-1 samples of
leachate and unbubbled and bubbled distillate. These samples were extracted
with 40 ml methylene chloride for 2 h. The solvent layer was removed, and
5 ul sub-samples were injected directly into the GC.

For all samples the GC was equipped with a flame ionization detector
(F.I.D.) and a 2 m x 2 mm i.d. glass column packed with 3% OV-101 on 80-100
mesh Gas Chrom Q (injector T = 250° » detector T = 300 °c). The column was
programmed from 100-250 C at 4 /min for the tissue analyses and 1-1 dis-
tillate sample and from 60-200°C at 4°/min for the 2-1 leachate and unbubbled
and bubbled distillate samples.


31.

32.

33.

34.

35.

36.

37.

REFERENCES (CONT.)

Stadelmann, P. 1974. Biomass estimation by measurement of adenosine
triphosphate. In: R.A. Vollenweider (ed.), A manual on methods for
measuring primary production in aquatic environments. Blackwell,

London. pp. 26-30.

Strickland, J.D.H. and T.R. Parsons. 1968. A practical handbook of
seawater analysis. Bulletin 167, Fisheries Research Board of Canada,

Ottawa. 311 pp.

Thompson, S. and G. Eglinton. 1976. The presence of pollutant hydro-
carbons in estuarine epipelic diatom populations. Estuarine and Coastal
Marine Science 4: 417-425.

Vandermeulen, J.H. and T.P. Ahern. 1976. Effect of petroleum hydro-
carbons on algal physiology: review and progress report. In: A.P.M.
Lockwood (ed.), Effects of pollutants and aquatic organisms. Cambridge
University Press. pp. 107-125.

Vollenweider, R.A. (ed.) 1974. A manual on methods for measuring
primary production in aquatic environments. IBP Handbook No. 12, 2nd
edition. Blackwell, London. 225 pp.

Walsh, G.E., K.A. Ainsworth, and L. Faas. 1977. Effects and uptake of
chlorinated naphthalenes in marine unicellular algae. Bulletin of
Environmental Contamination and Toxicology 18: 297-302.

Winters, K., R. O'Donnell, J.C. Batterton, and C. Van Baalen. 1976.

Water-soluble components of four fuel oils: chemical characterization
and effects on growth of microalgae. Marine Biology 36: 269-276.

68


A (ug/l)
BS

— =
oO N

CHLOROPHYLL
©

Figure 30.

a
@ J DISTILLATE TREATED

5 ]eontrots
® -LAKE

Effects of 5% v/v distillate on Clearwater Lake phytoplankton,
September 1977.

53


SECTION 1

INTRODUCTION

Increasing reliance on the use of coal as an energy source will
necessarily involve the shipment and storage of extremely large quantities
of this fuel. Much of the coal burned in the western and midwestern United
States is low-sulfur coal derived from recently expanded mining activities
in Montana, Wyoming, and North Dakota. For example, in Superior, Wisconsin,
a new transshipment facility has been built on a 200-acre (81-hectare) site
to help meet the energy needs of southeastern Michigan. Coal is shipped by
rail from Decker, Montana, to the Superior facility where it is stored in
open air holding piles. Eventually it is transferred to lake-going vessels
and carried to power plants in Michigan. In all, approximately 200 million
tons of Decker coal will be delivered to Michigan power plants, with ship-
ments reaching 8 million tons per year by 1980.

Environmental hazards associated with the shipment and storage of this
coal are largely unexplored. In spite of sophisticated dust suppression and
water treatment systems, the movement of large quantities of coal will in-
evitably release coal dust into the atmosphere; and during times of high
rainfall or snowmelt, runoff from coal storage areas may enter receiving
bodies of water. Spontaneous heating within coal storage piles may also
release volatile organic compounds which eventually enter the aquatic
environment. Although recent work with marine algae indicates that low
molecular weight aromatic hydrocarbons (benzenes, xylenes, naphthalenes) are
the primary toxicants associated with fuel oils, no comparable studies have
been conducted with coal.

The objectives of this study were to assess the effects of coal-derived
contaminants on freshwater algae and to identify the types of compounds in
coal leachates and distillates which stimulate or inhibit algal growth.
Although coal for the study was obtained from the Superior transshipment
facility, no attempt was made to provide a site-specific or impact study of
this facility. Instead, algal bioassays were chosen to provide information
of a more widely applicable nature and to compare the results of different
bioassay procedures.


Vi4

CHLOROPHYLL A (g/cm?)

A
5 T T T T T T T 1000
. . -
ab a 4 £ 800
a a
3 3
A= :
3r Oo 4 w 600
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8 o x
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DAY
Figure 10.

<

L,

ro
i .
e ]eeactate TREATED
°
Q
O Jeowrons
®
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10 12 14 16
DAY

Effects of 17% v/v leachate on laboratory stream periphyton, September 1977.

A- Chlorophyll a; B- Ash-free dry weight.



Figure 34,

A

300+

100 F

500-
400
@
Q

S LAKE
8 ]controis

s ] DISTILLATE TREATED

250

CELLULAR ORGANIC CARBON (yg/I)

150

100

a __g 8
° . ,
®,

200 \ 4
; |
N

50 4
~ __.

Effects of 20% v/v distillate. on Clearwater Lake
planktonic biomass, June 1978.
organic carbon were calculated from ATP analyses.
A- Whole water samples; B- Water samples filtered

6 7 8 9
oay

through a 48-y net.

60

Values for cellular


determined. Our own analyses indicated that phenolic compounds were present
in both leachates and distillates. Since some algae are known to degrade
phenols (Ellis, 1977), a stream bioassay was performed using phenol concen-
trations of 0.35 and 1.1 mg/l. These concentrations are 2-7 times higher
than the concentration measured in a sample of coal leachate. No effect on
periphyton growth was found. Thus, the effects of dissolved organic com-
pounds in leachates are probably quite small, but a more complete investi-
gation of this topic is needed. For example, it is possible that the.pheno-
lic compounds in leachates are substituted forms which are more toxic than
the unsubstituted phenol tested in the stream bioassay or that the indige-
nous periphyton in the laboratory streams were able to utilize organics
more efficiently than the bacterial-algal association present in the bottle
tests.

Coal particulates in leachates are suspected of causing algal responses
in both stream and bottle bioassays. In the streams, particles of less than
1 wm diameter were observed to be consistently and intimately associated
with growing periphyton cells. The effects of these particles, perhaps in
combination with bacterial activity, are unknown. In bottle tests, the tox-
icity of membrane filtered leachates was apparently correlated with tur-
bidity and not with conductivity, suggesting an important role for extremely
fine (<0.45 ym) coal particles. Tests indicated that the effects of parti-
cles in leachates were not simply a result of light attentuation in either
stream or bottle bioassays. Adsorption or chelation of trace nutrients by
particulate or dissolved organics in coal could conceivably contribute to
reduced growth in bottle tests, but it is not clear why similar effects did
not occur in stream bioassays, nor is significant adsorption or chelation
likely in bottle tests employing the synthetic nutrient medium, Of interest
in this regard are studies conducted by Ishio et al. (1972a, b) of the marine
alga Porphyra tenera growing on sea bottom mud heavily polluted by wastes
discharged by the coal chemical industry. These authors reported that sus-—
pended solids in the mud are the cause of a cancerous disease of the alga.
In addition, Coward et al. (1978) found that coal particles from western
coal may diminish the growth of Lemna minor. Our data similarly suggest,
but do not prove, that suspended fine particulates in coal leachates are the
likely cause of algal growth stimulation in stream bioassays and of inhibi-
tion in bottle bioassays.

All of our leachate bioassays were conducted with centrifuged or fil-
tered leachates. Thus, uncontrolled runoff from coal storage piles could
result in concentrations of suspended solids many times higher than those
tested in these experiments. Reduction of light penetration and loss of
suitable substrates for periphyton growth are additional effects which could
become important under these conditions.

The bioassays with coal distillates are perhaps more easily interpreted
than those with leachates. Bottle bioassays in which both bubbled and un-
bubbled distillates were tested showed that volatile organics were almost
certainly responsible for the toxic effects of distillates, The majority
of the compounds volatilized from coal appear to be alkanes, alkylbenzenes,
and alkylnaphthalenes (Carlson et al., 1978). A frequent result was

62


toxicity at higher concentrations of volatile organics (unbubbled distil-
lates) and growth stimulation at lower concentrations (bubbled distillates).
Growth stimulation observed with bubbled distillates could be interpreted as
resulting from C02 introduced during the bubbling process rather than from
low concentrations of volatile organics (Figures 27 and 28), and we
recommend nitrogen gas for stripping organics in future work. However, since
total growth in foam stoppered control flasks was not significantly greater
than in neoprene stoppered control flasks, we believe this alternative inter-
pretation is unlikely. Our experiments also demonstrated species specific
differences in algal responses and the need to tightly stopper all bioassay
flasks containing volatile organic materials. Neoprene rubber stoppered
flasks often showed distillate toxicity while foam stoppered flasks were
similar to controls, a finding similar to that of Atkinson et al. (1977).

All of these results are supported by the now extensive literature dealing
with the effects of fuel oils and low molecular weight hydrocarbons on algae
(e.g-, Gordon and Prouse, 1973; Kauss et al., 1973; Pulich et al., 1974;
Dunstan et al., 1975; Kauss and Hutchinson, 1975; Soto et al., 1975a; Parsons
et al., 1976; Prouse et al., 1976; Winters et al., 1976). Low boiling point,
soluble aromatics are thought to be mainly responsible for the high initial
toxicity of crude oils. For example, Kauss et al. (1973) found marked sti-
mulation of Chlorella growth in some oil extracts after the loss of toxic
compounds through volatilization. These authors suggest that such stimu-
lation results from an ability of some algae to use hydrocarbons in oils as
metabolites or from by-products of bacterial metabolism (C02, NH3). However,
Vandermeulen and Ahern (1976) speculate that stimulation may instead be the
result of carcinogenic stimulatory activity.

Field bioassays with distillates showed slight growth stimulation.
The failure of distillates to inhibit phytoplankton growth in Clearwater
Lake may be explained partly by the fact that the field enclosures were
routinely bubbled with air to ensure mixing prior to sampling. However, UV
absorbance measurements before and after bubbling indicated that loss of
volatiles as a result of this treatment was slight. Increased bacterial
populations in distillate enclosures may also have contributed to reduced
distillate toxicity in the field. Similar increases of algae and bacteria
in response to crude oil have been reported for freshwater ponds (Schindler
et al., 1975). The detailed interpretation of the ATP analyses in these
experiments is complicated and not fully understood. Increases in ATP in the
second bioassay probably reflect increases in bacteria or rotifer growth
(Figure 32), while declines in the third bioassay appear to be related to
zooplankton mortality (Figure 34).

In the vicinity of coal storage piles, the effects of volatile organics
will depend on both temperature and the opportunity for evaporative loss, as
both of these variables will affect the rate of volatilization of toxic
compounds (Vandermeulen and Ahern, 1976). As in the case of oil pollution
(Prouse et al., 1976), the toxic effects of hydrocarbons from coal are like-
ly to be short-lived unless inputs are maintained; and the interpretation
of bioassay results is complicated by changing composition and concentrations
of hydrocarbons with time, so that it is “impossible to ascribe a measured
effect to a specific hydrocarbon’or concentration."

63


1?

ag -
e JLEACHATE TREATED

QO

ie}

|contaots
°o

Z

“A

Sx!
—

A
3.0FT T T T T T T 1000
a .
JUEACHATE TREATED
° °
25+ 0 4
fo) ]contaots 800 ;-
° IN
=
‘a —
20h oo 4 $
z 3
5 ~ 600-
~ =x
= O° 6
rd
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) w 400F
5 £
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.
° 200 +
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32 34~«O368stC=<CHSC“‘a‘SC SSC
DAY
Figure 8.

40
DAY

42

Effects of 17% v/v leachate on laboratory stream periphyton, December 1976 and January

1977.

A- Chlorophyll a; B- Ash-free dry weight.


laboratory streams and the short-term bottle tests is not known. There is a
need to critically examine different algal bioassay techniques to help
resolve such inconsistencies in bioassay results.

This report was submitted in fulfillment of Grant No. R803932 by the
University of Minnesota under the sponsorship of the U.S. Environmental
Protection Agency. This report covers the period from July 15, 1975, to
July 14, 1978, and work was completed as of July 14, 1978.


ABSTRACT

The objective of this research was to assess the effects on the aqua-
tic environment of materials derived from coal storage piles, and specifi-
cally, the effects of these materials on freshwater algae. Coal leachates
and distillates were prepared in the laboratory from low-sulfur western coal
from Montana. Leachates prepared from this coal had conductivities of 70-
350 ymhos/cm and contained appreciable quantities of sodium and sulfate ions,
organic phosphorus, and coal dust. The pH values for leachates varied
between 7.0 and 7.7, and concentrations of metals and volatile organics were
low. Coal distillates, prepared by boiling a coal/water mixture and collect-
ing the condensate, had conductivities of 5-30 wmhos/cm, pH values of 4.5-
5.5, and contained volatile organic compounds. Total organic carbon in dis-
tillates ranged from 5-34 mg/l.

Three types of bioassays were conducted to determine the effects of
coal leachates and distillates on algal growth:

1) A laboratory stream facility was designed and constructed which
supported periphyton communities of 50-80 species growing on artificial sub-
strates. These communities generally showed stimulation of growth and some
species composition changes in response to leachate concentrations of 3% v/v
and higher. Concentrations of 15-20% v/v distillate inhibited the growth of
stream periphyton. Periphyton exposed to distillates accumulated aliphatic
hydrocarbons.

2) Short-term laboratory bottle tests with test species of algae
generally showed growth inhibition with leachate and distillate concentra-
tions of 5-20% v/v. However, when distillates were bubbled to remove vola-
tile organic compounds, growth stimulation frequently was observed.

3) Three in situ experiments in a small lake were conducted with dis—
tillates at concentrations of 1, 5, and 20% v/v. Increases in algal biomass
and bacteria populations in distillate-treated enclosures were observed in
each of these tests. Although nearly all zooplankters were killed at dis-
tillate concentrations of 20%, reduced zooplankton grazing was probably not
the cause of the observed increases in algal biomass.

Different samples of coal produced leachates and distillates which were
highly variable in their characteristics. The stimulatory and toxic respon-
ses to coal distillates were apparently due to the presence of volatile or-
ganic compounds. Bioassays with these materials must be conducted and inter-
preted with care since many toxic compounds may escape to the atmosphere
when test containers are bubbled, agitated, or are not tightly sealed. In
the case of leachates, the reason for the differing algal responses in the

iv


SECTION 4
PREPARATION AND CHARACTERISTICS OF COAL LEACHATES AND DISTILLATES

All coal for the preparation of leachates and distillates was collected
from storage piles at the transshipment facility in Superior, Wisconsin.
Collections were made several times a year in clean polyethylene bags.
Because of the extremely large coal storage area at the facility and the con-
tinuous activity of bulldozers and conveyor belts, it was not possible to
obtain random or representative samples from the piles. Thus, the coal
samples used in this study were by no means uniform in their physical and
chemical characteristics.

COAL LEACHATES

Coal leachates for heavy metal analyses were prepared by pumping double-
distilled, deionized water through samples of ground coal which had been
sieved to remove particles smaller than 0.25 mm and greater than 2.0 mm
diameter. A Wiley mill and stainless steel sieves were used to grind and
sieve the coal. Coal samples were held in a plexiglass tube while a peris-
taltic pump circulated the water from a polyethylene reservoir through the
sample and back to the reservoir. The coal was leached continuously for
65 h, and the resulting leachate was filtered through washed membrane filters
(0.45 ») to remove suspended coal particles. Leachates were prepared using
coal/water weight ratios of 0.004 and 0.016. These leachates were quite low
in concentrations of trace metals and other ions (Table 2).

All other leachates and all leachates used in bioassays were prepared
using a coal/water weight ratio of 0.08. Ground, unsieved coal and double-
distilled, deionized water were added to a polyethylene carboy and mixed
continuously with an electric stirrer for at least 24 h. For most stream
bioassays this leachate was centrifuged in a continous-flow centrifuge
(Sorvall SS-3) at 12,000-14,000 rpm. In several stream bioassays the cen-
trifuged leachate was filtered through Whatman GF/F glass fiber filters,
and for most bottle tests the leachate was not centrifuged but was filtered
through membrane filters (0.45 1). Characteristics of these leachates are
reported in Table 3. In addition, a single measurement of complexing capa-
city indicated that 1 1 of leachate will complex 128 ug of copper ion.

Considerable variation in leachate characteristics was found among

leachates prepared from different batches of coal. However, most leachates
appear to contain relatively high concentrations of sodium and sulfate ions

10


ACKNOWLEDGEMENTS

The Support of the Duluth Environmental Research Laboratory, U.S.
Environmental Protection Agency, is gratefully acknowledged. Dr. Donald
Mount, Director, and Dr. Kenneth Biesinger provided research space and
equipment for the laboratory streams and useful discussions during the course
of the project.

We thank Dr. Robert Carlson and his staff, Department of Chemistry,
and Duane Long, Lake Superior Basin Studies Center, University of Minnesota,
Duluth, for providing chemical characterization of coal leachates and dis-
tillates and tissue analyses of algal cells.

Test algae for the bottle tests were supplied by William E. Miller,
Corvallis Environmental Research Laboratory, U.S. Environmental Protection
Agency. The enclosure bottles for the field bioassays on Clearwater Lake
were designed by Thomas E. Davis, Lake Superior Basin Studies Center,
University of Minnesota, Duluth.

viii


FD. RESPONSE

5
z
i}
a
g
=
a
=
to 5 OG,
° 10 20 30 40 0 10 20 30 40
TIME (MINUTES) TIME (MINUTES)
c
rm
|
Zz
9
a
&
r-
=|
J
tot po
° 10 20 30 40
TIME (MINUTES)
Figure 4. GC fractionation of coal distillate. A- Aliphatic

fraction; B- Aromatic fraction; C- Polar fraction.

16


United States Environmental Research EPA 600 3-80-051
Environmental Protection Laboratory May 1980
Agency Duluth MN 55804

Research and Development

@EPA Algal Bioassays With
Leachates and
Distillates From
Western Coal

>> >


CHLOROPHYLL A (g/cm?)

w

v.

A T T T T T T T T
r T T T ¥ T T oO 100 4
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September 1977.

n
14 16
DAY

12

calculated from ATP analyses; C- Ash-free dry weight.

39

Effects of 15% v/v distillate on laboratory stream periphyton,
A- Chlorophyll a; B- Cellular organic carbon


SECTION 3

BIOASSAY TECHNIQUES

LABORATORY STREAMS

A laboratory stream facility was constructed to test the effects of
coal leachates and distillates on periphyton communities growing on artifi-
cial substrates. This facility used raw Lake Superior water as "stream"
water and as a source of periphyton organisms. Gerhart et al. (1977) have
described in detail the initial design of the streams and also provided
information concerning the replication of species composition, community
structure, and biomass measurements. In some of the work presented here,
several aspects of the design and operation of the streams differed from
methods reported previously, and these are outlined below.

In August 1976, the stream facility was expanded to include twelve
streams. Lighting was provided by lines of 40-watt Gro-Lux wide spectrum
fluorescent lamps running parallel to the stream channels. Light intensity
was 4,000 lux at the surface of the water; current speed was 16 cm/sec. The
bottoms of the stream channels were insulated with foam sheeting.

In November 1976, the valves which had previously controlled lake water
flow to the streams were replaced with capillary tubes in two secondary
headboxes, each of which served six streams. These headboxes, polyethylene
pails of 4-1 capacity, were each fitted with six capillary tubes in their
bases and an overflow tube which maintained the water level above the
openings of the capillary tubes. The water from a capillary tube dripped
into an open funnel (to prevent air locks) and flowed through polyethylene
tubing to one of the streams. The flow in each tube was maintained at 16 ml/
min by adjusting its height in the headbox. The headboxes and capillary
tubes were blackened on the outside to prevent the growth of algae which
might obstruct water flow. This arrangement allowed accurate control of low
flows of incoming lake water, which in turn permitted higher concentrations
of leachates and distillates to be tested. A second modification was the
replacement of the unglazed porcelain growth substrates, used previously,
with 10 x 10-cm quarry tile plates of fired clay.

A final procedural change was initiated in August 1977 and concerned
the colonization of the substrates by periphyton organisms. In earlier
experiments periphyton algae in the incoming lake water were allowed to
extablish themselves naturally on the substrates. However, at low headbox
flow rates, colonization by this method proved to be slow and erratic,
leading to poor replication of experimental communities. To ensure an


SECTION 2

CONCLUSIONS AND RECOMMENDATIONS

The results of algal bioassays with laboratory prepared coal leachates
and distillates have indicated that uncontrolled runoff from coal storage
piles may pose a hazard to the aquatic environment. At coal storage facili-
ties leachates are produced as rainwater percolates through coal, while dis-
tillates are a likely result of spontaneous heating within coal piles. Both
types of materials produced inhibition and stimulation of algal growth, the
response depending on the concentrations of volatile organic compounds and
coal particulates, the species and growth habit of the test algae, and the
type of bioassay experiment. Zooplankters were more sensitive to coal dis-
tillates than phytoplankters, while bacterial growth was enhanced by dis-
tillates. Bioaccumulation of aliphatic compounds was observed in periphyton
cells exposed to coal distillates.

The algal bioassays suggest that dissolved, volatile organic compounds
and coal particulates are the most hazardous materials which may enter the
aquatic environment from coal piles. Additional information is needed to
quantify the amounts of these and other materials entering receiving waters
from various types of coal storage facilities and to determine the extent of
their bioaccumulation. In addition, more work is needed to resolve incon-
sistencies in the results obtained from different algal bioassay techniques.


Bubbling was effective in reducing the concentration of dissolved or-
ganics in distillate. Absorbance measurements at 254 nm decreased 28-65%
as a result of bubbling (Figures 27-29).

Field Bioassays

Two field bioassays with 1% and 5% v/v coal distillate were conducted
on Clearwater Lake during the fall of 1977. The lake was isothermal during
these bioassays, with temperatures dropping from 14°c to 6°C. The phyto-—
plankton community was dominated by Dinobryon spp. The experimental de-
sign and treatments tested are listed in Table 10.

TABLE 10. EXPERIMENTAL DESIGN FOR FIELD BIOASSAYS

Bioassay Dates Treatments No. of Carboys
per Treatment

1% distilled water

1 Sept. 28- 1% coal distillate 2
Oct. 11, 1977 5% distilled water
5% coal distillate

5% distilled water
5% coal distillate

Oct. 19- 5% distilled water,
2 Nov. 4, filtered 2
1977 5% coal distillate,
filtered
June 15- 20% distilled water
3 June 24, 20% coal distillate 2
1978

During the first bioassay no toxic effects of 1% or 5% v/v distillate
were observed. However, on days 5 and 7 chlorophyll a levels were some-
what higher in bottles treated with 5% distillate than in control bottles
(Figure 30). In addition, bacteria populations on day 13 were generally
higher in distillate carboys than in control carboys and higher in control
carboys than in lake water (Table 11). Nitrate and phosphate concentra-
tions, pH, and other chemical parameters were similar in treatment and
control carboys.

52


ALIPHATIC AROMATIC

A
Hy
mall
rn Il
22, ge
\
|
3
f-}
2 el
=
6
; \
‘ Lawl ° Leh
\
; | |
!
alt J
A)
o 10 20 30040 °o 10 20 300 «40
TIME (MINUTES) TIME (MINUTES)
Figure 19. GC analysis of periphyton cells exposed to

12% v/v distillate for 10 days. A- Coal
distillate; B- Distillate exposed periphyton;
C- Control periphyton.

35


and increased importance of Cymbella spp. and Nitzschia acicularis. These
trends were confirmed by cell counts on day 37. A statistical analysis (F-
test and t-test using log transformations) indicated that the means of all
these groups except Nitzschia palea were significantly different at the 95%
level in treatment and control streams. The fact that Nitzschia palea was
consistently less abundant in leachate streams on two sampling days makes it
seem likely that this species too responded to the presence of leachate.

A second experiment with 17% v/v leachate was conducted in September
1977. To be certain that stimulatory effects observed previously were not a
consequence of large amounts of particulates in coal leachate, the centri-
fuged leachates used in this experiment were filtered through Whatman GF/F
glass fiber filters, thus reducing the concentration of suspended solids to
approximately 22 mg/l. Stream water temperatures were 13-15°C, and all
streams were enriched with 12 ug/l POy4-P. The conductivity of control
streams averaged 94 ymhos/cm, while leachate streams averaged 102 umhos/cm.
Chlorophyll a and ash-free dry weight analyses again indicated higher peri-
phyton biomass in leachate streams (Figure 10). On day 17 two of the treat-
ment streams showed higher frustule counts than the control streams, but
differences in total counts were not significant (Figure 11). Non-diatoms
were not counted. Cymbella minuta var. minuta was significantly more
abundant in leachate than control streams; the reverse was true of
Tabellaria fenestrata.

Several generalizations may be made regarding the stream bioassays with
coal leachates. First, the pH of treatment streams was never significantly
different from control streams. The pH of coal leachates was generally
between 7.5 and 8.0, and this range is nearly identical to the pH range for
Lake Superior water. Second, while dominance shifts and changes in algal
species composition were observed in leachate streams, significant shifts
in community structure (determined by plotting log-normal curves) were never
detected. Third, total and total dissolved phosphorus concentrations were
usually greater in leachate than control streams. Soluble reactive phos-
phorus concentrations, however, were not consistently greater in leachate
streams.

Bottle Tests

Beginning in September 1977, a total of 11 smali-volume bottle tests of
the effects of coal leachates were conducted using test algal species. The
control bottles in these experiments received volumes of double-distilled,
deionized water equivalent to the test volumes of leachate. Most of the
tests were conducted with membrane filtered (0.45 u) leachates having
turbidities of 3-14 NTU.

Three batch culture tests with centrifuged and membrane filtered
leachates employed the alga Selenastrum capricornutum growing in synthetic
nutrient medium (Environmental Protection Agency, 1971). Light intensity
was 2,200 lux. At a concentration of 20% v/v both centrifuged and filtered
leachates inhibited growth, measured by haemocytometer counts of algal cells
(Figure 12). Centrifuged leachates contained greater amounts of coal

23


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DISTILLATE TREATED

Effects of 15% v/v distillate on species composition and abundance of

laboratory stream periphyton, September 1977, day 15.

of figure indicate total frustule counts.

0 ° A 3° o r) n °
a a nn a - -
ob zueysoimienag

40

Figure 21.


ddS WITZ9WAD
VIANIW VITIEWAD

3 WN1VONOTZ “AVA 3NN3L YWOLVIG
be, AVIMZHONVA VIdVTOVes
a Va1Vd VIHDSZLIN 5
VWISSILNNIW S3HLNYNHOV
adS VaGaNAS
AVIMSHONVA ViIavTOVas
| WNLVDNOTS “AVA 3NNFL VWOLVIG 4
=) adS viuvuovad 2
ol VLINNIW W11ZEWAD -
wn adS Vad3NAS FS
V31Vd VIHOSZLIN oO
VWISSILNNIW S3HLNYNHOV
‘adS VTIZGWAD
3 ddS WidvUoOVad
| VWISSILNNIW S3HINVNHOV
x] 3VIMaHONVA VidvUoSVad
wo) VINNIW W1T3aWAD
Wa1Vd VIHOSZLIN
‘ddS VaGINAS
V21Vd VIHDSZLIN
3 SIBVINDIDV VIHDSZLIN
r) ‘ddS VTIAEWAD a
8 WNLVONOT AVA 3NNIL VWOLVIG a
o| VWISSILNNIW S3HINYNHOV S$
aa VIANIW v1138WAD =
i= ‘ddS VaG3NAS ww
Va1Vd VIHDSZLIN =
° SIAVINDIDV VIHDSZLIN x=
b=] ads VTI3EWAD g
oS WNLVONOTA “VA 3NNIL VWOLVIG wi
z= VWISSILANIW S3HLNVNHOV
- VLONIW VTIR8WAD
fddS VEGINAS
| ar L L L = ben mn
w i=] mo fo} w fo} wm fo) wm
t Tv a ” Nn N = had

(Obx,WO /SaTNIsnud

22

Effects of 17% v/v leachate on species composition and abundance of laboratory
Numbers at top of figure indicate

stream periphyton, January 1977, day 41.

Figure 9.

total frustule counts.


T T T T “T T T T T T 7
Be @ LAKE -
S J controts e
- ‘ ] DISTILLATE TREATED 7
= 68 oN =
=
a
7
: e ° ]
5 4b @ —_—~_ ° 4
z Pe a
= a
g 8 7
=
oO

Figure 33. Effects of 20% v/v distillate on Clearwater Lake phytoplankton,
June 1978.

59


CONTENTS

FOLEWOTd. cece ce cece reece eee cee eee e anes cee scenes eeesesssssssecseeens elit
Abstract....... eee
Acknowledgements... ..eccee ee cce cece ec cce cece e nee ee enc eeeerceceseeene ec eViL

L. Introduction. ...cecse sees cece ccc ceeceeee ences
2. Conclusions and Recommendations
3. Bioassays Techniques...
Laboratory Streams
Bottle Tests.....
Field Bioassays......
Biomass Measurements..
Chemical Analyses......... dee e cee ceeereeecees

4. Preparation and Characteristics of Coal Leachates
and Distillates..ccccesesecsesceresssevearerreeces
Coal Leachates....
Coal Distillates.............
5. Experimental Procedures and Results.
Coal Leachates...........
Laboratory Streams.
Bottle Tests.....
Coal Distillates.
Laboratory Streams.
Bottle Tests..........
Field Bioassays....
6. Discussion.....sscceeecceccccccccsececcsecceccescesescecesens OL

References.......eccec ccc ce eet e cece ese cee ects eteeceeceeseesssenseeeeeee OF

vii


late (Figure 5).

*
TABLE 3. CHARACTERISTICS OF 0.08 W/W COAL LEACHATES

Parameter

Approximate Range Approximate Mean

pH

Conductivity (ymhos/cm)
Suspended Solids
Turbidity (NTU)
Alkalinity (as Caco,)
Calcium

Magnesium

Sodium

Potassium

Chloride

Sulfate

Nitrate nitrogen

Ammonium nitrogen
Dissolved organic nitrogen
Reactive phosphorus

Total dissolved phosphorus
Total phosphorus

Phenols

Total Organic Carbon

7.0-7.7
70-350
8-140
20-65
5-25
0.1-0.3
0.05-0.15
30-90
0.7-0.8
2.4-3.0
40-120

0.02-0.12

<0.001-0.008
0.01-0.10

0.02-0.12

13-27

7.4
200
highly variable
52
15
0.2
0.10
53

0.8

63
0.07
0.15
1.35
0.001
0.05
0.07
0.16

19

*
All concentrations are mg/l. Centrifuged leachate samples were filtered
through glass fiber or membrane filters for all analyses except suspended

solids, turbidity, and total phosphorus.

indicates that only one sample was analyzed.

13

A dash (-) in the "Range" column


B-TEST 2, NITZSCHIA

CHLOROPHYLL A (yg/!) SE

5 NEOPRENE FOAM 125
DAY 4
60 Nay 7 100
| al l
NN 4
45h « I \ n M\ 47
Tr \ \\ i
\
\ Nh \ «
at ny N \ Nt 0 fi
iN I \ iN iN Wey ow
\ \ \ \
ee PY Pa Pu ye
\ y \ \ =
if i i \\ No}
0 NN aNN NN Na NN oo.
Vy
DAY 5 Zz
g
60 100
— 2
l I My [gl g
45h 0 \ l I nl 1” x
\ =
y \\ uN
\ \
304 i qt i i \t 4s0
Nh I NN NN \ vy
NN yt \ in
15} i it i nH NN 25
iy ‘My \ NN VA
y \ \\ VN NN
0 u ut MN NN Yo,
CONTROL UNBUBBLED BUBBLED CONTROL UNBUBBLED
(0.157) (0.113) (0.157)

Figure 28B. Effects of 20% v/v unbubbled and bubbled distillate on the growth
of Nitzschia palea in Lake Superior water in batch cultures.
Each treatment had 3 replicate flasks. Error bars indicate one
standard deviation. Numbers in parentheses indicate absorbance
values of distillate in l-cm cells at 254 nm. Both foam and
neoprene stoppered flasks were employed.

50


1500 a
a
1300 ° DISTILLATE TREATED 4
5 ] CONTROLS
= | @ - LAKE e
$ |
Zz
9 900 4
a
3
Vv ®
Zz
<
@
o 600 |
4 @ Oo
< ta
3 ®
wu
° ®
300 - 4
fe) J an i J 1 i 1 |
) 2 4 6 8 10 12. «14
DAY

Figure 32. Effects of 5% v/v distillate on Clearwater Lake planktonic biomass,
October 1977, unfiltered carboys. Values for cellular organic
carbon were calculated from ATP analyses.

57


SELENASTRUM A

25
@ 5% CONTROL
© 5% LEACHATE
20+ 0 10% CONTROL
@ 10% LEACHATE
O 20% CONTROL oO
sk M@ 20% LEACHATE —————
3 a —}
—_——
ol ASI 4
= ————., om
Z 5F ——— *]
<
x . —=:
3 oul 1 1 1 1
x
= 100 NITZSCHIA B
a
°
2
=
Oo 80F ® 7
——
e}
60 8 ——%
40F- ' +
20 Zz j— .
r z — ae a
0 a- 1 1 1 L 1
0 1 2 3 4 5
DAY
Figure 15. Effects of 5%, 10%, and 20% v/v leachate on the growth of

Selenastrum capricornutum (A) and Nitzschia palea (B) in
Lake Superior water in 50%/day replacement cultures. Each
treatment had 3 replicate flasks. Error bars indicate one
standard deviation, No error bars are shown where the bar
is smaller than the symbol. Leachate conductivity was 143
umhos/cm; turbidity was 11-12 NTU.

30


250 C: CONTROL FL: FILTERED LEACHATE

|SELENASTRUM| NITZSCHIA weno

c
200 + a
a “I
150 - |
100 + | 4
c
sob I FL | ‘LL |
| I
0 | |

Figure 16. Effects of 20% v/v leachate on the growth of Selenastrum
capricornutum, Nitzschia palea, and Anabaena flos-aquae
in Lake Superior water in batch cultures, day 5. Each
treatment had 3 replicate flasks. Error bars indicate
one standard deviation. Leachate conductivity was 103-132
umhos/em; turbidity wag 3-5 NIU.

CHLOROPHYLL A (ng/!)



9”

CHLOROPHYLL A (u9/1)

800 rr

600

400F

200F

()

Figure

| @ DISTILLATE TREATED

© CONTROLS

a“

0 rr) 1 n 1

1 2 3 4
DAY

26.

replicate flasks.
B- 5% v/v distillate.

CHLOROPHYLL A (¥9/1)

800

600

400

200

Le—? 1 A A 1

@ DISTILLATE TREATED —e_|
7

O CONTROLS

is}
a |

0 1 2 3 4 5 6
DAY

Effects of 2% and 5% v/v distillate on the growth of Selenastrum capricotnutum in
synthetic nutrient medium in 50%/day replacement cultures.
Error bars indicate one standard deviation.

Each treatment had 4
A- 2% v/v distillate;


A 8B

T T T T T 18 T T T T
be | a a
os @ Jteacuare TREATED @ JHEACHATE TREATED
o S
9 Jcontrots WE oo |eouous 7
asf 4 °
4 .
a -
£ 04b — 4 EOF 4
= 4 a
< z
3
g | 3
z oap ey 3
2 o = 06h 4
g ZA g
5 a S é e
02- fo} 4 =
° co
e
. © 03 e o o 7
ok w4 4 wo to
o eas a
id 8
oo Lo
——3—o °
oo LOS 1 1 1 n ool Omit 1 1
8 10 12 14 16 18 20 10 4 16 18

12
15 t 4
& JLeAcHATE TREATED WA
bd ry

5 ]eonrrots a
12F 9 - LEACHATE + TM/ 4
09k . 4
/ p
. °
ost L/L / fo 4
2
— ok”

0.0

CHLOROPHYLL A (yg/em*)

DAY

Figure 6. Effects of 3.2% v/v leachate on laboratory stream periphyton.
A- June 1976; B- July 1976; C- September 1976.

19


6%

A-TEST 1, NITZSCHIA

NEOPRENE FOAM
2.5 125 .
Ma
DAY 3 \ N
20- 100 \\
- a
= Pr
315 475 2
<i Zz
= 1b I NN iy I al l 450 2
3 gt \\ \\ ml ¢
sos NN iN i NV + 8
\\ Wt \\ v\ Nn z
\\ \\ \\ \\ \\
00 NN ul M ul UJ,
CONTROL UNBUBBLED BUBBLED CONTROL UNBUBBLED
(0.118) (0.071) (0.118)

Figure 28A. Effects of 20% v/v unbubbled and bubbled distillate on the growth of Nitzschia
palea in Lake Superior water in batch cultures. Each treatment had 3 replicate
flasks. Error bars indicate one standard deviation. Numbers in parentheses

indicate absorbance values of distillate in l-cm cells at 254 nm.
and neoprene stoppered flasks were employed,

Both foam


A final field bioassay was conducted in June 1978 using 20% v/v dis-
tillate (Table 10). The distillate used in this bioassay had a pH of 4.7
and a conductivity of 11 umhos/cm. UV absorbance of this distillate at 254
nm was only half that of distillate used in the fall 1977 experiments. The
results of chlorophyll analyses and bacterial plate counts (Figure 33, Table
11) were similar to those observed in the previous tests, with distillate
carboys showing higher chlorophyll concentrations on days 5 and 7 and much
higher bacterial counts than control carboys. On day 5 when chlorophyll
reached peak values in distillate carboys, photosynthetic capacity also
appeared to be somewhat greater in distillate carboys. The percent increases
in fluorescence after the addition of DCMU were 152 and 163 in distillate
carboys and 70 and 95 in control carboys on this day. In spite of higher
chlorophyll and bacteria counts in distillate carboys, ATP analyses indicated
lower total biomass in these enclosures (Figure 34). This was true for all
sampling days when whole water samples were analyzed (Figure 34A), but only
for days 3 and 5 when samples which had been filtered through a 48-» net
were analyzed (Figure 34B). Both ATP and ash-free dry weight analyses
indicated that a precipitous decline in biomass occurred in the lake during
the course of the experiment. Field and laboratory observations of zoo-
plankton samples collected in 80-y net indicated that nearly all zooplankters
were killed in distillate carboys, while zooplankters appeared in good
condition in control carboys. The pH in distillate carboys was significantly
lower (avg. = 5.9) than in control carboys (avg. = 6.5) or in lake water
(avg. = 6.6).

58


DISCLAIMER

This report has been reviewed by the Duluth Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publi-
cation. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor does

mention of trade names or commercial products constitute endorsement or
recommendation for use.

di


TABLE 9. NUMBER OF DIATOM SPECIES OBSERVED IN STREAMS TREATED
WITH 15% COAL DISTILLATE AND 15% DISTILLED WATER, DAY 15*

Total Frustules No. Species
Treatment Strean Counted Observed
1 7,731 70
15% Coal
Distillate 2 7,652 68
3 4,970 81
1 7,325 55
15% Distilled
Water 2 7,241 58
3 8,051 52

*The data represent the combined counts from three substrate plates in each
stream.

Bottle Tests

A total of 15 small-volume bottle tests of the effects of coal distil-
late were conducted using test algal species. The control bottles in these
tests received volumes of double-distilled, deionized water equivalent to
the test volumes of distillate. All flasks were stoppered with foam plugs
except where specifically indicated otherwise.

An initial test of the effects of coal distillate on Chlorella sp. was
conducted in April 1977. The growth of Chlorella in enriched, membrane
filtered Lake Superior water containing 20% v/v coal distillate was moni-
tored using in vivo fluorescence measurements. The calculated chlorophyll a
values indicated initial inhibition of growth by distillate (Figure 22).

From September to December 1977 different concentrations of distillate
were tested using the alga Selenastrum capricornutum growing in synthetic
nutrient medium. Four batch culture tests with 20% v/v distillate showed
slight inhibition of growth, determined by haemocytometer counts of algal
cells (Figure 23). When these tests were repeated with daily replacement of
50% of the culture volume, the effects were more pronounced (Figure 24).

38


A similar 10 day dosage with 15% v/v distillate was conducted in May
1978. While chlorophyll a and other measures of biomass showed no signifi-
cant differences between treatment and control streams, in vivo fluorescence
measurements showed greater percent increases in fluorescence in control
streams after the addition of DCMU (Table 7). These results suggest higher
photosynthetic rates in control streams (Samuelsson et al., 1978).

TABLE 7. PERCENT INCREASE IN IN VIVO FLUORESCENCE AFTER ADDITION
OF DCMU IN STREAMS TREATED WITH 15% COAL DISTILLATE AND
15% DISTILLED WATER FOR 10 DAYS*

Treatment Stream Substrate % Increase in Fluorescence
Plate
1 1 55.3
2 53.3
15% Coal
Distillate 2 1 63.0
2 60.4
3 1 51.4
2 62.9
1 1 65.8
2 70.0
15% Distilled
Water 2 1 70.4
2 71.4
3 1 74.5
2 66.7

*Samples were collected 11 days after the start of the dosage.

Much more dramatic effects on growth were obtained when periphyton were
exposed to 15% v/v distillate from the very beginning of a stream bioassay.
Such an experiment was conducted in September 1977 when stream temperatures
were 14-15'C. UV absorbance values for this distillate were approximately
twice those obtained for other distillate samples, indicating higher concen-
trations of dissolved organics. Chlorophyll a, ash-free dry weight, and ATP
data indicated severe inhibition of growth in distillate streams (Figure 20).
Ratios of dry weight to cellular organic carbon (based on ATP) were general-
ly much higher in distillate than control streams (Table 8), indicating that
some non-living organic matter accumulated on the substrates in distillate
streams. Diatom frustule counts-on day 15 also showed reduced growth in
distillate streams (Figure 21). However, the percent abundance of the

36


SECTION 6
DISCUSSION

Although coal leachates usually stimulated periphyton growth in labo--
ratory streams, filtered leachates inhibited the growth of test algae in
bottle tests. At present, this contradiction between the results of stream
and bottle bioassays cannot be adequately explained. Important differences
between the two bioassay types include algal growth habit, the presence of a
multispecific association of bacteria and algae in the stream bioassays, and
the use of polyethylene and plexiglass in the laboratory stream structure
vs. glass enclosures for the bottle bioassays. However,in tests with copper
and nickel, Gerhart and Davis (1978) likewise found that algae in laboratory
cultures showed more extreme responses to additions of toxicants than algae
in field enclosures, and copper was occasionally toxic in laboratory experi-
ments but stimulatory in the field. This study employed multispecific commu-
nities of algae contained in glass enclosures in both field and laboratory.
Additional work with coal leachates using in situ lake enclosures would be
desirable.

Some speculation is possible concerning the components of leachates
which produced these growth responses. Simple leaching of western coals
with pure water has been shown to remove only small amounts of metals
(Coward et al., 1978), and our own analyses also suggest that metals were
unimportant. Organic phosphorus in leachates was initially suspected as a
source of phosphorus for algal growth in stream bioassays. However, no
evidence in support of this theory was obtained, and in one experiment
growth in leachate and control streams was equally slow before the addition
of inorganic phosphorus to the lake water. In addition, no evidence of
stimulation was observed in bottle tests conducted at low phosphorus concen-
trations. It seems likely that the different responses of the algae in
stream and bottle bioassays occurred in response to the same component of
coal leachate. There are many examples of algae responding by both growth
enhancement and inhibition to the same material under different conditions
(e.g., Niemi, 1972; Kauss et al., 1973; Gordon and Prouse, 1973; Dunstan et
al., 1975). Both metals and organic compounds have been observed to produce
these dual effects. Such studies clearly suggest that algae may respond to
some chemical stresses with a generalized increase in growth.

Non-polar aromatic organic compounds were not important in leachates.
Carlson et al. (1978) found concentrations of these compounds in leachates
to be similar to background concentrations in Lake Superior. Polar materials
were also present in leachates, but their kinds and concentrations were not

61


MA NEOPRENE STOPPER

FLOAT # 4
LINE SCREW CLAMP
XK
AIR LINE

— en

+
SHER

JAR CLAMP

\i
PVC TUBING

Figure 2. Detail of field enclosure bottle.

6


8y

B-TEST 2, SELENASTRUM

120

%m FLUORESCENCE INCREASE
ao
°
t

SS |
~——6

-—e

O CONTROL J

@ UNBUBBLED (0.242)

© BUBBLED (0.117) _ __—
ce)

aa e a

40F
te)

= 75}

=

o

3

<|

= 50F

al

>

=x

a

Q 25+

°

—_a

=

1s}
()
0

Figure 27B.

1 2 3 4 5
DAY

Effects of 20% v/v unbubbled and bubbled distillate on the growth of
Selenastrum capricornutum in Lake Superior water in batch cultures.

Each treatment had 3 replicate flasks. Error bars indicate one standard
deviation. No error bars are shown where the bar is smaller than the

symbol. Numbers in parentheses indicate absorbance values of distillate
in l-cm cells at 254 nm. Test 2 employed neoprene stoppered flasks only.


TABLE 11. BACTERIA PLATE COUNTS, FIELD BIOASSAYS

Bioassay Treatment Carboy Colonies / ml Avg.
1% distilled water 1 380 400
2 640 700 530
1% coal distillate 1 1,500 1,840
2 900 - 1,413
1 5% distilled water 1 960 740
(Day 13) 2 780 600 770
5% coal distillate 1 1,560 1,580
2 1,500 980 1,405
Lake water - 52 107 80
5% distilled water 1 520 700
2 1,820 1,580 1,155
5% coal distillate 1 3,120 3,100
2 TNC* TNC* -
2 5% distilled water, 1 2,700 2,540
(Day 14) filtered 2 1,280 1,660 2,045
5% coal distillate, 1 3,580 2,880
filtered 2 3,660 3,260 3,345
Lake water - 194 248 221
20% distilled water 1 3,800 3,800
2 4,700 4,800 4,275
3 20% coal distillate 1 61,700 65,000
(Day 9) 2 58,900 37,900 55,875
Lake water - 130 104 117

*TNC = Too numerous to count.

54


algal cells, periphyton samples from stream substrates were centrifuged and

dried at 50°C to constant weight.

An 0.8000 g portion was removed, mixed

with 10 g sodium sulfate, and Soxhlet extracted for 5 h with 200 ml 50:50

methylene chloride/hexane.
pended in hexane, and concentrated to 10 ml.

TABLE 1.

The extract was concentrated to dryness, resus—
One ml was then removed for

METHODS FOR CHEMICAL ANALYSES

Analysis

Method

Reference

Major cations
Trace metals

Chloride

Reactive phosphorus

Total phosphorus
Nitrate nitrogen
Ammonium

Dissolved organic
nitrogen

Sulfate

Total alkalinity

Turbidity
Total organic carbon

Phenol

Chemical oxygen demand

Organics

Organics

Complexing capacity

Flame atomic absorption
Flameless atomic absorption

Ferric thiocyanate spectro-
photometric

Ascorbic acid/molybdate

Persulfate digestion
UV spectrophotometric
Nesslerization

Sulfuric acid digestion

Turbidimetric

Bromcresol green/methyl red
indicator

Nephelometric
Combustion, IR analysis

4-aminoantipyrene spectro-
photometric

Dichromate reflux

UV analysis

Gas chromatography

Modified

American Society of Test-
ing and Materials, 1976

American Public Health
Association, 1975

_ Mrkva, 1969; Mattson et

al., 1974
See text

Chau and Lum-Shu-Chan,
1974 .


EPA-600/3~-80-051
May 1980

ALGAL BIOASSAYS WITH LEACHATES AND
DISTILLATES FROM WESTERN COAL

by

David Z. Gerhart, Joseph E. Richter, Sidney J.
Curran, and Thomas E. Robertson
Department of Biology and
Lake Superior Basin Studies Center
University of Minnesota, Duluth
Duluth, Minnesota 55812

Grant No. R803932

Project Officer

William E. Miller
Special Studies Branch
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330

ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804


|. @ CONTROL Sy9/IP ° J
50F OCONTROL 200ng/1P
O LEACHATE 5y9/IP
= @ LEACHATE 200p9/1P
B 40r 4
<i
: er : i ]
a
2 aol a
2
=
° te —~
10F
olt 1 L ——— ee
B
150
ws
120 $
8 o———
2 90- ob. i
3 ——4s
&
4 60F —_ 4
oe
3 t
2 t
* ° ° la NN |
out n n _—— 26 a)
° 1 2 3 4 5
DAY
Figure 14. Effects of 20% v/v leachate on the growth of Selenastrum

capricornutum in Lake Superior water in 50%/day replacement
cultures, Test 2, Each treatment had 4 replicate flasks.
Error bars indicate one standard deviation. No error bars
are shown where the bar is smaller than the symbol. Leachate
conductivity was 275 wmhos/cem., A- Chlorophyll a; B- Photo-
synthetic capacity.

29


UNBUBBLED

BUBBLED

FIL.D. RESPONSE

n [as en ae Se ee

1
0 10 20 30 40
TIME (MINUTES)

Figure 5. GC analysis of 2-1 samples of unbubbled and
bubbled distillate. Bubbling time was 60 min.

17


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development. U.S. Environmental
Protection Agency. have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields
The nine series are:

Environmental Health Effects Research

Environmental Protection Technology

Ecological Research

Environmental Monitoring

Socioeconomic Environmental Studies

Scientific and Technical Assessment Reports (STAR)
Interagency Energy-Environment Research and Development
“Special” Reports

Miscellaneous Reports

OCONANSWNH

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.

This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.


ar OT rn es ee | T Lm |
100 - J
|

80+ @ DISTILLATE TREATED T 4
O CONTROL 7
60 7

4ob 4

20 ; =
|

10+ 4

CHLOROPHYLL A (ng/I)

‘ad
2u 1 ==
0 2

Figure 22, Effects of 20% v/v distillate on the growth of Chlorella sp.
in Lake Superior water in batch cultures, Each treatment had
6 replicate flasks. Error bars indicate one standard deviation.

41


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sysusuojos> oy10j}G03
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suapo] %f Opnuyws opjoquik>

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Bjpydazossw soyjuouyry OT} Mi —<—|

1 340000

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BjoryUO) MIYDszEN

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800000

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ynujw 30a OpNU;W _YFEqQUIA>

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500 000

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* sysusuojos> Dys0j16024

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‘sJsuauojos> D1sDj16045
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30

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10;
5
04

25

Numbers at top

LEACHATE TREATED

CONTROLS
Effects of 17% v/v leachate on species composition and abundance of

laboratory stream periphyton, September 1977, day 17.

of figure indicate total frustule counts.

Figure 11.


2,510,000

1,520,000

L

(AVIGIOWDIS 8 3VSNLAO) ddS VIHDSZLIN
(AVLVTOJINV1) ddS VIHDSZLIN
JAVINIAID NOIGIW

ddS VITIZEWAD

AVIMSHONVA VINV1SVad

VLANIW VTTSEWAD

VWISSILANIW SSHLNVNHOV
ddS Vad3NAS

ddS V1IaEWAD
(3VAdIOWDIS ¥ 3VSNLGO) ddS VIHDSZLIN
VINNIW W11aaWAD
auvINDUID NOIGaW
aVIMZHONVA VIVID vad

dd I
WISSILNNIW 8 VIHOS
S3HLNVNHOV
1 1 ae r 1
to) 2) o nm o wo o wo
N ° o ~ o + ” v

(0hx WO/SaTNISNad

DISTILLATE TREATED

CONTROL

Numbers at top of figure indicate total frustule

abundance of laboratory stream periphyton, March 1977,

Effects of 12% v/v distillate on species composition and
day 36.

Figure 18.

counts.

34


58,000 25,000 33,000

58,000

SISNINOLOYD VINVIOVAS
VIVUINOTD VTTZLOIDAD

“‘ddS VUGINAS
VIVINNVUD VUISOTIN
uaHLO

SVIMBHINVA WiNVOVEd

‘ddS VUGINAS

VAIV4EWOTD VIIRLO01DAD
VIVIANVES VUISOTAW
waHLO

CONTROLS

SISNINOLOUD VIEVISVAS
VIV4aWO1D VT1L019A9
“‘ddS WaG3aNAS
VAVIANVED VEISOTAW
wIHLO

SISNINOLOYD VINV1OVed
VIVIANVES VaISOTaW
“ddS VUG3NAS

VIVEINOTD VITZLO1SAD

LEACHATE TREATED

Olt WO/SaTNISNed

43H10

VINSHOAVA VINVIOVES
VIVEAWOTD VTITRLOIDAD

‘ddS VUGaNAS

VIVHd3508DIW SIHINVNHIV
43HLO

VOINOdVT VIBVTIOVEd

13,000
LEACHATE +

COPPER

623,000

‘ddS V8G3NAS
VIVHd30NDIW S3HINVNHOV

4uaHLO

SISNINOLOYD VidVOVEs

‘ddS ViNVUOVEs

‘ddS Vud3NAS

VIVHd3D0NDIW S3HINVNHOY

CONTROLS

578,000

waH1O
SISNINOLOUD VidvTOVed
‘ddS ViINVIUSVUd
VIVHda2089IW SIHLNVNHOV
‘ddS VGINAS

1,020,000

waHLO-
WNLVONOT3 “YVA 3NNIL VWOIVIG

‘dds viuvtsvud
VIVHdID085IW SSHINVNHSY

LEACHATE TREATED

886,000

‘ddS VUG3NAS
1 1

6 °

ry + o
or * WD / SaTNISNYs

Effects of 3.2% v/v leachate on species composition and

Figure 7.

A- June 1976,
Numbers at top of figures

abundance of laboratory stream periphyton.

day 13; B- September 1976, day 24.
indicate total frustule counts.

20


adequate supply of viable cells to colonize the substrates at low headbox
flows, the streams were seeded in the following manner. Nearshore peri-
phyton communities were collected from Lake Superior, homogenized to form a
slurry, and filtered through an 80-y plankton net. On the first day of an
experiment each stream was seeded with 100 ml of the slurry, added at the
stream outlets. This technique resulted in rapid colonization and well-
replicated communities. Species composition and community structure were
found to be similar in the slurry and in the communities which developed on
the substrates.

BOTTLE TESTS

Short-term (3-7 day) bottle tests were conducted in 300-ml Erlenmeyer
flasks at 25-27°C. Culture volume was 100 ml. The flasks were maintained
on a rotary shaker at approximately 80 rpm. Continuous illumination of
2,200-3,700 lux was provided by "cool-white" fluorescent lamps. Bioassays
were conducted both in a synthetic nutrient medium (Environmental Protection
Agency, 1971) and in phosphate-enriched Lake Superior water which had been
filtered through Whatman GF-F glass fiber filters. In some cases the bottles
were treated as batch cultures, with no additions during the course of an
experiment. In other "replacement" tests the bottles were treated as semi-
continuous cultures, with 50% of the culture volume removed and replaced
daily with fresh medium and test additions. Tests were conducted in both
foam and neoprene stoppered flasks.

The test algae were maintained in the synthetic nutrient medium, and
test flasks were inoculated with 1 ml of exponentially growing stock culture.
The algae were not washed prior to inoculation. The test algae included a
local greenhouse strain of Chlorella and the following algae obtained from
the EPA Corvallis Environmental Research Laboratory: Selenastrum capricor-
nutum, Nitzschia palea, and Anabaena flos-aquae. All other procedures were
those of the EPA Algal Assay Procedure (Environmental Protection Agency,1971).

FIELD BIOASSAYS

Three field bioassays with coal distillate were conducted on a small
mesotrophic lake using in situ enclosures. Clearwater Lake, located 24 km
north of Duluth, Minnesota, occupies a sheltered basin less than 0.1 em? in
area and contains clear, unstained water. The lake has a maximum depth of
6 m and a secchi disk value of approximately 3 m. Total alkalinity is only
4 mg/1 as CaCO3, conductivity is approximately 31 wmhos/cm, and the pH
ranges from 6.5-6.8. Several species of Dinobryon were the dominant phyto-
plankters when bioassays were conducted.

Eight 21-1 Pyrex carboys were suspended individually from buoys at a
depth of 1.5 m (Figure 1). The buoys were anchored on separate anchor lines
in 4-5 m of water. Each carboy was fitted with a neoprene stopper held
tightly in place with metal clamps (Figure 2). Two tubes passed through

each stopper: an air exhaust/inlet tube and a sampling tube. A hand pump
was fitted with polypropylene tubing and PVC elbows at each end and used

4


A
35 T T T T T T TI

by ] DISTILLATE TREATED

[> 30k 4

3 5 ] CONTROLS a

<i ® -LAKE oF
25+ @ - 4

4 ® a7

z 20 ot 4

ie} a _—

ao e | Q

° 5 ZL, ZA, o=—

= nl i | _

(3) ® 25

DAY
B
35 T T TT Tt T
: ] DISTILLATE TREATED
_ 30 4
5 5 ]controis _
Z25b ® -LAKE 4
@ 738
a
> 4 o
= 20 Pa Za 4
2 é
J — ¢ —
Q 15- at 4
=
v : go
10 -
l L 1 es re | l
0 2 4 #6 8 10 12 14

Figure 31. Effects of 5% v/v distillate on Clearwater Lake
phytoplankton, October 1977. A~ Enclosure water
unfiltered; B- Enclosure water filtered through
an 80-y plankton net.

56


rad

CELLS /MLx10°

C: CONTROLS
TEST 1 TEST2

D: DISTILLATES
TEST 3 TEST 4

6-- rq]

sf |e
4-4 | Cc
<
5 D
34 9 D
2
2+
15 Cc
D
o+_—
3 6 3 6

DAY

Figure 23. Effects of 20% v/v distillate on the growth of Selenastrum capricornutum in
synthetic nutrient medium in batch cultures. Each treatment had 6 replicate
flasks. Error bars indicate one standard deviation,



Tests of lower concentrations of distillate with 50%/day replacement
cultures were inconsistent. In September two tests were performed with
concentrations of 2%, 5%, and 10% v/v distillate. Light intensity was 2,200
lux, and growth was measured by haemocytometer cell counts. In the first
test fresh distillate was used, and all concentrations were toxic (Figure
25, Test 1). Distillate used in the second test was stored under refriger-
ation for one week prior to the test. Concentrations of 5% and 10% v/v
again appeared toxic, but the 2% treatment showed no effect (Figure 25,

Test 2). The experiments with 2% and 5% v/v distillate were repeated in
December using distillate prepared from the same coal sample used in Septem-
ber. Light intensity was 3,700 lux, and growth was measured by cell counts
and in vivo fluorescence. No inhibitory effects were observed at either
concentration, and 2% v/v distillate may have been slightly stimulatory
(Figure 26).

Five additional batch tests were conducted in filtered Lake Superior
water enriched with 200 yg/1 POy-P. In these tests chlorophyll a was deter-
mined by in vivo fluorescence measurements after the addition of DCMU, and
the percent increase in fluorescence after the addition of DCMU was used as
an indicator of photosynthetic capacity. Two tests were conducted with
Selenastrum capricornutum, two with Nitzschia palea, and one with Anabaena
flos-aquae. In each case the effects of bubbled and unbubbled 20% v/v dis-
tillate were examined. In addition, some flasks were stoppered with foam
plugs, while others were sealed tightly with neoprene stoppers to prevent
the escape of volatile organic compounds. Bubbled distillate was prepared
by bubbling distillate samples with filtered air through an airstone for 30-
60 minutes. UV spectrophotometric measurements of coal distillate were made
in l-cm far UV quartz cells.

Chlorophyll determinations in neoprene stoppered flasks indicated that
the growth of Selenastrum was inhibited by unbubbled distillate but may have
been stimulated slightly by bubbled distillate (Figures 27A and 27B). In
addition, increases in fluorescence after the addition of DCMU were smaller
in unbubbled distillate than in control cultures, indicating a reduction in
photosynthesis in these flasks. In contrast, unbubbled distillate had lit-
tle effect in foam stoppered flasks (Figure 27A).

Similar trends were observed using Nitzschia as the test alga
(Figures 28A and 28B). Anabaena, however, showed no inhibition with unbub-
bled distillate in neoprene stoppered flasks and may even have been stimu-
lated by this treatment (Figure 29). Unbubbled distillate was, however,
inhibitory in foam stoppered flasks. Bubbled distillate had no effect on
Anabaena.

Considerable increases in pH were occasionally evident in neoprene
stoppered bottles, indicating carbon limitation. The magnitude of these
increases depended upon the test spec’es and the total yield of algal cells.
The highest pH values were recorded in the test with Anabaena. Final pH's
in this test (day 5) were 8.6-8.7 in foam stoppered flasks and 10.7-10.8 in
neoprene stoppered flasks.

44


both to withdraw and return water to the carboys. For sampling, the elbow
on the output end of the pump was inserted into the sampling tube of the
carboy, the clamp on the air exhaust/inlet tube was opened, and 15-20 pump
strokes of air were pumped into the carboy to mix its contents thoroughly.
The pump was then immediately reversed, and 18% of the carboy's contents

was removed. This water was used for sample water for all analyses. A
standard volume of distilled water (control carboys) or coal distillate
(treatment carboys) was then poured into the carboy through the sampling
tube, the pump was again reversed, and lake water was pumped from a depth of
1.5 m into the carboy to restore the original volume (18 liters). This scheme
permitted all sampling operations to take place without removing the carboys
from the Lake. Samples were collected three days per week. In the first

two bioassays (September and October, 1977) 18% of the carboy volume was
replaced daily with fresh lake water and the appropriate volume of distilled
water or coal distillate. In the third bioassay (June 1978) 33% of the
carboy volume was replaced three times per week when the bottles were sampled.

BIOMASS MEASUREMENTS

Chlorophyll a analyses were conducted in vivo with a Turner Model 111
fluorometer or on ‘samples filtered through Whatman GF/F glass fiber filters
and extracted in 90% acetone. In the latter case, either fluorometric
determinations or spectrophotometric measurements at 663 nm were employed.
All chlorophyll a and phaeopigment methods were those of Strickland and
Parsons (1968). Fluorometric measurements of in vivo samples before and
after the addition of 10 uM DCMU were used to ‘estimate photosynthetic capaci-
ty and algal viability (Slovacek and Hannan, 1977; Samuelssonet al., 1978).

Ash-free dry weight analyses of periphyton and plate counts of bacteria
populations were performed using methods described by the American Public
Health Association (1975). ATP was extracted from algal samples with boiling
Tris buffer after filtration onto glass fiber filters (Holm-Hansen and Booth,
1966; Rudd and Hamilton, 1973). The extracted ATP was frozen until analysis
with a DuPont Bioluminescence Photometer. The cellular organic carbon
content of algal samples was found by multiplying ATP concentrations by 286
(Stadelmann, 1974).

Cell counts of periphyton diatoms were made by filtering a known
volume of sample through a 25-mm membrane filter (0.45 um) and then washing
the filter with 5 ml of 50% ethanol and 5 ml of absolute ethanol. After
drying, the filter was mounted on a glass slide in creosote and the edges of
coverslip were sealed with melted paraffin. Generally, 8,000 cells were
counted per sample. Samples for counts of algae other than diatoms were
preserved in 1% acid Lugol's solution and counted using an inverted micros-
cope technique (Vollenweider, 1974).

CHEMICAL ANALYSES

Methods used in the chemical analysis of water samples and coal
leachates and distillates are listed in Table 1. For tissue analysis of

7