FINAL REPORT FOR COOPERATIVE AGREEMENT # CR810774-01-0
DEVELOPMENT OF TOXICITY TEST PROCEDURES FOR THE MARINE
RED ALGA CHAMP LA PARVULA
by
Glen B. Thursby
Botany Department
University of Rhode Island
Kingston, R.I. 02881
Project Officer
Richard Latimer
Environmental Research Laboratory
U.S. Environmental Protection Agency
Narragansett, R.I. 02882
November 1984
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DISCLAIMER
This document has not been peer and administratively reviewed
within EPA and is for internal Agency use/distribution only.
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OVERVIEW
New and exotic chemicals, as well as many known compounds,
are being released into Che air and. water daily. Most of these
compounds find their way into the marine environment and need to
be tested as potential toxicants to different trophic levels of
marine organisms. Standard toxicological test procedures have
been developed for fish, invertebrates, amphibians, and
niicroalgae. However, very little work has been done toward the
development of a standard procedure for testing the effects of
toxicants on macroalgae. Macroalgal tissue residues have been
used to monitor pollution of heavy metals in the field. This type
of study requires the use of relatively tolerant species because
the goal is to have an alga that will accumulate a pollutant
without dying. In toxicological studies, the opposite is true.
One looks for a relatively sensitive organism; since, by
protecting sensitive organisms, tolerante species should also be
preserved.
A toxicity test method has been developed for the marine red
alga Champia parvula to assess chronic effects of pollutants to
"marine macroalgae. The method has been used to generate toxicity
data for water quality criteria. The test method has previously
been" evaluated with heavy metals, cyanide, arsenate and arsenite.
These results, plus those from this study, indicate that this
procedure is comparible to the most sensitive marine animal
chronic tests. These results also demonstrate the importance of
conducting chronic tests with the previously overlooked
macroalgae.
Currently, the only other algal toxicity tests used routinely
employ planktonic microalgae. The level of expertise required to
use Ghampia parvula is approximately equal to that necessary to
run a. microalgal toxicity test. The test with C. parvula is less
labor intensive because axenic cultures are not required. This
procedure, however, is not intended to replace microlagal tests,
but rather to complement them by adding to the types of plants
included in marine toxicity testing.
Champia parvula is a common species in many parts of the
world. It has been reported from Mexico and Southern California
on the west coast of North America, and from the Carribean to
Massachusetts on the east coast. It has also been reported from
France, Spain, Korea, Brazil and Southern Australia. Because of
its sensitivity and wide distribution, C. parvula may be a useful
surrogate species for determining the relative toxicity of a
variety of chemicals. Because of its Europena distribution, £._
parvula could also become a standard test species for the
Organization for Economic Co-operation and Development (OECD).
The Environmental Protection Agency, under the mandate of the
Toxic Substance Control Act, is required to identify and control
chemicals that are hazardous to human health or the environment.
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An important aspect of this responsibility is to develop
methodology for testing potential hazards to different trophic
levels of marine organism. According to EPA's Office of Research
and Development planning documents, data are needed for 40 to 65
toxic pollutants for which little or no chronic toxicity
information exists. These data must be both scientifically sound
and legally defensible. At the same time, it is necessary for the
methods to be as simple and cost efficient as possible. All of
the above requirements are met by the test methods developed for
the marine red alga Champia parvula.
This report covers work completed during the time period from
May 23, 1983 through October 31, 1984. The specific objectives of
the cooperative agreement were:
1. verification of the static toxicity test with
Champia parvula using organic compounds;
2. write test guidelines and support document;
3. develop artificial seawater medium for Champia
-• parvula so that toxicity tests can be performed in
a chemically defined medium;
4. test two other isolates of Champia parvula to
determine if test results with the standard isolate
are representative;
5. test two other species of red algae to determine if
the results with Champia parvula are representa-
tive of other red algae;
6. compare sensitivity of a Champia mini-chronic
test with that of the existing two week test;
7. test the feasability of the above mini-chronic for
use in ERLN's Effluent Testing Program.
Because of the relatively short time required to complete a
given toxicity test and the ease in which the organims is
maintained in culture, C. parvula has the potential to become a
rapid screening tool for chronic effects among the macroalgae.
The short-term, "mini-chronic" test in which the exposure period
to the toxicant may be as short as 24 hours, shows promise for use
in EPA's Effluent Monitoring Program. This procedure may prove
also to be useful for testing single compounds. The sensitivity of
the standard isolate of Champia compared favorably with that of
two other isolates of Champia, as well as two other species of red
alga. An acceptable artificial seawater recipe has also been
recommended for use in the testing procedure. This will make the
test available to laboratories with out access to clean natural
seawater.
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TABLE OF CONTENTS
Page
PART I. EFFECTS OF ORGANIC COMPOUNDS ON GROWTH AND
REPRODUCTION IN THE MARINE RED ALGA CHAMPIA
PARVULA. 6
PART II. CHAMPIA PARVULA TEST GUIDELINE 30
PART III.- CHAMP IA TEST GUIDELINE SUPPORT
DOCUMENT.
PART IV. OPTIMUM GROWTH OF THE MARINE RED ALGA
CHAMP LA PARVULA IN NATURAL AND ARTIFICIAL
SEAWATERS 66
PART V. CHAMPIA TESTING: ARTIFICIAL SEAWATER
AND OTHER ISOLATES AND SPECIES 82
PART VI. A SHORT-TERM EXPOSURE TEST FOR CHAMPIA
PARVULA FOR USE IN EFFLUENT TESTING 105
PART VII. DRAFT PROTOCOL FOR EFFLUENT FIELD TEST
WITH THE MARINE RED ALGA CHAMPIA PARVULA 121
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PART I.
EFFECT OF ORGANIC COMPOUNDS ON GROWTH AND REPRODUCTION
IN THE MARINE RED ALGA CHAMPIA PARVULA
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INTRODUCTION
A relatively fast, simple and inexpensive toxicity test
method has been developed to assess chronic effects of pollutants
on the marine red macroalga Champia parvula (Steele and Thursby,
1983). Currently, the only other algal toxicity tests used
routinely employ planktonic microalgae. Microalgae are considered
to be less sensitive to toxicants than animals (Kenaga and
Moolenaar, 1979; Kenaga, 1982). The test method with C. parvula
has been evaluated previously with heavy metals and cyanide, and
the results demonstrate that the sensitivity of C. parvula is
comparable to that of the most sensitive marine crustacean or fish
life cycle test (Steele and Thursfay, 1983). However, its
sensitivity to organic compounds needs to be determined. To
address this, ten organic compounds were tested to determine C.
parvula*s sensitivity,-and therefore its usefulness as a standard
test species.
Test endpoints quantified effects on vegetative growth,
sexual reproduction, and formation of tetrasporangia (asexual
spore production). Earlier work with metals and cyanide has shown
sexual reproduction to be the most sensitive endpoint. The
'results of this study will be used to determine if for organic
chemicals sexual reproduction can be used as the sole endpoint to
further simplify the test procedure.
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MATERIALS AND METHODS
Test procedures were those previously established for the
red alga Champia parvula (C. Agardh) Harvey by Steele and Thursby
(1983), except EDTA was oinmitted from the medium and vitamins were
added. Unialgal stock, cultures of males, females and
tetrasporophytes were maintained in 1000 mL, aerated Erlenmeyer
flasks containing 800 mL of a modified medium 'f' (Guillard and
Ryther, 1962). The medium consisted of 37.4 mg NaN03, 2.5 mg
NaH2P04.H20, 10.4 ug Fe (as Cl~2) and 'f'/4 levels of vitamins
(0.24 ug B12. 0-24 ug biotin and 50 ug thiamine-HCl) per liter of
filtered seawater (15 um charcoal filter and 0.3 urn Balston*
filter). Flasks were illuminated with 75 to 80 uE.m~2.s~l of cool
white fluorescent light on a 16h:8h, light:dark cycle. Temperature
and salinity were 22 to 24°C and 30 ppt, respectively. Media were
changed once each week during culturing.
Toxicity test lasted 11 or 14 days for tetrasporophytes (11
days when tetrasporangia were counted) and 14 days for females.
Tests were performed with 400 mL of medium in screw-capped, 500 mL
Erlenmeyer flasks. The medium was one-fourth strength of that for
the stock cultures. In addition, 150 mg/L sodium bicarbonate was
'added in lieu of aeration. Flasks were shaken on a rotary shaker
at 100 rpm. Media were replaced on days 7 and 11. All other
conditions were the same as those for stock cultures.
After stock plants were rinsed in sterile seawater to remove
traces of old medium, 2- to 3-mm branch tips were cut to serve as
innocula for toxicity tests. Five tips of each life history stage
in question were placed into each test flask. One male branch
(about 1 cm long), visibly producing spermatia, was added to each
flask containing females. Replicate flasks were used for each
treatment. At the termination of a toxicity test, females were
examined for the presence of cystocarps (evidence of sexual
reproduction) and tetrasporophytes were examined for the presence
of tetrasporangia (site of meiosis). For 7 of the 10 chemicals
tested, a second experiment was run inwhich numbers of cystocarps
and numbers of tetrasporangia were counted. Vegetative growth of
females and tetrasporophytes was recorded as final dry weight
after drying 48 hours at 80 C.
Chronic values were calculated using two different criteria
for determining significant differences. Statistical significance
for differences in vegetative growth and number of reproductive
structures per mg dry weight was calculated using analysis of
variance followed by Dunnett's multiple range test (Steel and
Torey, 1960); alpha level was 0.05. Difference was also
determined by absence of reproductive structures. Results from
replicate flasks within an experimental run were pooled for
statistical analysis. Controls and carrier controls did not
differ statistically and were pooled. Although stimulation of
either growth or reproduction was evident for lower concentrations
of some compounds, only inhibition was considered for this study.
* Mention of trade names does not imply the endorsement
of the U.S. Environmental Protection Agency.
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Maximum allowable toxicant concentration (MATC) values were
calculated as the geometric mean of the lowest concentration that
resulted in a significant decrease from the control and the next
lowest concentration (Buikema et al., 1982). By definition the
MATC refers to the most sensitive endpoint, therefore, the end-
point used to calculate the MATC may vary from chemical to
chemical.
Six of the ten test chemicals required an organic solvent.
Stock solutions of pentachlorophenol, 2,4,5-trichlorophenol,
pentachloroethane, endosulfan, naphthalene, and
2,4-dichlorophenoxyacetic acid (2,4-D) were prepared in
triethylene glycol (TEG). Phenol was dissolved in deionized water
and toxaphene was dissolved first in acetone and then diluted with
TEG to give a final acetone to TEG ratio of 1 mL in 20, Only one
stock solution per chemical was prepared to minimize dilution
errors. Therefore, solvent concentrations were proportional to
toxicant concentrations in test solutions. Isophorone and benzene
were dispensed directly from their bottles. All toxicant
concentrations were obtained by dispensing from stock solutions
with adjustable micropipets.
Range-finding and definitive experiments were performed for
-each toxicant. Range-finding experiments covered a broad range of
concentrations (eg 1000 to 100,000 ug/L). The highest
concentration in the definitive run was selected to cause death or
a near death response (except for 2,4-D, where the highest
range-finding concentration was used as the highest definitive
concentration). In the definitive run the dilution factor was
0.6. Carrier controls (1200 uL/L—the highest concentration of
carrier used with a toxicant) were used with those toxicants that
required a carrier for solubility. Water samples were not
chemically analyzed for toxicant concentrations, therefore, all
concentrations are concentrations added. For isophorone, benzene
and pentachloroethane (all liquids) density was used to calculate
the weight of the compound in solution.
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RESULTS
Effects of the ten test chemicals on Champia parvula sexual
reproduction, tetrsporangia formation and female and
tetrasporophyte growth are shown in Figures 1 to 10. All results
are expressed as percent of the control. In general, response
curves for females and tetrasporophytes are similar for all ten
compounds tested. Each compound also showed a good concentration
response curve.
There was considerable variation in the average dry weight of
the control plants among the experiments (Table 1). This
reflected the seasonal change in the quality of seawater from
lower Narragansett Bay. Champia generally exhibits pooer growth
in water collected during the months of June, July and August than
during the rest of the year. A complete explanation for this
phenomenon has not been found, although the effect is more
pronounced following rain storms. This may be due to increased
river flow into the bay, carrying with it larger amounts of
pollution from the upper bay region. However, when replicate
experiments were performed (at different time of the year)
response curves were similar.
, The upper limit for chronic values for females and
tetrasporophytes are listed in Table 2 for growth, number of
repro'ductive structures per mg dry weight and absence of
reproductive structures. The compounds are listed in order of
most to least toxic. There was no endpoint that was consistently
more sensitive than the others. Chronic values based on statistical
differences in growth of females were less than or equal to those
for tetrasporophytes for only 6 of the 15 experimental runs.
Chronic values for number of tetrasporangia per mg dry weight were
less than or equal to those for number of cystocarps for 6 of the
7 chemicals for which this data was gathered. Number of
cystocarps resulted in chronic values that were always less than
or equal to those for absence of tetrasporangia. Concentrations
of test chemicals that resulted in absence of reproductive
structures were generally greater than or equal to those for the
other four end points. Chronic values for aquatic animals and
microalgae are compared to MATC valuse for Champia in Table 3.
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DISCUSSION
Comparison of Ghampia endpoints
No endpoint was consistantly more sensitive than any other.
Although, concentrations of organic chemicals where reproductive
structures in females were absent were less than or equal to those
for tetrasporophytes (except for 2,4-D). The ranking of the
compounds from most to least toxic was-similar reguardless of
which endpoint was used. This suggested that the sensitivities
of females and tetrasporophytes to these toxicants were similar.
In contrast sexual reproduction was the most sensitive endpoint
in Champia parvula for heavy metals and cyanide (Steele and
Thursby, 1983).
Seasonal variation in the quality of the water form lower
Narragansett Bay has been documented (Smayda, 1974; Hanisak,
1979), although the months of lesser quality were not always the
same as for Champia. Similarity in Champia response curves from
different times of the year suggested that the variation in the
jwater quality may not have played a major role in this study.
However, an artificial seawater medium suitable for the growth of
Champia would eliminate the possibility of water quality
interference, as well as make the test procedure available to
laboratories without ready access to clean natural seawater.
Although none of the end points were difficult to quantify,
counting cystocarps was less labor intensive than counting
tetrasporangia; there are as many as 1000 tetrasporangia per
tetrasporophyte vs usually less that 25 cystocarps per female.
Cystocarps could be counted with the unaided eye. However,
tetrasporangia required the use of a stereo microscope.
Tetrasporophytes were also more troublesome to maintain in
culture. They continuously release tetraspores, some of which
attach and germinate on the parent thallus. Eventually the
resulting gametophytes become indistinguishable from branches of
the tetrasporophyte. Therefore, there is the potential for
confusion when cutting branch tips for toxicity tests.
Although chronic -values for the absence of cystocarps were
not smaller than those for either number of cystocarps per mg dry
weight or growth of females, they were often the same or close.
Absence of cystocarps is the simplest endpoint to quantify, as it
does not require time-consuming counting or drying and weighing.
Production of cystocarps as an endpoint also does not leave any
doubt as to whether the observed difference was biologically
significant, since absence of cystocarps directly eliminates
sexual reproduction in the species. Thus absence of cystocarps
may be a useful endpoint for screening toxicants for relative
toxicities, particularly since the ranking of the organic
toxicants from most to least toxic by this endpoint was simialr
to that for the other endpoints.
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Sensitivity of Champia Relative to Other Species
Direct comparison of the sensitivity of Ghampia with those of
other species is difficult because of the different methods used.
Many of the animal tests were performed in flow-through systems in
which the actual concentrations of the toxicants were measured
(USEPA, 1980a-i). Champia parvula tests were static and exposure
concentrations were not mesured. However, it is important to note
that the animals tested were more sensitive than Champia for only
phenol and pentachloroethane. Champia was among the most
sensitive for isophorone, benzene, naphthalene and 2,4-D. In
addition, the geometric mean for the MATC for Champia could not be
calculated for six of the test chemicals because the effect
concentration was the lowest tested. Thus, Champia may even be
more sensitive than indicated.
As with the animal data, is is difficult to compared
senstivities of microalgae with that of Champia parvula. For
microalgae a variety of end points were used; including EC50's
for cell number, chlorophyll a content and oxygen evolution
(USEPA, 1980a-i, Walsh, 1972). Champia was generally more
sensitive than the microalgae that have been tested, and except
for 2,4,5-trichlorophenol and toxaphene absence of cystocarps was
a more sensitive end point than those used for microalgae.
- These results demonstrate the importance of conducting
chronic toxicity tests with previously overlooked macroalgae. The
test is easily conducted, sensitive and reproducable. Champia is
as sensitive to organic chemicals and metals as animals and almost
always more sensitive than phytoplankton.
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Table 1. Means for controls of the various endpoints for
Champia parvula. A and B for tetrasporophyte growth refer to
duplicate experiments. Experiment B only lasted 11 days (this
is the experiment from which the tetrasporangia were counted).
ENDPOINT RANGE MEAN
(AVERAGE + 3D)
GROWTH
females
tetrasporophytes
4.83-19.64
A 4.46-12.35
B 2.20- 5.24
8.02 + 3.98
7.40 + 2.61
3.10 + 1.14
REPRODUCTIVE STRUCTURES/
MG DRY WT
^
cystocarps 2.2-5.4 3.6 + 1.5
tetrasporangia 146-221 181+29
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TABLE 2. Chronic values (yg/L) for Champia parvula based on growth (final
dry weight) and reproduction of females and tetrasporophytes. Numbers represent
the lowest concentration tested that was statistically different from the
control (this is the upper limit for the chronic value, the next lowest
concentration was 60% of this value). A and B refer to replicate experiments
with the same compound.
Compound
Toxaphene
Endosulfan
Pentachlorophenol
Naphthalene
2 , 4 ,5-Trichlorphenol
Pentachloroethane
Phenol
2 , 4-Dicholrophenoxy-
acetic acid
Benzene
Isophorone
GROWTH
FEMALE TETRA
A 23a
B 39
47a
360
A 2210
B nd
A 1290
B nd
A 7900
B 4700
A 21,600
B 21,600
21,600
A 57,100
B 94,890
A 83,070
B 83,070
23a
14a
130
240
2210
695a
2040
1300
4700
7900
13,000
7800a
36,000
34,260a
57,100
49,840
49,840
#CYSTO.
ndb
39
nd
nd
nd
2210
nd
1290
nd
4700
nd
7800a
nd
nd
57,100
nd
83,070
REPRODUCTION
#TETRASPL +CYSTO.
nd
65
nd
nd
nd
1160
nd
780a
nd
1680
nd
7800a
nd
nd
34,260a
nd
49,840
108
180
360
600
2210
nd
5960
nd
13,100
13,100
36,000
21,600
MOO, 000
94,890
94,890
83,070
138,450
4-TETRASP .
180
180
600
1100
2210
3220
9460
10,000
13,100
21,840
60,000
60,000
100,000
57,100
94,890
138,450
138,450
a Lowest concentration tested.
b No data.
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Table 3. Summary of chronic data for animals and data on microalgae (mostly for US
Environmental Protection Agency's Water Quality Criteria Documents, 1980) compared
with MATC values for Champia parvula. The MATC values for Champia were calculated
based on the lowest chronic value for each chemical from Table 2.
Compound ANIMALS MICROALGAE
# Species MATC(ug/L)a $Species Resultsb( ug/L)
Toxaphene 20
Endosulfan 4
Pentachlorophenol 10
Naphthalene 2
2 , 4 ,5-Trichlorophenol
Penta'chlprae thane 2
Phenol ' 4
2,4-Dichlorophenoxy-
acetic acid 1
Benzene 1
Isophorone 1.
0.037-15.2
0.28-108
3.2-510
620-1000
ndd
281-1100
70-3120
49,000
>98,000
110,000
7C 0.15-1000
2C 1000-10,000
5 7.5-293
2 5000-43,400
3 890-10,000
2 58,200-134,000
4 20,000-1,500,000
4 50,000-75,000
5 20,000-525,000
2 105,000-126,000
CHAMPIA
MATC (ug/L)
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Figure 1. Effect of toxaphene on growth (mg dry weight) of females
and tetrasporophytes in duplicate tests A and B (solid lines)
and on number of cystocarps and tetrasporangia per me dry weight
(dashed lines) for Champia parvula. The lowest concentration
resulting in a response statistically less than the control id
denoted by an asterisk. Cystocarps and tetrasporangia were not
counted in experiment A. However, the concentration where -
females failed to produce cystocarps is noted by NC, and where
tetrasporophytes failed to produce tetrasporangia by NT.
Figure 2. Effect of endosulfan on growth (mg dry weight) of females
and tetrasporophytes of Champia parvula. The lowest concentration
resulting in a response statistically less than the control is
denoted by an asterisk. Cystocarps and tetrasporangia were not
counted. Hwever, the concentration where females failed to
produce cystocarps is noted by NC, and where tetrasporophytes
failed to produce tetrasporangia by NT.
Figure 3. Effect of pentachlorophenol on growth (mg dry weight) of
females and tetrasporophytes of Champia parvula. The lowest
concentration resulting in a response statistically less than
the control is denoted by an asterisk. Cystocarps and tetrasporangia
were not counted. However, the concentration where females
failed to produce cystocarps is noted by NC, and where
tetrasporophytes failed to produce tetrasporangia fay NT.
Figure 4. Effect of naphthalene on growth (mg dry weight—solid
lines) of females and tetrasporophytes (A and B refer to duplicate
tests) and number of cystocarps and tetrasporangia per mg dry
weight (dashed lines) for Champia parvula. Tetrasporangia were
not counted in experiment A, however, the concentration where
tetrasporophytes failed to produce tetrasporangia is denoted by NT,
Figure 5. Effect of 2,4,5-trichlorophenol on growth (mg dry weight—
solid lines) of females and tetrasporophytes (A and B refer to
duplicate tests) and number of cystocarps and tetrasporangia per
mg dry weight (dashed lines) for Champia parvula. Tetrasporangia
were not counted in experiment A, however, the concentration
where tetrasporophytes failed to produce tetrasporangia is
denoted by NT.
Figure 6. Effect of pentachloroethane on growth (mg dry weight) of
females and tetrasporophytes in duplicate tests A and B (solid
lines) and on number of cystocarps and tetrasporangia per tag dry
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PAGE 17
weight (dashed lines) for Champia parvula. The lowest concentration
resulting in a response statistically less than the control is
denoted by an asterisk. Cystocarps and tetrasporangia were not
counted in experiment A. However, the concentration where
females failed to produce cystocarps is noted by NC, and where
tetrasporophytes failed to produce tetrasporangia by NT.
Figure 7. Effect of phenol on growth (rag dry weight) of females and
tetrasporophytes in duplicate tests A and B (solid linesO and on
number of cystocarps and tetrasporangia permg dry weight (dashed
lines) for Champia parvula. The lowest concentration resulting
ina responce statistically less than the control is denoted by
an asterisk. Cystocarps and tetrasporangia were not counted in
experiment A. However, the concentration where females failed
to produce cystocarps is noted by NC, and where tetrasporophytes
failed to produce tetrasporangia by NT,
figure 8. Effect of 2,4-diclorophenoxyacetic acid on growth (mg dry
weight) of females and tetrasporophytes of Champia parvula. The
lowest concentration resulting in a response statistically less
than the control is denoted by an asterisk. Cystocarps and
tetrasporangia were not counted. However, the concentration
where females failed to produce cystocarps is noted by NC, and
where tetrasporophytes failed to produce tetrasporangia by NT.
Figure 9. Effect of benzene on growth (mg dry weight) of females and
tetrasporophytes in duplicate tests A and B (solid lines) and on
number of cystocarps and tetrasporangia per mg dry weight (dashed
lines) for Champia parvula. The lowest concentration resulting
in a response statistically less than the control is denoted by
an asterisk. Cystocarps and tetrasporangia were not counted in
experiment A. However, the concentration where females failed
to produce cystocarps is noted by NC, and where tetrasporophytes
failed to produce tetrasporangia by NT.
Figure 10. Effect of isophorone on growth (mg dry weight) of females
and tetrasporophytes in duplicate tests A and B (solid lines)
and on number of cystocarps and tetrasporangia per mg dry weight
(dashed lines) for Ghampia parvula. The lowest concentration
resulting in a response statistically less than the control is
denoted by an asterisk. Cystocarps and tetrasporangia were not
counted in experiment A. However, the concentration where
females failed to produce cystocarps is noted by NC, and where
tetrasporophytes failed to produce tetrasporangia by NT.
-------
120
100
80
60
40
O 20
p-
§ °
o
FEMALE
i i i i i i i
20 30 40 50
100
200
TETRASPOROPHYTE
o
10 20 30 50
TOXAPHENE
100
Figure 1.
-------
100
80
60
40
O 20
cr
K
§ o
CJ
£100
80
60
40
20
0 L
50
FEMALE
100 200 300 500
TETRASPOROPHYTE
50 100 200 300
ENDOSULFAN (>/g/L)
500
Figure 2.
-------
CONTROL
U.
0
52
100
80
60
40
20
0
100
80
60
40
20
n
*^^
•x. FEMALE
\
\
\NC
200 300 500 1000 2000
-
*^ . TETRASPOROPHYTE
\
\
*\.
*\
\ NT
t 1 T 1 1 t ! ) 1 f A i
200 400 500 1000 2000
PENTACHLOROPHENOL ( >/q/L)
Figure 3.
-------
FEMALE
2 345
NAPHTHALENE (103>/g/L)
Figure 4.
-------
100
80
60
40
_i
O 20
tu
°
o 100
80
60
40
20
2 345
TETRASPOROPHYTE
i i i
.71 2345 10
2,4,5-TRlCHLOROPHENOL (l03^yg/L)
Figure 5.
-------
20
100
80
60
40
o
fe
- 0
FEMALE
00
80
60
40
20
* B
20 30 40
TETRASPOROPHYTE
2345
PENTACHLOROETHANE
20 30 40
Figure 6.
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o
tr
o
o
140
120
100
80
60
40
20
100
80
60
40
20
0
FEMALE
10
20 30 40 50
100
TETRASPOROPHYTE
10 20 30 40 50
PHENOL (l03^/g/L)
00
Figure 7.
-------
100
80
60
40
.0 20
o u
o
o 100
80
60
40
20
10
0 L ^
I I 1
10
FEMALE
slight reduction
in number of
cystocarps
t t i i t t i
20 30 40 50 100
^ TETRASPOROPHYTE
NT
i 1 1 1 1 1 1
20 30 40 50
100
2,4-DlCHLOROPHENOXYACETlC ACID (!0°//g/L)
Figure 3.
-------
O
-------
160
140
120
100
80
60
§ 40
8 20
u.
o n
100
80
60
40
20
0
30 40 50
FEMALE
100
200 300
TETRASPOROPHYTE
30 4050 100
ISOPHORONE (I03
200 300
Figure 10.
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PAGE 28
REFERENCES
Buikema, A.L., Jr., B.R. Niederlehner, and J. Cairns, Jr. 1982.
Biological Monitoring. Part IV, Toxicity Testing; Water
Res. 16:239-262.
Guillard, R.R.L. 'and J.H. Ryther. 1962. Studies on marine
planktonic diatoms: I. Cyclotella nana Hustedt and Detonula
confervaces (Cleve) Gran. Can J. Microbiol. 8:229-239"!
Kenaga, E.E. and R.J. Moolenaar. 1976. Fish and Daphnia
toxicity as surrogates for aquatic vascular plants and algae.
Environmental Sci. Technol. 13:1479-1480.
Kenaga, E.E. 1982. The use of environmental toxicology and
chemistry data in hazard assessment: Progress, needs,
challenges. Environ. Toxicol. Chem. 1:69-79.
Kusk, K.O. 1981. Effect of hydrocarbons on respiration,
photosynthesis and growth of the diatom Phaeodactylum
tricornutum. Bot. Mar. XXIV:413-418.
^
Steel, R.G. and J.H. Torrie. 1960. Principles and procedures
"of statistics. 481 pp.
Steele, R.L. and G.B. Thursby. 1983. A toxicity test using
life stages of Champia parvula (Rhodophyta). pp. 73-89 (in)
W.E. Bishop, R.G. Cardwell and B.B. Heidolph (eds) Aquatic
Toxicology and Hazard Assessment: Sixth Symposium. ASTM STP
802. American Society for Testing and Materials,
Philadelphia.
U.S. Environmental Protection Agency. 1980a. Ambient Water
Quality Criteria for Isophorone. EPA report 440/5-80-056.
U.S. Environmental Protection Agency. 1980b. Ambient Water
Quality Criteria for Benzene. EPA report 440/5-80-018.
U.S. Environmental Protection Agency. 1980c. Ambient Water
Quality Criteria for Naphthalene. EPA report 440/5-80-059.
U.S. Environmental Protection Agency. 1980d. Ambient Water
Quality Criteria for Phenol. EPA report 440/5-80-066.
U.S. Environmental Protection Agency. 1980e. Ambient Water
Quality Criteria for Chlorinated Phenols. EPA report
440/5-80-032.
U.S. Environmental Protection Agency. 1980f. Ambient Water
Quality Criteria for Pentachlorophenol. EPA report
440/5-80-065.
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PAGE 29
U.S. Environmental Protection Agency. 1980g. Ambient Water
Quality Criteria for Endosulfan. EPA report 440/5-80-046.
U.S. Environmental Protection Agency. 1980h. Ambient Water
Quality Criteria for Chlorinated Ethanes. EPA report
440/5-80-029.
U.S. Environmental Protection Agency. 1980i. Ambient Water
Quality Criteria for Toxaphene. EPA report 440/5-80-076.
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PAGE 30
PART II.
CHAMPIA PARVULA TEST GUIDELINE
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PAGE 31
CHAMPIA CHRONIC TOXICITY TEST
I. PURPOSE
This guideline describes procedures for obtaining laboratory
data concerning adverse effects of a chemical or mixture of
chemicals on life history stages of the marine red alga Champia
parvula. Plants are exposed to a range of concentrations of a
chemical for 11 to 14 days in a static renewal system. Vegetative
growth as well as sexual and asexual reproduction are measured as
endpoints. For the purpose of this document toxicity tests with
females and tetrasporophytes are considered as separate test runs.
II. SIGNIFICANCE
This document provides guidance for designing toxicity test
•with macroalgae. Macroalgae represent a different ecological
niche from microalgae. They are generally sessile and also
represent a different food source from microalgae, contributing
primarily to detrital food chains. Currently, the only other
algal toxicity tests routinely employ planktonic microalgae. The
level of expertise required to use Champia parvula is
approximately equal to that necessary to run microalgal toxicity
tests. The Champia chronic test is not intended to replace
microalgal tests but rather to complement them by increasng the
number of types of plants included in marine toxicity testing.
Results from this test will provide information on the
relative sensitivity of macroalgae in relation to test species
from other trophic levels. The toxicity test with Champia can
evaluate simultaneously the effects of compounds on vegetative
growth (haploid and diploid life history stages) and on
reproduction. The test is relatively fast and inexpensive—only .
to 4 man hours are needed each week to maintain Champia stock
cultures, and only 10 to 12 are required to complete the testing
of any given toxicant. The test requires only a small volume of
water. Clonal material is used and is available year round from
stock cultures. Prior tests indicate that Champia is often as
sensitive or more so than the most sensitive aquatic animal, and
is almost always more sensitive than microalgae. These results
demonstrate the importance of conducting chronic tests with the
previously overlooked macroalgae.
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PAGE 32
III. TEST CONDITIONS
A. Test Species
1. Selection
The test species for all tests is Champia parvula (C.
Agardh) Harvey. Most toxicity tests to date have been performed
with one standard Champia clone, although it may not be essential
to have a standard clone.
2. Source
New clones can be started from field material, however, some
experience is required to isolate material into unialgal culture.
Vegetatively propagated clones of the standard isolate are
available from Glen Thursby and Richard Steele at the U.S.
Environmental Protection Agency, South Ferry Road, Narragansett,
RI 02882. Plans also are being made to store life history stages
of this clone at the culture collection of algae at the University
of Texas (Starr, 1978).
3. Maintenance of Stock Cultures
Unialgal stock cultures of each of the life history stages
are maintained in 800 mL of natural seawater in aerated 1000 mL
Erlenmeyer borosilicate flasks. The nutrients added to the
seawater are listed in Table 1. Seawater for culture media should
be collected on an incoming tide if collected from coastal areas.
Champia will not grow well in most artificial seawater recipes,
however, the seawater recipe and medium listed in Tables 2-4 are
acceptable. Cultures are gently aerated through sterile,
disposable, polystyrene 1 mL pipettes. Cultures are illuminated
from the side with 75 uE.m~^.s~^ of cool-white fluorescent light
on a 16:8, light:dark cycle. The temperature is 22 to 24 C.
Media are changed once a week. All three plants of the life
history can be maintained in separate unialgal cultures. New
cultures can be started from excised branches, making is possible
to maintain clonal material indefinitely. Thus, plant material
can be available at any time for toxicity tests.
In order to keep a constant supply of plant material
available it is recommended that several cultures of each life
history stage be maintained simultaneously. Each culture should
be at different stages of development (i.e., with different
amounts of tissue per flasks). Initial stock cultures should be
started weekly with ca. 20 0.5- to 1.0-cm branch tips. To reduce
the amount of biomass as the plants grow, about half of the plants
should be discarded (or placed into another culture vessel) with
each weekly medium change. At the end of three weeks plants will
be ready to use in toxicity test. Readiness is defined as having
enough plant material to perform at least one toxicity test. With
this procedure, actively growing plants will be continuously
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PAGE 33
available. The total number of cultures maintained will, of
course, depend on the expected frequency of toxicity testing.
B. Test Procedure
1. Range-Finding Tests
The range for definitive tests will normally be chosen from a
range-finding test. Range-finding experiments generally cover a
broad range of concentrations (eg. 10 to 10,000 ug/L). These
test are set up and run similar to definitive tests, but are
usually shorter (3 to 7 days), with no media renewals and with
only one flask per treatment. The end point examined is the
presence of necrotic tissue or death.
2. Definitive tests
a). Test Concentrations
The highest concentration in the definitive run is selected
to cause death or a near-death response. All other concentrations
should be at least 60% of the next highest concentration. At
least 5 concentrations are used (plus control).
b). Loading
All plants for a toxicity test should come from the same
stock culture to minimize differences in pre-conditioning. After
plants are rinsed in sterile seawater, to remove traces of old
media, 2- to 3-mm branch tips are cut to serve as innocula for a
toxicity test. The tips are easily cut with fine-point, stainless
steel forceps. A stereomicroscope is not necessary, but can help
minimize tip-to-tip variation .
Five tips of the life history stage in question are
transferred by Pasteur pipette into each test flask. All test are
performed in duplicate. One male branch (approximately 1.5 cm
long), visably producing spermatia, is added to each flask
containing females. To assure that any fertilization of females
takes place in the presence of the toxicant males must be added
after the toxicant. When media are changed, the male plants ae
clipped back to ca 1.5 cm , and any plants that are sticking
together are separated.
A culture of Champia should not be used as a source of test
material if:
(1) the tips of the branches turn pink relative to
the older tissues;
(2) the entire plant turns pale yellow;
(3) there is evidence of necrotic tissue (white areas);
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PAGE 34
(4) stock cultures become contaminated with microalgae;
(5) cystocarps are present on females.
c). Controls
Controls are required for every test. Carriers should only
be used when they are necessary to solubilize hydrophobic
compounds. Triethylene glycol (TEG) has been used with success
with Champia. At least one carrier control treatment must be run
using the highest volume of carrier that is used in the toxicant
treatments. A control without TEG should also be run as a check
on the carrier control.
d). Endpoints
The endpoints examined at the termination of a toxicity test
are: number of tetrasporangia per mg dry weight (asexual
production of spores by meiosis); number of cystocarps per mg dry
weight (evidence of sexual reproduction); and vegetative growth
or tetrasporophytes and females (as measured as final dry weight).
The toxicant concentration that completely inhibits reproduction
is also noted.
Tetrasporangia and cystocarps are counted by placing
tetrasporophytes or females between the inverted halves of a
polystyrene petri dish with a small volume of seawater (to hold
the entire plant in one focal plane). Using a stereomicroscope,
individual reproductive structures can "be easily observed. After
the above examinations are completed, plants are rinsed with
deionized water, oven-dried at 80 C (24 to 48 h), and weighed
+0.01 mg. Separation of individual plants must be maintained so
number of reproductive structures per mg dry weight can be
calculated for each plant.
e). Unacceptable Tests
Test runs should be repeated if:
(1) more than two of the ten control plants die;
(2) control plants in either flask exhibit any of
the characteristics deemed unacceptable in
stock cultures;
(3) the average final dry weight of pooled female
control plants is less than 4.00 mg;
(4) the average final dry weight of tetrasporophyte
pooled control plants is less than 3.00 mg;
(5) no sexual or asexual reproduction occurrs in
controls (this test must be repeated);
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PAGE 35
(6) the two replicate flasks at the toxicant
concentration determined to be the effe'ct
level are statistically different;
(7) carrier controls and controls are not statistically
equal.
D. Facilities
1. Exposure System
All range-finding and definitive test are performed in 400 mL
of medium in screw-capped 500 mL Erlenmeyer flasks (caps are
fitted with a teflon liner). However, silicone-rubber-stoppered
flasks can be used. Flasks are shaken on a rotary shaker at 100
rpm. Toxicity tests last 11 days for the tetrasporophytes and 14
days for the females. All the seawater medium for a given test is
mixed in a single batch to eliminate nutrient variation among the
flasks. The medium for the toxicity test in natural seawater is
1/4-strength of that listed in Table 1. For artificial seawater
the medium listed in Table 2 is used. The media are renewed on
day 7 for the tetrasporophyte and days 7 and 11 for the female.
2. Dilution Water
The seawater used for testing should be the same source as
that for stock cultures.
3. Cleaning Glassware
Organic toxicants
(1) rinse with acetone from squeeze bottle (10 to 15 mL);
use fume hood.
(2) empty acetone into metal safety waste can
(3) fill flask with tap water and rinse three times
(4) rinse flasks with 10 to 15 mL of concentrated sulfuric
acid, followed by tap water rinses
(5) wash flasks in mild detergent followed by deionized
water rinses
(6) soak flasks in 10% HC1 followed by deionized water
rinses (this removes any detergent residue which
can be toxic to Champia). (note: teflon liners
of caps are also acid stripped in 10% HC1, but
screw-caps are not acid stripped. Acid treatment
followed by autoclaving makes the plastic brittle.
Caps are just rinsed well with deionized water.)
(7) Autoclave loosely capped for 10 min
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PAGE 36
Heavy Metals
Only steps 5 through 7 above are necessary with the following
exception; flasks are soaked in 10% HC1 overnight (at step 6).
E. Environmental Conditions
1. Light
Light is supplied at 75 uE.nT^s"1 (ca 500 ft candles) with
cool-white fluorescent lamps on a 16h:8h, light:dark photoperiod.
Light irradiance is measured among the test flasks at the platform
surface of the shaker.
2. Temperature
The temperature should be maintained between 22 to 24°C.
3. Salinity
The salintity should be between 28 to 30 ppt.
4. pH
The pH should be between 7.8 and 8.2.
F. Reporting
1. Calculating Chronic Values
Chronic values are calculated as the geometric mean of the
lowest concentration that results in a significant difference from
the control and the next lowest concentration. If the lowest
concentration that results in a significant difference is the
lowest concentration tested, then one can only report that
concentration. Chronic values are determined for vegetative
growth and number of reproductive structures per mg dry weight
using statistical significance as the criterion for difference
(analysis of variance and mean separation test). Significant
difference from the control can also be determined as absence of
reproductive structures. Maximum allowable toxicant concentration
(MATC) is the chronic value from the most sensitive end point
measured.
2. Documentation
The following should be provided for each test:
(1) Name of investigator, laboratory and test dates.
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PAGE 37
(2) A description of the test chemical including
source, composition and chemical and physical
properties.
(3) Identification of carrier, if any, and maximum
concentration used.
(4) The source of dilution water.
(5) The source of Champia used.
(6) Analytical methods, used to verify toxicant
concentrations, if analyzed.
(7) Dry weight, number of reproductive structures
and number of reproductive structure per mg
dry weight for each plant.
(8) Calculated chronic values and reference to the
statistical method used.
(9) Any deviation from these procedures.
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PAGE 38
Table 1. Recipe for nutrient medium for growth of Champia parvula
in natural seatwater. Both EDTA and trace elements have been
omitted.
COMPOUND CONCENTRATION PER LITER
FINAL SOLUTION CONCENTRATED STOCK3
NaN03
NaH2P04'H20
37.4 rag
2.48 mg
3.74 grams
0.25 grams
Ironb 10 ug 1.00 mg
Vitamins
B12 0-24 ug
Biotin 0.24 ug 10
Thiamine-HCl 50 ug
a Use 10 mL/L for final concentrations. Add
150 mg/L sodium bicarbonate when cultures are not aerated.
A stock solution of 60 mg/mL NaH2C03 is prepared by auto-
claving sodium bicarbonate as a dry powder and then
dissolving it in sterile deionized water. Use 2.5 mL/L
for the final concentration.
b Iron stock solution prepared by dissolving 1 g iron powder
in 10 mL concentrated HC1 and diluting to 1 liter with
deionized water.
c Vitamin stock solution autoclaved separately in 10 mL
sub-samples. Each 10 mL contains 24 ug B^2, 24 ug biotin
and 5 mg thiamine'HCl.
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PAGE 39
Table 2. Recipe for artificial seawater based on major salts from
Provasoli's ASP-1 (Provasoli, 1963).
COMPOUND GRAMS/LITER
NaCl
KC1
MgS04'7H20
MgCl2'6H20
CaCl2-2H20
24.0
0.6
6.0
4.5
1.5
Sodium bicarbonate (300 mg/L) is added as a pH buffer and carbon
source in lieu of aeration. The bicarbonate is added after
autoclaving the ASP-1 (this prevents precipitation in the ASP-1),
A stock solution of 60 mg/mL NaH2C03 is prepared by autoclaving
sodium bicarbonate as a dry powder and then dissolving it in
sterile deionized water.
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PAGE 40
Table 3. Recipe (PES-M) of the modified Provasoli's enrichment
solution (Provasoli, 1968) for culturing Champia in artificial
seawater.
COMPOUND AMOUNT PER LITER
FINAL SOLUTION CONCENTRATED STOCKa
17.5 mg 7.0 grams
NaH2P04'H2Ob 1.1 mg 0.44 grams
Na2EDTA'2H20 2.08 mg 830 mg
Iron0 25 ug 50 mg
PII Trace Elements
FeCl3'6H20
MnS04'H2Od
ZnS04'7H20
CoS04'7H20
H3B03
Vitamins
B12
Biotin
Thiamine-HCl
61 ug
154 ug
28 ug
6 ug
1425 ug
0.5 ug
0.25 ug
25 ug
24.5 mg
61.5 mg
11.0 mg
2.4 mg
0.57 grams
10 mLe
a Use 2.5 mL per liter for final concentrations
k Original recipe uses sodium glycerophosphate
c From a 1 mg/mL iron stock solution. One g iron powder
dissolved in 10 mL concentrated HC1 and diluted to 1
liter with deionized water. Original recipe called for
Fe(NH4)S04'6H20.
d Original recipe called for MnS04.4H20.
e 200 ug B12, 100 ug biotin, and 10 mg thiamine'HCl per
10 mL of vitamine stock. Autoclaved, separate from rest
of nutrients, only 1 minute (i.e., just bring autoclave up
to pressure).
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PAGE 41
Table 4. Recipe for SII trace elements (Provasoli, 1963) for
culturing Champia in artificial seawater.
COMPOUND CONCENTRATION PER LITER
FINAL SOLUTION CONCENTRATED STOCK3
KBr
KI
NaMo04'2H20
LiCl
SrCl2'6H20
RbCl
14.8 mg
13.1 ug
1.3 mg
1.2 mg
6. 1 mg
0.28 mg
745 mg
654 ug
63 mg
61 mg
304 mg
14 mg
a Use 10 mL per liter for final concentrations. Add 1 mL
concentrated HC1 to stock solution before autoclaving.
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PAGE 42
PART III.
CHAMPIA TEST GUIDELINE SUPPORT DOCUMENT
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PAGE 43
TABLE OF CONTENTS
PAGE
I. PURPOSE 44
II. SCIENTIFIC ASPECTS 44
A. General 44
B. Range-Finding Tests 46
C. Definitive Tests 46
III. TEST CONDITIONS 46
A. Test Species 46
1. Selection 46
2. Sources 47
3. Maintenance of Test Species 47
(a). Stock Cultures 47
(b). Nutrition—natural seawater 49
(c). Nutrition—artificial seawater 49
B . Test Procedures 50
1. Test Concentrations 50
2. Loading 50
3. Controls 51
4. Carrier Controls 52
C. Facilities 52
1. Exposure System 52
2. Dilution Water 53
3. Cleaning Glassware 53
D. Environmental Conditions 53
1. Light 53
2. Temperature 54
3. Salinity 54
4. pH 54
E. Reporting 54
IV. REFERENCES 56
Tables 58
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PAGE 44
TECHNICAL SUPPORT DOCUMENT OF CHAMPIA CHRONIC
TOXICITY TEST
I. PURPOSE
The purpose of this document is to provide the scientific
background and rationale used in the development of the Champia
Chronic Toxicity Test. The test procedure uses life stages of the
marine red macroalga Champia parvula to evaluate the toxicity of
chemical substances. The document describes studies to develop a
static-renewal exposure system and to examine the utility of
various end points. The test results encompass vegetataive growth
and both sexual and asexual reproduction.
II. SCIENTIFIC ASPECTS
A. General
New and exotic chemicals, as well as many known compounds are
being released into the air and water daily. Most of these
compounds find their way into the marine environment and need to
be tested as potential toxicants to different trophic levels of
marine organisms. Standardized toxicological test procedures have
been developed for fish, invertebrates, amphibians, and microalgae
(USEPA 1974, USEPA 1978, ASTM 1980). However, very little work
has been done toward the development of a standard procedure for
testing the effects of toxicants on macroalgae. Macroalgae have
been used to monitor pollution of heavy metals in the field
(Melhuus et al. 1978, Eide and Myklestad 1980, Levine and Wilce
1980). This type of study requires the use of relatively tolerant
species since the goal is to have an alga that will accumulate a
pollutant without dying (Phillips 1977). In toxicological
studies, the opposite is true. One looks for a relatively
sensitive organism; since, by protecting sensitive organism,
tolerant species should be protected.
In the open ocean phytoplankton are the primary producers,
however, inshore or estuarine production depends heavily on
contributions from macroalgae. Macroalgae represent a different
ecological niche than microalgae. They are generally sessile, and
as such may be more vulnerable to pollution than planktonic algae.
Macroalgae also represent a different food source than microalgae,
contributing primarily to detrital food chains (Mann 1972).
Macroalgae act as the basis for communities of other plants and
animals in providing living space and in some cases ameliorating
the effects of stress, both natural and man-made.
A relatively fast, simple and inexpensive static-renewal
toxicity test method has been developed for the marine red
macroalga Champia parvula. to assess chronic effects of pollutants
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PAGE 45
to marine seaweeds. The method has been used to generate toxicity
data for water quality criteria. It has previously been evaluated
with heavy metals and cyanide (Steele and Thursby, 1983), arsenite
and arsenate (Thursby and Steele, 1984), as well as with ten
different organic compounds (see Part I, this report). A static
test was chosen for several reasons. Flow-through systems are
generally more expensive, complicated and require more volume of
dilution water and toxicant. Flow-through systems also can not be
kept algal-free. At the light and temperature to grow Champia,
diatoms and blue-green algae become a serious biological
contamination problem. These in turn compete with Champia for
nutrients and light, thereby decreasing the growth of Champia.
Currently, the only other algal toxicity tests used routinely
employ planktonic microalgae. Microalgae are considered to be
less sensitive to toxicants than animals (Kenaga and Moolenaar,
1979; Kenaga, 1982). However, the above work with Champia shows
that it is as sensitive or more so than the most sensitive aquatic
animal, and is almost always more sensitive than microalgae. The
level of expertise required to use Champia is approxiamtely equal
to that necessary to run microalgal toxicity tests. The Champia
toxicity test is not intended to replace microalgal tests, but
rather to compliment them by increasing the number of types of
plants included in marine toxicity testing.
The toxicity test with Champia can simultaneously evaluate
the effects of compounds on vegetative growth (both haploid and
diploid life history stages) and on reproduction (both sexual and
asexual production of spores). An algistatic response has been
proposed by Payne and Hall (1979) as the best endpoint for
toxicity tests with microalgae. This endpoint would not be
useful with macroalgae since asexual and sexual reproduction can
be imparied before growth ceases (Steele and Thursby, 1983;
Thursby and Steele, 1984; see Part 1, this report). Similar
effects have been reported for oil in relation to sexual
reproduction in the brown algae Fucus edentatus and Laminaria
saccharina and for Champia parvula (Steele and Hanisak 1978,
Steele and Thursby 1981). Pollution assessment with macroalgae
must take into consideration reproduction if an accurate picture
of the potential harm is to be drawn.
The Environmental Protection Agency, under the mandate of the
Toxic Substance Control Act, is required to identify and control
chemicals that are hazardous to human health or the environment.
An important aspect of this responsibility is to develop
methodology for testing potential hazards to different trophic
levels of marine organisms. Data from these test must be both
scientifically sound and legally defensible. At the same time, it
is necessary for the methods used to be as simple and cost
efficient as possible. All of these requirements are met by the
test method developed for Champia parvula. In addition, because
of the relatively short time required to complete a given toxicity
test (in both man hours and total elasped time), Champia has the
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PAGE 46
potential to become a rapid screening tool for chronic effects
among the macroalgae.
B. Range-Finding Tests
Some idea of the concentration range for definitive tests can
come from the literature or from personal experience. However, .
Champia's sensitivity can vary from that of other species,
especially microalgae. In addition, little or no toxicological
information may be available for a particular compound.
Therefore, the range for definitive tests will normally be chosen
from a range-finding test. Range-finding experiments generally
cover a broad range of concentrations (eg. 10 to 10,000 ug/L).
These test are set up and run similar to definitive tests, but are
usually shorter (3 to 7 days), with no media renewals and with
only one flask per treatment. The endpoint examined is the
presence of necrotic tissue or death.
C. Definitive Tests
The results of the definitive test will be used to establish
any statistically significant difference between the treatments
and the control(s) pertaining to growth and reproduction. The
highest concentration in the definitive run is selected to cause
death or a near-death response. It is recommended that all other
concentrations be at least 60% of the next highest concentration
(ASTM 1980). At least 5 concentrations are used (plus control).
All definitive experiments are performed with duplicate flasks at
each treatment.
The endpoints examined at the termination of a toxicity test
are: number of tetrasporangia per mg dry weight (asexual
production of spores by meiosis); number of cystocarps per mg dry
weight (evidence of sexual reproduction); and vegetative growth
of tetrasporophytes and females (as measured as final dry weight).
Early tests showed that the dose response of the male was similar
to that of the femalee, therefore data from the growth of males
was judged unnecessary. In addition, females grow faster than
males, presumably because males put a lot of energy into
production of spermatia. Spore germination tests were found to be
too tedious, as well as less sensitive than reproduction, for use
in the final test protocol.
III. TEST CONDITIONS
A. Test Species
1. Selection
The test species for all tests is Champia parvula (C.
Agardh) Harvey. Plants are bushy and 5 to 10 cm tall in the
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PAGE 47
field. Main axes and branches are cylindrical, hollow and
septate. Champia has been chosen because of its ease of
maintenance in culture, its short life history and because it is
relatively sensitive to many toxicants.
Champia parvula is a common species in many parts of the
world (Taylor 1957, Abbott and Hollenberg 1976, Lewis 1973,
Reedman and Womersley 1976). It has been found in Mexico and
Southern California on the west coast of North America, and from
the Carribean to Massachusetts on the east coast. It has also
been reported from France, Spain, Korea and Southern Australia.
Because of its wide distribution, Champia may be a useful
surrogate species for determining the relative toxicity of a
variety of chemicals.
Three different isolates of Champia parvula, as well as two
other species of marine red algae (Agardhiella subulata and
Grinnellia americana), have been compared for their sensitivity to
phenol, toxaphene and isophorone (see Part V, this report).
Results from these comparisons show that the sensitivity of the
standard isolate of Champia (Steele and Thursby, 1983; Thursby
and Steele, 1984; see Part I, this report) is not restricted to
that particular isolate, and may be representative of red algae in
general.
2. Sources
Most Champia parvula toxicity tests to date have been
performed with one standard clone isolated from Rhode Island in
1979. Comparing results with one other clone from Rhode Island
isolated in 1981, as well as with an isolate from South Korea,
suggests that it is not essential to have a standard clone.
However, some experience is required to isolate new clones from
the field into unialgal culture. Vegetatively propagated clones
of the standard isolate are available from Glen Thursby and
Richard Steele at the U.S. Environmental Protection Agency, South
Ferry Road, Narragansett, RI 02882. Plans are being made to store
life history stages of this clone at the culture collection of
algae at the University of Texas (Starr, 1978). This would
guarantee future access to this clone.
3. Maintenance of Test Species
(a). Stock cultures
As with many red algae, Champia parvula has three free-living
isomorphic plants in its life history; haploid male and female
gametophytes and a diploid tetrasporophyte (Fig. 1). In culture
the entire life history takes approximately six weeks to complete.
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PAGE 48
All three plants of the life history can be maintained in separate
unialgal cultures. New cultures can be started from excised
branches, making is possible to maintain clonal material
indefinitely. No special preconditioning is required to induce
reproduction. Under the conditions described below, male
gametophytes produce spermatia continuously, female gametophytes
are always receptive, and tetrasporophytes constantly produce
tetrasporangia. Thus, plant material can be available at any time
for toxicity tests.
Laboratory cultures of Champia provide test plants of similar
preconditioning. All individuals used in a test should be from
the same stock culture to minimize variability of test results.
Unialgal stock cultures of each of the life history stages are
maintained in aerated 1000 mL Erlenmeyer borosilica flasks
containing 800 mL of natural seawater (30 ppt salinity). The
choice of these culture vessels is one of preference rather than
necessity. Champia will grow equally well in polycarbonate or
flint glass vessels. Seawater is autoclaved in 20 liter linear
polyethylene carboys for 20 min at 15 psi after the salinity in
adjusted with deionized water (if necessary) to 30 ppt. The
culture flasks are autoclaved dry, capped with aluminum foil, for
10 min. Culture medium is made up by dispensing seawater from the
20 liter carboys into the sterile flasks and adding nutrients from
a sterile stock solution (see below). Sterile seawater, flasks
and nutrient stock soultion are used to prevent microalgal
contamination, not to keep cultures bacteria free. Cultures are
gently aerated through sterile, cotton-plugged, disposable,
polystyrene 1 mL pipettes. Cultures are illuminated from the side
with 75 uE'm *s~l of cool-white fluorescent light on a 16:8,
light:dark cycle. The temperature is 22 to 24°C. Media are
changed once a week.
In order to keep a constant supply of plant material
available it is recommended that several cultures of each life
history stage be maintained simultaneously. Each culture should
be at different stages of development (i.e., with different
amounts of tissue per flasks). Initial stock cultures should be
started weekly with ca. 20 0.5- to 1.0 cm branch tips. To reduce
the amount of biomass as the plants grow, about half of the plants
should be discarded (or placed into another culture vessel) with
each weekly medium change. At the end of three weeks plants will
be ready to use in toxicity test. Readiness is defined as having
enough plant material to perform at least one toxicity test. With
this procedure, actively growing plants will be continuously
available. The total number of cultures maintained will, of
course, depend on the expected frequency of toxicity testing.
A culture of Champia should not be used as a source of test
material if the plants appear stressed or undernourished. Under
conditions of stress the tips of the branches will turn "pink" and
the older tissues will generally be much paler. Under conditions
of nutrient deficiency (resulting usually from too much plant
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PAGE 49
material in the culture flask or too long since the last medium
change) the entire plant will turn pale yellow. If the stress is
severe enough the older tissues (main axes) or occasionally the
branch tips will turn white (evidence of necrotic tissue).
Cultures also should not be used if they become contaminated with
microalgae. These will compete for nutrients in the test flasks
and reduce the light levels as they multiply, thus effecting the
growth rate of Champia. If cystocarps are present on females in
maintenance cultures then plants are not suitable for testing.
This would occur as a result of contamination with male tissue or
spermatia.
These criteria are designed to maximize the potential for a
successful test run. Although, Champia can usually recover in a
few days from most non-fatal causes of stress, stressed plants may
be extra sensitive to toxicants. Any enhanced sensitivity would
bias the results.
(b). Nutrition—natural seawater
The optimum natural seawater nutrient medium for stock
cultures (and Champia toxicity tests) is listed in Table 1.
Optimum nutrient concentrations are based on what is essential
from Guillard and Ryther's medium 'f (Guillard and Ryther, 1962),
Culture experiments followed procedures designed for toxicity
testing. Details of the results from these experiments are not
necessary here, and are reported elsewhere (see Part V, this
report).
(c). Nutrition—artificial seawater
Champia parvula experiences seasonal effects on growth and
vigor in culture,presumably due to changes in the quality of the
natural seawater used. The availability of an artificial seawater
medium would allow more consistency in water quality among
different experiments, as well as among different laboratories.
In addition, the use of an artificial medium would make toxicity
tests with Champia more useful by making it available to
laboratories without ready access to clean natural seawater. The
artificial seawater recipe used for Champia consist of the major
salts from Provasoli's ASP-1 (Table 2).
The bulk of artificial marine media have been designed for
work on microscopic algae, although several macroalgae have been
successfully grown in artificial seawater. The nutrient medium
designed for growth of Champia parvula in natural seawater is not
adequate for artificial seawater (Table 3). In addition, plants
in artificial seawater do not have the correct morphology; i.e. ,
septa formation is inhibited. Another nutrient medium, PES-M
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PAGE 50
(Table 4) gave slightly better results, but the morphology was
still incorrect (Table 3). In the presence of SII trace elements
(Table 5) Champia exhibited the correct morphology, although the
optimum natural seawater medium was still insufficient for good
growth in ASP-1 (Table 3).
The main difference between the optimum natural seawater
medium (which yields poor growth in ASP-1) and PES-M (which yields
good growth in ASP-1) is the presence of Pll trace elements, EDTA
and more iron in the later. Qualitative artificial seawater
experiments showed that adding all three of these to the optimum
natural seawater nutrients yielded plants similar in size,
morphology and reproductive capacity to plants grown in PES-M (in
the presence of SII trace elements for both media). Omitting any
one of the three 'extra1 components results in less than optimum
growth.
B. Test Procedure
1. Test Concentrations
Toxicants are added directly to treatment flasks and are
replenished when the media are renewed. Only one stock solution
is prepared for each toxicant, to minimize dilution errors. All
of the test concentrations are obtained by dispensing from this
solution with adjustable micropipets using disposable tips.
2. Loading
All plants for a toxicity test should come from the same
stock culture to minimize differences in pre-conditioning. After
plants are rinsed in sterile seawater, to remove traces of old
media, 2- to 3-mm branch tips are cut to serve as innocula for a
toxicity test. The tips are easily cut with fine-point, stainless
steel forceps. A stereomicroscope is not necessary, but can help
minimize tip-to-tip variation by making it easier to cut tips of a
uniform length. A stereomicroscope also helps in selecting branch
tips that do not have branch primordia (young branches) near the
tip. The presence of branch primordia gives the branch tip more
than one growing point at the beginning of the test. This gives
these tips a "head start" on those tips which form their first
branch only after on or two days into the test. The coefficient
of variation is less if all tips either have a branch primordia or
if young branches are absent from all innocula. The latter is
easier to achieve. Without a stereomicroscope the innoculum is a
mixture of the two types of tips, increasing the plant-to-plant
variation in final dry weight.
One three-week-old plant can usually supply all the tips
needed for a given test. Five tips of the life history stage in
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PAGE 51
question are transferred by Pasteur pipette into each test flask.
All tests are performed in duplicate. One male branch
(approximately 1.5 cm long), visably producing spermatia, is added
to each flask containing females. To assure that any
fertilization of females takes place in the presence of the
toxicant males must be added after the toxicant. Even though the
females are only a few mm long at the beginning, they already
possess mature trichogynes (receptive hairs that spermatia attach
to). Attachment of spermatia to trichogynes can take place within
seconds.
When media are changed, the male plants are clipped back to
ca 1.5 cm to minimize their competition for nutrients with the
female plants. During the first week of the test, the tips have a
tendency to attach to one another by the cut surface. At the
first media change these can easily be clipped apart with the
fine-point forceps.
3. Controls
Controls are required for every test to insure that the
observed effects are due to the test substance and not to other
factors. Occasionally, one or two plants in a test flask will die
for no obvious reason while all other plants in the flask are
alive and growing well. If more than two of the ten control
plants (two flask with five plants each) die, then the test should
be repeated. If at any time during a test plants in either
control flask exhibit any of the characteristics deemed
unacceptable in stock cultures, then that test must be repeated.
Because of seasonal variation in seawater quality the average
final dry weight of controls can vary greatly from experiment to
experiment. It is difficult to say what is a "bad" final dry
weight. For females particularly, an increase in seawater quality
can actually decrease final dry weight. The better the water
quality, the more cystocarps produced. The presence of cystocarps
appears to reduce the number of branches formed. Fewer branches
mean fewer growing points and therefore, smaller final dry
weights. However, in general, if the average final dry weight of
the combined female controls is less than 4.00 mg the female test
run should be repeated. If the average combined dry weight of the
tetrasporophytes is less than 3.00 mg, then the tetrasporophyte
test run should be repeated. If no sexual or asexual reproduction
occurs in a test, then the respective test must be repeated.
Plants in replicate flasks vary to different degrees in their
growth responses. Often there is little or no difference in
average dry weight between replicates. However, if the two flasks
from the toxicant concentration determined to be the effect level
are suspected of being significantly different, then a Student's t
test should be performed to test for difference. If the two sets
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PAGE 52
of data can not be pooled, then the test must be repeated. If carrier
controls (see next section) and controls are not statistically equal
(t test), then the test should be repeated.
4. Carrier Controls
Carriers should only be used when they are necessary to
solubilize hydrophobic compounds. Triethylene glycol (TEG) is a
general carrier of choice due to its low volatility and toxicity,
as well as to its ability to dissolve many organic compounds. TEG
has been successfully used as a carrier with Champia at
concentrations as high as 1200 uL per liter (see PaFt I, this
report). With one compound, toxaphene, the toxicant was first
dissolved in acetone and then diluted (1 mL to 100 mL) with TEG.
Champia grows equally well in the presence of TEG (or TEG plus 1 %
acetone) as it does without. At least one carrier control
treatment must be run using the highest volume of carrier that is
used in the toxicant treatments. A control without TEG should
also be run as a check on the carrier control.
C. FACILITIES
1. Exposure System
All range-finding and definitive test are performed in 400 mL
of medium in screw-capped 500 mL Erlenmeyer flasks (caps have
teflon liners). Silicone-rubfaer-stoppered flasks can be used, but
the seal is more easily broken with handling than with the
screw-capped flasks. This is important when dealing with volatile
toxicants that are potentially harmful to humans. All the
seawater medium for a given test is mixed in a single batch to
eliminate nutrient variation among the flasks. The medium for the
toxicity test is 1/4-strength of that listed in Table 1.
Definitive tests last 11 days for the tetrasporophytes and 14 days
for the females. After 11 days the tetrasporangia start to become
too numerous to easily count. Fourteen days are need for tests
with females to give ample time for cystocarps to develop. The
media are renewed on day 7 for tetrasporophytes and days 7 and 11
for females.
Flasks are shaken on a rotary shaker at 100 rpm. Champia
grows better with aeration than with shaking, but the latter was
selected as a standard method to minimize the loss of volatile
toxicants. Some early data suggest that hand-swirling may be just
as effective as shaking. However, experiments have to be done to
determine if hand-swirled plants remain in log growth over the
course of the test period. In any event, some sort of continuous
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PAGE 53
or daily agitation is necessary for two reasons. (1) Red algae do
not have motile spermatia, therefore, in order for sexual
reproduction to take place there must be water motion to carry the
spermatia to the female. (2) Water motion minimizes the thickness
of the unstirred or boundary layer of seawater at the surface of
the plants. The rate of uptake of nutrients (or toxicants) is
inversely proportional to the thickness of this layer. Thus
growth rate is partially dependent on the thickness of this layer.
2. Dilution Water
The seawater used for cultures and tests should be from the
same source. Natural seawater should be collected on an incoming
tide if collected from coastal areas. Champia will not grow well
in most artificial seawater recipes, however, the seawater recipe
listed in Table 2 is acceptable.
3. Cleaning glassware
Before use, flasks are washed; acid stripped; rinsed with
18 megaohm deionized water; and autoclaved at 1.1 kg/cm2 (15 psi)
for 10 min. Specific details for the cleaning procedure differ
slightly for heavy metals and organic compounds. These are listed
in Table 6.
D. Environmental Conditions
1. Light
The light conditions for toxicity tests are similar to those
for stock cultures. Flasks are illuminated from above at ca 75
uE-m~2-s~l with very-high-output cool-white fluorescent lights
(approxiamtely 500 ft candles). Light reading are taken among the
flasks on the shaker near the surface of the shaker platform.
The growth rate of Champia is not saturated at 75 uE'm~^'S~^.
This level is a compromise between higher levels that result in
maximum dry weight gain and those that are easiest to maintain
uniformly over the test area on the shaker. The photoperiod used
is 16H:8H, light:dark cycle. Other light levels and photoperiods
may be just as useful for toxicity testing, but should be checked
out before being used. Under the above light condiitons Champia
remains in log growth for the duration of the test (Fig. 2).
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PAGE 54
2. Temperature
Champia grows well over a wide range of temperatures (6 to
30°C) with its optimum at ca 24°C. Temperature can effect the
metabolic rate of plants and therefore, presumably their response
to toxicants. Test temperature should be maintained between 22
and 24°C.
3. Salinity
The preferred salinity for both stock cultures and toxicity
tests is 30 ppt. Although cultures have been successfully
maintained at 20 ppt, no growth experiments have been run at other
than 30 ppt. Thus, use of salinities other than 30 ppt should be
done with caution.
4. pH
The pH. of the test medium should be between 7.8 and 8.2.
With the above culture conditions no pH buffer is necessary other
than sodium bicarbonate.
E. Reporting
For each set of data, chronic values are calculated for dry
weight and number of cystocarps for females, and dry weight and
number of tetrasporangia for tetrasporophytes. The toxicant
concentration that completely inhibits reproduction is also noted.
Tetrasporangia are counted by placing tetrasporophytes between the
inverted halves of a polystyrene petri dish with a small volume of
seawater (to hold the entire plant in one focal plane). Using a
stereomicroscope, individual tetrasporangia can be easily observed
on both sides of the translucent thallus. Females are also
examined under a stereomicroscope to enumerate the emergent
cystocarps, which are easily distinguished from young branches
because they possess an apical ostiole (opening) and darkly
pigmented spores. After the above examinations are completed,
plants are rinsed with deionized water, oven-dried at 80°C (24 to
48 h), and weighed + 0.01 mg. Separation of individual plants must
be maintained so that number of reproductive structures per mg dry
weight can be calculated for each plant.
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PAGE 55
Chronic values are calculated using two different criteria
for determining significant differences. Chronic values are
determined for vegetative growth and number of reproductive '
structures per mg dry weight using statistical significance as the
criterion for difference. The suggested method for statistical
significance is anlaysis of variance (ANOVA) followed by Dunnett's
mean separation test (alpha = 0.05) for comparison of treatments
with a control (Steel and Torrie, 1960). The results from
replicate flasks within a test run generally can be pooled for
statistical analysis. Control and carrier controls are also
pooled. Significant difference from the control can also be
determined as absence of reproductive structures.
Chronic values are calculated as the geometric mean of the
lowest concentration that results in a significant difference from
the control and the next lowest concentration (Buikema et al.
1982). If the lowest concentration that results in a significant
difference is the lowest concentration tested, then the chronic
value can not be calculated. One can only report the concentration
that gave the significant difference. Maximum allowable toxicant
concentration (MATC) is the chronic value from the most sensitive
endpoint measured. The endpoint used for the MATC can vary from
chemical to chemical and even between replicate test if the
chronic values among the endpoints are close.
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PAGE 56
IV. REFERENCES
Abbott, I.A. and Hollenberg G.J. 1976. Marine algae of California,
Stanford University Press, pp.564-565.
American Society for Testing and Materials (ASTM). 1980.
Standard practice for conducting acute toxicity tests with
fishes, macroinvertebrates, and amphibians. ASTM E-729-80.
Buikema, A.L., Niederlehner, B.R., and Cairns, J. 1982. Biological
monitoring: Part IV. Toxicity testing. Water Res.
16:239-262.
Eide, I. and Myklestad, S. 1980. Long-term uptake and release of heavy
metals by Ascophyllum nodosum (L.) Le Jolis. Environmental
Pollution 23:19-28.
Guillard, R.R.L. and Ryther, J.H. 1962. Studies on marine planktonic
diatoms: I. Cyclotella nana Hustedt and Detonula
confervaces (Cleve) Gran. Can. J. Microbion8:229-239.
Kenaga, E.E. 1982. The use of environmental toxicology and
chemistry data in hazard assessment: Progress, needs,
challenges. Environ. Toxicol. Chem. 1:69-79.
Kenaga, E.E. and Moolenaar, R.J. 1976. Fish and Daphnia toxicity as
surrogates for aquatic vascular plants and algae. Environ.
Sci. Technol. 13:1479-1480.
Levine, H.G. and Wilce, R.T. 1980. Ulva lactuca as a bioindicator of
coastal water quality. Pub. 119, Water Resources Research
Center, University of Massachusetts at Amherst.
Lewis, E.J. 1973. The protein, peptide and free amino acid
composition in species of Champia from Sauashtra Coast,
India. Botanica Marina. XVI:145-147.
Mann, K.H. 1972. Introductory remarks, Procedding of the
IBP-UNESCO Symposium on detritus and its role in aquatic
ecosystems. Mem. Irt. Ital. Idrobiol. (Suppl.) 29:13-16.
Melhuus, A., Seip, K.L., and Seip, H.M. 1978. A preliminary study of the
use of benthic algae as biological indicators of heavy metal
pollution in Sorfjorden, Norway. Environmental Pollution
15:101-109.
Payne, A.G. and Hall, R.H. 1979. A method for measuring algal toxicity
and its application to the safety assessment of new
chemicals. Aquatic Toxicology ASTM STP 667. Marking LL,
Kimerle RA (eds). American Society for Testing and
Materials, pp. 171-180.
Phillips, D.J.H. 1977. Use of biological indicator organisms to
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PAGE 57
monitor trace metal pollution in marine and esturaine
environments: A review. Environmental Pollution 13:281-317.
Provasoli, L. 1963. Growing marine seaweeds. Proc. Int.
Seaweed Symp. 4:9-17.
Provasoli, L. 1968. Media and prospects for the cultivation of
marine algae. In: Cultures and collections of algae, pp.
63-75, Watanabe A, Hattori A (eds). Japan Conf. Hakone,
September 1966. Jap. Soc. Plant Physiol.
Reedman, D.J. and Womersley, H.B.S. 1976. Southern Australian species of
Champia and Chylocladia (Rhodymeniales: Rhodophyta). Trans.
R. Soc. Aust. 100:75-104.
Starr, R.C. 1978. The culture collection of algae at the
University of Texas at Austin. J. Phycol. 14
suppl.:47-100.
Steel, R.G. and Torey, J.H. 1960. Principles and procedures of
statistics. McGraw Hill, 481 pp.
Steele, R.L. and Hanisak, M.D. 1978. Sensitivity of some brown algal
reproductive stages to oil pollution. Proc. Ninth Int.
Seaweed Symp, pp. 181-190.
Steele, R.L. and Thursby, G.B. 1981. Development of a bioassay using the
like cycle of Champia parvula (Rhodophyta). (Abstract)
Phycologia 20:114.
Steele, R.L. and Thursby, G.B. 1983. A toxicity test using life stages
of Champia parvula (Rhodophyta). In: Aquatic Toxicology and
Hazard Assessment, Sixth Symp., pp. 73-89. Bishop, W.C.,
Cardwell, R.D., Heidolph, B.B. (eds). ASTM STP 802. American
Society of Testing and Materials, Philadelphia.
Taylor, W.R. 1957. Marine algae of northeastern coast of North
America. University of Michigan Press, Ann Arbor, pp. 289.
Thursby, G.B. and Steele, R.L. 1984. Toxicity of arsenite and arsenate
to the marine macroalga Champia parvula (Rhodophyta).
Environmental Toxicology and Chemistry 3:391-397.
U.S. EPA. 1974. U.S. Environmental Protection Agency. Marine
algal assay procedure: Bottle test. National Environmental
Research Center, Corvallis, Ore.
U.S. EPA. 1978. U.S. Environmental Protection Agency. Bioassay
procedures for the Ocean Disposal Permit Program. EPA report
600/9-78-010.
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PAGE 58
Table 1. Recipe for nutrient medium for growth of Champia parvula
in natural seawater. Both EDTA and trace elements have been
omitted.
COMPOUND CONCENTRATION PER LITER
FINAL SOLUTION CONCENTRATED STOCKa
NaN03
NaH2P04'H20
Ironb
Vitamins
B12
Biotin
Thiamine-HCl
37.4 mg
2.48 mg
2.5 ug
0.24 ug
0.24 ug
50 ug
"3.74 gr ams
0.25 grams
1.00 mg
10 mLc
a Use 10 mL/L for final concentrations. Add 150 mg/L
sodium bicarbonate when cultures are not aerated.
A stock solution of 60 mg/mL NaH2C03 is prepared by auto-
claving sodium bicarbonate as a dry powder and then
dissolving it in sterile deionized water. Use 2.5 mL/L
for the final concentration.
b Iron stock solution prepared by dissolving 1 g iron powder
in 10 mL concentrated HC1 and diluting to 1 liter with
deionized water.
c Vitamin stock solution autoclaved separately in 10 mL
sub-samples. Each 10 mL contains 24 ug B12, 2^ u§
and 5 mg thiamine-HCl.
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PAGE 59
Table 2. Recipe for artificial seawater based on major salts from
Provasoli's ASP-1 (Provasoli, 1963).
COMPOUND GRAMS/LITER
NaCl
KC1
MgS04-7H20
MgCl2'6H20
CaCl2'2H20
24.0
0.6
6.0
4.5
1.5
Sodium bicarbonate (300 mg/L) is added as a pH buffer and carbon
source in lieu of aeration. The bicarbonate is added after
autoclaving the ASP-1 (this prevents precipitation in the ASP-1).
A stock solution of 60 mg/mL NaH2C03 is prepared by autoclaving
sodium bicarbonate as a dry powder and then dissolving it in
sterile deionized water.
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PAGE 60
Table 3. Comparison of growth and morphology of Champia parvula
grown in artificial seawater (ASP-1) with various nutrient media.
MEDIUM MG DRY WEIGHT3 (n=10)
Optimum Natural
Seawater Medium (4X)b C2.59 + 0.53
PES-M C4.21 + 1.08
Optimum Natural Seawater
Medium + SII Trace Elem. 1.82 + 0.79
PES-M + SII Trace Elements 5.23 + 1.01
a mean + s.d.
b Earlier experiments showed that the 'normal' concentration
of this medium was insufficient.
c No septa and stubby
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Table 4. Recipe (PES-M) of the modified Provasoli's enrichment
solution (Provasoli, 1968) for culturing Champia in
artificial seawater.
COMPOUND AMOUNT PER LITER
FINAL SOLUTION CONCENTRATED STOCKa
NaN03
NaH2P04'H2Ob
Na2EDTA'2H20
17.5 mg
1.1 mg
2.08 mg
7.0 grams
0.44 grams
830 mg
Ironc 25 ug 50 mg
PII Trace Elements
FeCl-,'6H90
j LA
MnS04'H2Od
ZnS04'7H20
CoS04'7H20
H3B03
Vitamins
B12
Biotin
Thiamine-HCl
61 ug
154 ug
28 ug
6 ug
1425 ug
0.5 ug
0.25 ug
25 ug
24.5 mg
61.5 mg
11.0 mg
2.4 mg
0.57 grams
10 mLe
a Use 2.5 mL per liter for final concentrations
b Original recipe uses sodium glycerophosphate
c From a 1 mg/mL iron stock solution. One g iron powder
dissolved in 10 mL concentrated HC1 and diluted to 1
liter with deionized water. Original recipe called for
Fe(NH4)S04'6H20.
d Original recipe called for MnS04.4H20.
e 200 ug B12, 100 ug biotin, and 10 mg thiamine'HCl per
10 mL of vitamine stock. Autoclaved, separate from rest
of nutrients, only 1 minute (i.e., just bring autoclave up
to pressure).
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PAGE 62
Table 5. 'Recipe for SII trace elements (Provasoli, 1963) for
culturing Champia in artificial seawater.
COMPOUND CONCENTRATION PER LITER
FINAL SOLUTION CONCENTRATED STOCKa
KBr
KI
NaMo04*2H20
LiCl
SrCl2'6H20
RbCl
14.8 mg
13.1 ug
1.3 mg
1.2 mg
6.1 mg
0.28 mg
745 mg
654 ug
63 mg
61 mg
304 mg
14 mg
a Use 10 mL per liter for final concentrations. Add 1 mL
concentrated HC1 to stock solution before autoclaving.
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PAGE 63
Table 6. Instructions for cleaning glassware for toxicity tests
with Champia.
Organic Toxicants
(1) rinse with acetone from squeeze bottle (10 to 15 mL);
use fume hood.
(2) empty acetone into metal safety waste can
(3) fill flask with tap water and rinse three times
(4) rinse flasks with 10 to 15 mL of concentrated sulfuric acid,
followed by tap water rinses.
(5) wash flasks in mild detergent followed by deionized water
rinses
(6) soak flasks in 10% HC1 followed by deionized water rinses
(this removes any detergent residue which can be toxic to
Champia). (note: teflon liners of caps are also acid
stripped in 10% HC1, but screw-caps are not acid stripped.
Acid treatment followed by autoclaving makes the plastic
brittle. Caps are just rinsed well with deionized water.)
(7) Autoclave loosely capped for 10 min
Heavy Metals
Only steps 5 through 7 above are necessary with the following
exception; flasks are soaked in 10% HC1 overnight.
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PAGE 64
Figure 1. Life history of the marine red alga Champia parvula.
tetrasporangia—*
TETRASPOROPHYTE
spermatic
fertilization
cystocarp
5mm
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PAGE 65
Figure 2. Growth curve for the marine red alga Champia parvula in
natural seawater. All culture conditions were those used in the
standard toxicity test procedure.
ii
c
30
20
5
4
3
CP
E
0.5
0.4
0.3
0.2
0.
0.05-
0.04Q
0.031-
i
0.02
0.01
• 1982
O 1983
6 8 10 12 14 !€ 18
TIME (days)
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PAGE 66
PART IV.
OPTIMUM GROWTH OF THE MARINE RED ALGA
CHAMPIA PARVULA IN NATURAL AND ARTIFICIAL SEAWATERS
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PAGE 67
INTRODUCTION
Champia parvula experiences seasonal effects -on growth and
vigor in culture, presumably due to changes in the quality of the
natural seawater used. The availability of an artificial seawater
medium would allow more consistency in water quality among
different experiments, as well as among different laboratories.
In addition, the use of an artificial medium would make toxicity
tests with C. parvula more useful by making it available to
laboratories without ready access to clean natural seawater.
The bulk of artificial marine media have been designed for
work on microscopic algae, although several macroalgae have been
successfully grown in artificial seawater. Individual algal
species and individual clones of a particular species can vary in
their nutritional and physiological requirements. For this reason
a particular formulation could not be assumed to be the best
medium for Champia parvula. This report describes work on
determining the best nutrient additions in both natural and
artificial seawaters for Champia parvula.
MATERIALS AND METHODS
Tests to determine optimum culture conditions were similar to
procedures previously established for the red alga Champia parvula
(C. Agardh) Harvey (Steele and Thursby, 1983). Unialgal stock
cultures of males and females were maintained in 1000 mL, aerated
Erlenmeyer flasks containing 800 mL of filtered seawater (15 urn
charcoal filter and 0.3 urn Balston filter). The nutrient medium
(Table 1) consisted of medium 'f '/4, minus silica (Guillard and
Ryther, 1962). Flasks were illuminated with 75 to 80 uE-nT^s"1
of cool white fluorescent light on a 16h:8h, light: dark cycle.
Temperature and salinity were 20 to 22 C and 30 ppt, respectively.
Media were changed once a week.
Culture experiments lasted 14 days and were performed with
400 mL of medium in screw-capped, 500 mL Erlenmeyer flasks.
Flasks were shaken on a rotary shaker at 100 rpm, and sodium
bicarbonate (150 mg/L) was added in lieu of aeration. Media were
replaced on days 7 and 11. Light, salinity and temperature were
the same as for stock cultures. Except for experiments with
artificial seawater, all water was from lower Narragansett, RI,
collected on an incoming tide. The artificial seawater medium
used (Table 2) consisted of the major salts from Provasoli's ASP-1
(Provasoli, 1963). All seawaters were autoclaved in 16 to 18
liter batches (20 min, 15 psi) and nutrients were added from
autoclaved stock solutions.
Starting plants for any given experiment came from the same
stock culture. After stock plants were rinsed in sterile seawater
to remove traces of old medium, 2- to 3-mm branch tips were cut to
serve as innocula for an experiment. Five female branch tips were
placed into each test flask. One male branch (about 1.5 cm long),
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PAGE 68
visibly producing spermatia, was added to each flask for
experiments in which sexual reproduction was also examined. All
experiments were performed in duplicate. Vegetative growth was
recorded as final dry weight after drying 24 to 48 hours at 80 C.
For natural seawater, optimal nutrient concentrations were
based on determining what was essential from medium 'f. Various
concentrations of vitamins, trace metals, iron, nitrate (NaN03),
and phosphate (NatL^PO^'H20) were tested for optimal growth of C.
parvula. Vitamins (thiamine.HCl, biotin and B12), as well as
trace elements (Co, Cu, Mo, Mn and Zn), were tested as a unit.
The optimal nutrients from the natural seawater experiments were
also tested in ASP-1. In addition, nutrients from a modified
Provasol's enrichment solution (PES-M—Table 3) were tested in
ASP-1. SII (Table 4) and PII (Table 3) trace elements were tested
as single units.
RESULTS
Natural Seawater
Neither trace metals nor EDTA were essential additions for
growth of Champia in natural seawater from Narragansett Bay
(Tables 5 and 6). Although, in the absence of vitamins EDTA could
stimulate growth. Early experiments showed that in the absence of
vitamins 100 ug/L EDTA was sufficient. Vitamins stimulated growth
whether or not EDTA was present. Preliminary experiments
suggested that the essential vitamin was B]^.
Owing to the large amount of iron in natural seawater, iron
deficiency could only be demonstrated with seawater precipitated
with ferric chloride (Steele, 1975). This precipitation removes
both iron and phosphate. Plants grown in minus iron media were
not only smaller, but also pink. One ug Fe/L was sufficient to
overcome the deficiency (Fig. 1).
Like iron, phosphorus deficiency could only be demonstrated
in the ferric chloride precipitated seawater. Phosphorus results
are expressed as ash-free dry weight (Fig. 2). At lower
phosphate concentrations a white precipitate formed on the plants
(presumably CaC03). Although 1 uM phosphate (f/64) was sufficient
to overcome phosphorus deficiency, 4.5 uM (f/16) was selected as
the optimal media concentration because the CaC03 precipitation
rarely formed at this concentration.
In the absence of nitrate Champia plants died (Fig. 3). At
28 and 55 uM of added nitrate, plants were pale yellow, a symptom
of nitrogen deficiency. The best concentration of nitrate was 110
uM (f/16).
Artificial Seawater
The nutrient medium designed for optimum growth of Champia
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PAGE 69
parvula in natural seawater (Table 7) was not adequate for
artificial seawater (Table 8). In addition, plants in ASW did not
have the correct morphology; septa formation was inhibited.
Complete medium 'f/4 and PES-M gave slightly better results, but
the morphology was still incorrect. In the presence of SII trace
elements Champia exhibited the correct morphology, although the
optimum natural seawater nutrients were still insufficient for
good growth in ASP-1 (Table 8).
The medium ASP-1 with PES-M and SII is sufficient for our
purposes, however, we are continuing our experiments to minimize
the medium. The main difference between the optimum natural
seawater nutrients (which yields poor growth in ASP-1) and PES-M
(which yields good growth in ASP-1) is the presence of PII trace
elements, EDTA, and more iron in the later. Qualitative artificial
seawater experiments showed that adding all three of these to the
optimum natural seawater nutrients yielded plants similar in size,
morphology and reproductive capacity to plants grown in PES-M (in
the presence of SII trace elements for both media). Omitting any
one of the three 'extra' components results in less than optimum
growth.
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Table 1. Recipe for complete medium 'f'M (based on Guillard and
Ryther, 1963) minus silica.
COMPOUND AMOUNT PER LITER FINAL SOLUTION
NaN03
NaH2P04'H20
Irona
Vitamins
B12
Biotin
Thiamine-HCl
Trace Elements
CuS04'5H20
ZnS04'7H20
CoCl2'6H20
MnCl2'4H20
Na2Mo04'2H20
37.5 mg
2.5 mg
325 ug
0.25 ug
0.25 ug
50 ug
4.9 ug
11 ug
5 ug
90 ug
3.2 ug
a A 1 mg/mL stock solution was prepared by dissolving 1 g
iron powder in 10 mL of concentrated HC1 and diluting to
1 liter with 18 megaohm deionized water.
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Table 2. Recipe for artificial seawater based on major salts from
Provasoli's ASP-1 (Provasoll, 1963).
COMPOUND GRAMS/LITER
NaCl
KC1
MgS04'7H20
MgCl2'6H20
CaCl2-2H20
24.0
0.6
6.0
4.5
1.5
Sodium bicarbonate (300 mg/L) is added as a pH buffer and carbon
source in lieu of aeration. The bicarbonate is added after
autoclaving the ASP-1 (this prevents precipitation in the ASP-1),
A stock solution of 60 mg/mL NaH2C03 is prepared by autoclaving
sodium bicarbonate as a dry powder and then dissolving it in
sterile deionized water.
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Table 3. Recipe (PES-M) of the modified Provasoli's enrichment
solution .(Provasoli, 1968).
COMPOUND
NaN03
NaH2P04-H2Ob
Na2EDTA'2H20
Ironc
PII Trace Elements
FeCl3'6H20
MnS04'H2Od
ZnS04'7H20
CoS04'7H20
H3B03
Vitamins
B12
Biotin
Thiamine-HCl
AMOUNT
FINAL SOLUTION
17.5 tag
1.1 mg
2.08 mg
25 ug
61 ug
154 ug
28 ug
6 ug
1425 ug
0.5 ug
0.25 ug
25 ug
PER LITER
CONCENTRATED STOCK.3
7.0 grams
0.44 grains
830 mg
50 mg
24.5 mg
61.5 mg
11.0 mg
2.4 mg
0.57 grams
10 mLe
a Use 2.5 mL per liter for final concentrations
b Original recipe uses sodium glycerophosphate
c From a 1 mg/mL iron stock solution. One g iron powder
dissolved in 10 mL concentrated HC1 and diluted to 1
liter with deionized water. Original recipe called for
Fe(NH4)S04'6H20.
Original recipe called for MnS04'4H20.
e 200 ug BIT, 100 ug biotin, and 10 mg thiamine'HCl per
10 mL of vitamine stock. Autoclaved, separate from rest
of nutrients, only 1 minute (i.e., just bring autoclave up
to pressure).
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Table 4. Recipe for SII trace elements (Provasoli, 1963).
COMPOUND CONCENTRATION PER LITER
FINAL SOLUTION CONCENTRATED STOCK3
KBr
K.I
NaMo04*2H20
LiCl
SrCl2'6H20
RbCl
14.8 mg -
13.1 ug
1.3 mg
1.2 mg
6. 1 mg
0.28 mg
745 mg
654 ug
63 mg
61 mg
304 mg
14 mg
a Use 10 mL per liter for final concentrations. Add 1 mL
concentrated HC1 to stock solution before autoclaving.
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PAGE 74
Table 5. Growth of Champia parvula after 2 weeks in natural
seawater with and without medium 'f trace elements. All other
nutrients were as presented in Table 1.
TREATMENT MG DRY WEIGHT3 (n=10)
+ Trace Elements 15.31 + 3.17
- Trace Elements 13.84 j- 2.61
a mean + s.d.
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PAGE 75
Table 6. Growth of Champia parvula after 2 weeks in natural
seawater with and without vitamins, and with and without EDTA.
All other nutrients were as presented in Table 1, except trace
elements were omitted.
TREATMENT MG DRY WEIGHT3 (n=10)
+ Vitamins
+ EDTA 10.61 + 1.55
- EDTA 12.47 + 3.01
- Vitamins
+ EDTA 5.86 + 2.35
- EDTA 2.32 + 0.62
a mean + s.d.
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Table 7. Recipe for optimum nutrient medium for growth of Champia
parvula in natural seatwater. Both EDTA and trace elements have
been omitted.
COMPOUND CONCENTRATION PER LITER
FINAL SOLUTION CONCENTRATED STOCK3
NaN03
NaH2P04'H20
9.35 mg
0.62 mg
3.74 grams
0.25 grams
Iron*5 2.6 ug 1.04 mg
Vitamins
B12 0.06 ug
Biotin 0.06 ug 10 mLc
Thiamine-HCl 12.5 ug
a Use 2.5 mL/L for final concentrations. Add 150 mg/L sodium
bicarbonate when cultures are not aerated.
b Iron stock solution prepared by dissolving 1 g iron powder
in 10 mL concentrated HC1 and diluting to 1 liter with
deionized water.
c Vitamin stock solution autoclaved separately in 10 mL
sub-samples. Each 10 mL contains 24 ug B^2> 24 ug biotin
and 5 mg thiamine-HCl.
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Table 8. Comparison of growth and morphology of Champia parvula
grown in artificial seawater (ASP-1) with various nutrient media.
MEDIUM MG DRY WEIGHT3 (n=10)
Optimum Natural
Seawater Medium (4X)b C2.59 + 0.53
Medium 'f'M C3.21 + 0.89
PES-M C4.21 + 1.08
Optimum Natural Seawater
Medium + SII Trace Elem. 1.82 + 0.79
PES-M + SII Trace Elements 5.23 +1.01
a mean +_ s.d.
b Earlier experiments showed that the 'normal1 concentration
of this medium was insufficient.
c No septa and stubby.
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Figure 1. Growth of Champia parvula at various concentrations of
iron. All other nutrients were at non-limiting concentrations.
EDTA and trace elements were omitted.
o
f/512 f/256 f/128
f/64
10
20 25
Fe
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PAGE 79
Figure 2. Growth of Chainpia parvula at various concentrations of
phosphate. All other nutrients were at non-limiting
concentrations. EDTA and trace elements were omitted.
CaC03 precipitation
f/64 f/32 f/16
f/8
0 2.5
7.5
10
NaH2P04-H20
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PAGE 80
Figure 3. Growth of Champia parvula at various concentrations of
nitrate. All other nutrients were at non-limiting concentrations.
EDTA and trace elements were omitted.
pale yellow
f/64 f/32 f/16 f/IO f/8
ii i i i
0 50 100 150 200 250
NaNO
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PAGE 81
REFERENCES
Guillard, R.R.L. and J.H. Ryther. 1962. Studies on marine
planktonic diatoms. I. Cyclotella nana Hustedt and Detonula
confervaceae (Cleve) Gran. Can. J. Microbiol. 8:229-39^
Provasoli, L. 1963. Growing marine seaweeds. Proc. Int.
Seaweed Symposium 4:9-17.
Provasoli, L. 1968. Media and prospects for the cultivation of
marine algae. (in) Watanabe, A. and Hattori, A. (eds).
Cultures and collections of Algae. Proc. US-Japan Conf.
Hakone, September 1966. Jap. Soc. Plant Physiol. pp.
63-75.
Steele, R.L. 1975. Growth requirements of Enteromorpha compressa
and Codium fragile, (in) Proceedings, Biostimulation and
Nutrient Assessment Workshop. EPA-600/3-75-034. U.S.
Environmental Protection Agency, Washington, D.C., pp.
213-225.
Steele, R.L. and G.B. Thursby. 1983. A toxicity test using
life stages of Champia parvula (Rhodophyta). (in) W.C.
Bishop, R.D. Cardwell, and B.B. Heidolph, eds., Aquatic
Toxicology and Hazard Assessment, Sixth Symposium. ASTM STP
802. American Society of Testing and Materials,
Philadelphia, pp. 73-89.
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PAGE 82
PART V.
CHAMPIA TESTING: ARTIFICIAL SEAWATER AND
OTHER ISOLATES AND SPECIES
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PAGE 83
TABLE OF CONTENTS
Page
STATEMENT OF THE PROBLEM AND OBJECTIVES 84
BACKGROUND 84
ARTIFICIAL SEAWATER 86
Introduction 86
Materials and Methods 86
Culture Experiments 86
Toxicity Testing 87
Results 87
Culture Experiments 87
Toxicity Testing 88
OTHER ISOLATES AND SPECIES 88
Introduction 88
Materials and Methods 88
Test Species 88
Maintenance of Stock Cultures 89
Toxicity Testing 89
Results 89
Feasibility of Using Agardhiella and
Grinnellia for Toxicity Testing 89
CONCLUSIONS 90
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STATEMENT OF TEE PROBLEM AND OBJECTIVES
Toxicity testing completed to date on Champia parvula
demonstrates its usefulness as a toxicity test species.However,
two main areas remain to be addressed to complete the work. An
artificial seawater medium needs to be developed for Champia so
that static tests can be performed in a chemically defined medium,
as well as be available to laboratories without ready access to
clean natural seawater. Other isolates of Champia and other red
algal species need to be tested to determine if the current
results with Champia are representative or if they are the result
of a "super sensitive" isolate.
This report documents the completion of the final work
necessary for writing the laboratory culture and toxicity testing
guidelines for Champia. The specific objectives of the work were:
I. Artificial Seawater
A. finalize formulation of an artificial seawater
medium.
B. compare sensitivity of Champia to three organic
toxicants in both natural and artificial seawaters.
II. Other Isolates and Species
A. compare the sensitivity of two other isolates of
Champia (one from Rhode Island and one from
Korea) with that of the standard isolate.
B. compare sensitivity of Champia with that of two
other red algal species (Agardhiella subulata
and Grinnellia americana).
BACKGROUND
In the open ocean phytopankton are the primary producers,
however, inshore or estuarine production depends heavily on
contributions from macroalgae. Macroalgae are generally sessile,
and as such may be more vulnerable to pollution than planktonic
algae. Macroalgae also represent a different food source than
microalgae, contributing primarily to detritial food chains (Mann,
1972). Macroalgae act as the basis for communities of other
plants and animals in providing living space and in some cases
ameliorating the effects of stress, both natural and man-made.
New and exotic chemicals, as well as many known compounds,
are being released into the air and water daily. Many of these
compounds find their way into the marine environment and need to
be tested as potential toxicants to different trophic levels of
marine organisms. Standardized toxicological test procedures have
been developed for fish, invertebrates, amphibians, and microalgae
(ASTM, 1980, USEPA, 1974; 1978). However, very little work has
been done toward the development of a standard procedure for
testing the effects of toxicants on macroalgae. Macroalgal tissue
residues have been used to monitor pollution of heavy metals in
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PAGE 85
the field (Melhuus et al., 1978; Eide, 1980). This type of study
requires the use of relatively tolerant species since the goal is
to have an alga that will accumulate a pollutant without dying
(Phillips, 1977). In toxicological studies, the opposite is true.
One looks for a relatively sensitive organism; since, by
protecting sensitive organisms, tolerant species should also be
preserved.
A relatively fast, simple and inexpensive toxicity test
method has been developed for the-marine red alga Champia parvula,
to assess chronic effects of pollutants to marine macroalgae
(Steele and Thursby 1983). The method has been used to generate
toxicity data for water quality criteria. The test method has
previously been evaluated with heavy metals and cyanide (Steele
and Thursby, 1983), arsenite and arsenate (Thursby and Steele,
1984), as well as ten different organic compounds (see Part I,
this report). The results of these tests indicate that Champia is
often as sensitive, or more so, than the most sensitive aquatic
animal test, and almost always more sensitive than microalgae.
The data also demonstrate the importance of conducting chronic
tests with the previously overlooked macroalgae.
The Environmental Protection Agency, under the mandate of the
Toxic Substance Control Act, is required to identify and control
chemicals that are hazardous to human health or the environment.
An important aspect of this responsibility is to develop
methodology for testing potential hazards to different trophic
levels of marine organisms. According to EPA's Office of Research
and Development planning documents, data are needed for 40 to 65
toxic pollutants for which little or no chronic toxicity
information is currently available. These data must be both
scientifically sound and legally defensible. At the same time, it
is necessary for the methods to be as simple and cost efficient as
possible. All of these requirements are met by the test method
developed for Champia. In addition, Champia parvula is a common
species in many parts of the world (Taylor, 1957; Abbott and
Hollenberg, 1978; Lewis, 1973; Reedman adn Womersley, 1976). It
has been found in Mexico and southern California on the west coast
of North America, and from the Carribean to Massachusetts on the
east coast. It has also been reported form France, Spain, Korea,
and Southern Australia. Because of its wide distribution, £._
parvula may be a useful surrogate species for determining the
relative toxicity of a variety of chemicals.
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ARTIFICIAL SEAWATER
Introduction
Champia parvula experiences seasonal effects on growth and
vigor in culture, presumably due to changes in the quality of the
natural seawater used. The availability of an artificial seawater
medium would allow more consistency in water quality among
different experiments, as well as among different laboratories.
In addition, the use of an artificial medium would make toxicity
tests with C. parvula more useful by making it available to
laboratories without ready access to clean natural seawater.
The bulk of artificial marine media have been designed for
work on microscopic algae, although several macroalgae have been
successfully grown in artificial seawater. Individual algal
species and individual clones of a particular species can vary in
their nutritional and physiological requirements. For this reason
a particular formulation could not be assumed to be the best for
Champia parvula. Nutrients that yield good growth when added to
natural seawater do not generally yield good growth when added to
an artificial seawater recipe. This report describes work on
determining the best nutrient additions in artificial seawaters
for Champia parvula, as well as on comparing the sensitivity of
Champia in natural and artificial seawaters.
Materials and Methods
Culture Experiments
Tests to determine optimum culture conditions in artificial
seawater were similar to procedures previously established for the
red alga Champia parvula (C. Agardh) Harvey (Steele and Thursby,
1983). Unialgal stock cultures of males and females were
maintained in 1000 mL, aerated Erlenmeyer flasks containing 800 mL
of filtered natural seawater (15 um charcoal filter and 0.3 urn
Balston filter). The nutrient medium used is described in Table
1. Flasks were illuminated with 75 to 80 uE-m~2-s~l of cool white
fluorescent light on a 16h:8h, light: dark cycle. Temperature
and salinity were 20 to 22°C and 30 ppt, respectively. Media were
changed once a week.
Culture experiments lasted 14 days and were performed with
400 mL of medium in screw-capped, 500 mL Erlenmeyer flasks.
Flasks were shaken on a rotary shaker at 100 rpm, and sodium
bicarbonate (150 mg/L) was added in lieu of aeration. Media were
replaced on days 7 and 11. Light, salinity and temperature were
the same as for stock cultures. The artificial seawater medium
used (Table 2) consisted of the major salts from Provasoli's ASP-1
(Provasoli, 1963). The nutrient medium designed for the optimum
growth of Champia in natural seawater (Table 3) was tested in
ASP-1. In addition, a modified Provasoli's enrichment solution
(PES-M—Table 4) was also tested in ASP-1. SII (Table 5) and PII
(Table 4) trace elements were tested both as single units and as
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PAGE 87
units minus each of the elements.
Starting plants for any given experiment came from the same
stock culture. After stock plants were rinsed in sterile seawater
to remove traces of old medium, 2- to 3-mm branch tips were cut to
serve as innocula for an experiment. Five female branch tips were
placed into each test flask. One male branch (about 1 cm long),
visibly producing spermatia, was added to each flask for
experiments in which sexual reproduction was also examined. All
experiments were performed in duplicate. Vegetative growth was
recorded as final dry weight after drying 24 to 48 hours at 80°C.
Toxicity Testing
Toxicity test procedures followed those for the culture
experiments. Tests were performed in both natural and artificial
(ASP-1) seawaters. The nutrient medium used was PES-M plus SII
trace elements. The endpoints measured were dry weight of
females and number of cystocarps per mg dry weight. Statistical
significance for differences was calculated using analysis of
variance followd by Dunnett's multiple range test; alpha level
0.05 (Steel and Torey, 1960).
Results
Culture Experiments
The nutrient medium designed for growth of Champia parvula in
natural seawater was not adequate for artificial seawater (Table
5). In addition, plants in artificial seawater did not have the
correct morphology; septa formation was inhibited. PES-M gave
slightly better results, but the morphology was still incorrect.
In the presence of SII trace elements Champia exhibited the
correct morphology, although the optimum natural seawater medium
was still insufficient for good growth in ASP-1 (Table 5). Among
the SII trace elements, bromine was the one responsible for the
control of septal formation (Table 6), although the overall
morphology of Champia was better with all the SII's than with
bromine alone.
The main difference between the optimum natural seawater
medium (which yields poor growth in ASP-1) and PES-M (which yields
good growth in ASP-1) is the presence of PII trace elements, EDTA
and more iron in the later. Qualitative artificial seawater
experiments showed that adding all three of these to the optimum
natural seawater nutrients yielded plants similar in size,
morphology and reproductive capacity to plants grown in PES-M (in
the presence of all SII trace elements for both). Leaving any one
of the three 'extra1 components out results in less than optimum
growth. Among the PII trace elements, boron was shown to be the
most essential (Table 7).
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Toxicity Testing
The results for the comparison of toxicity tests in natural
seawater and artificial seawater (expressed as percent of control)
are shown in Table 8. Results for chronic values (Table 9) were
similar in both seawaters for both growth and reproduction. There
was some variability in the results for toxaphene, but the
responses were within the range of results found in other
experiments for toxaphene with Champia (see Part I, this report;
and see below). In addition, the differences between the chronic
values for toxaphene in natural and artificial seawaters were only
one treatment concentration apart. These results indicate that
ASP-1 (with PES-M and SII nutrients) is an adequate defined medium
for toxicity tests with Champia.
OTHER ISOLATES AND SPECIES
Introduction
Champia parvula can frequently undergo spontaneous somatic
mutation in the laboratory. Therefore, it is important to
determine if the Champia isolate that has been selected for
routine use in toxicity testing is not a "super sensitive" mutant.
To examine this, two other isolates of Champia parvula were
selected for testing. One of the isolates was collected in 1981
from Rhode Island, USA (the standard isolate was collected in 1979
from Rhode Island), and the other isolate was collected from South
Korea. In addition to these two isolates two other species of red
algae, Agardhiella subulata and Grinnellia americana, were also
tested. The testing of these two species would also give some
indication as to how representative the Champia toxicity test data
is of red algae in general.
Materials and Methods
Test Species
Two isolates of Champia parvula (C. Agardh) Harvey were
used. The first (Champia II) was collected from Narragansett,
Rhode Island in November of 1981. The other isolate (Champia III)
was originally collected from Pusan, South Korea. The responses
of these two isolates were compared to that of the standard
isolate (Champia I) collected from Ninigret Pond, Rhode Island in
1979. Isolates of Agardhiella subsulata (C. Agardh) Kraft and
Wynne and Grinnellia americana (C. Agardh) Harvey were
established from plants collected from Narragansett, Rhode Island.
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Maintenance of Stock Cultures
Medium and culture conditions for male, female, and
tetrasporophytic plants were as described above for stock cultures
in artificial seawater experiments.
Toxicity Testing
Toxicity tests were performed as described above for test
with artificial seawater, except for the following modifications:
1) tests with Agardhiella females were run for 21 days with media
changes on days 7, 11, and 18; 2) for the blade-forming
Grinnellia, each flask contained one male blade (1 cm long)
visibly producing spermatia, 5 young whole female blades 5 mm long
(for dry weight determinations) and 5 pieces of indeterminately
growing procarp tissue 1 cm long (each consisting of about 30-40
fertilizable tips for asessment of effects on sexual
reproduction).
Results
Chronic values for toxicity tests with phenol, toxaphene and
isophorone using the other isolates and species are shown in
Tables 10, 11 and 12, respectively. No single isolate of Champia
stood out as consistantly the most sensitive. Most importantly,
the standard isolate (I) did not show up as super sensitive
relative to the other two Champia's. In addition, the species of
Agardhiella and Grinnellia were also similar in their
sensitivities (for both reproduction and growth) to the isolates
of Champia. Results from previous toxicity tests with a variety
of toxicants indicate that the standard isolate of Champia is as
sensitive or more so than the most sensitive aquatic animal test,
and is almost always more sensitive than microalgae (Steele and
Thursby, 1983;Thursby and Steele, 1984; see Part I, this report).
Our current results show that Champia's previous sensitivity is
not restricted to that particular isolate and may be
representative of red algae in general.
Feasibility of Using Agardhiella and Grinnellia
for Toxicity Testing
Agardhiella—Although the sensitivity of Agardhiella was similar
to the Champia isolates, the extra time required for the test (a
total of 21 days) makes this species less desirable for testing
than Champia.
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Grinnellia—Unlike Champia each Grinnellia plant consists of an
unbranched blade. Therefore, obtaining large numbers of uniform
plants for innocula was difficult and time consuming. Young
female blades had to be used for innocula and these had a long lag
period before being reproductive. We had to use indeterminately
growing procarpic tissue (female reproductive tissue) for testing
the effect of toxicants on sexual reproduction. Preliminary .
experiments showed that each branch tip of this abberent female
tissue rapidly formed a cystocarp in the presence of male tissue
when in aerated culture. Under the conditions of the toxicology
test, however, many of these tips reverted to producing blades and
were not reproductive. Thus, the present difficulties in using
Grinnellia in toxicity tests makes it less desirable than Champia.
CONCLUSIONS
1. A successful artificial seawater recipe has been
found for the culture of Champia parvula in the
laboratory.
2. The sensitivity of Ghampia to phenol, toxaphene
and isophorone is similar in both natural and artificial
seawater.
3. The Champia isolate selected as the standard isolate
for toxicity testing is not "super sensitive" relative
to other isolates of Champia or other species of
red algae.
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PAGE 91
Table 1. Recipe for optimum nutrient medium for growth of Ghanipia parvula
in natural seatwater. Both EDTA and trace elements have been
omitted.
COMPOUND CONCENTRATION PER LITER
FINAL SOLUTION CONCENTRATED STOCK3
NaN03
NaH2P04.H20
Ironb
Vitamins
B12
Biotin
Thiamine.HCl
9.35 mg
0.62 mg
2.6 ug
0.06 ug
0.06 ug
12.5 ug
3.74 grams
0.25 grams
1.04 mg
10 mLc
a Use 2.5 mL/L for final concentrations. Add 150 mg/L sodium
bicarbonate when cultures are not aerated. A stock solution of 60 mg/mL
NaH2C03 is prepared by autoclaving sodium bicarbonate as a dry powder
and then dissolving it in sterile deionized water. Use 2.5 mL/L for the
final concentration.
b Iron stock solution prepared by dissolving 1 g iron powder in
10 mL concentrated HC1 and diluting to 1 liter with deionized water.
c Vitamin stock solution autoclaved separately in 10 mL sub-samples.
Each 10 mL contains 24 ug B12, 24 ug biotin and 5 mg thiaraine.HCl.
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PAGE 92
Table 2. Recipe for artificial seawater based on major salts from
Provasoli's ASP-1 (Provasoli, 1963).
COMPOUND GRAMS/LITER
NaCl 24.0
KC1 0.6
MgS04.7H20 6.0
MgCl2.6H20 4.5
CaCl2.2H20 1.5
Sodium bicarbonate (300 mg/L) is added as a pH buffer and carbon
source in lieu of aeration. The bicarbonate is added after autoclaving
the ASP-1 (this prevents precipitation in the ASP-1). A stock solution
of 60 mg/mL NaH2C03 is prepared by autoclaving sodium bicarbonate as a
dry powder and then dissolving it in sterile deionized water.
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Table 3. Recipe (PES-M) of the modified Provasoli's enrichment
solution (Provasoli, 1968).
COMPOUND AMOUNT PER LITER
FINAL SOLUTION CONCENTRATED STOCK3
NaN03
NaH2P04.H2Ob
Na2EDTA.2H20
Ironc
PII Trace Elements
FeCl3.6H20
MnS04.H2Od
ZnS04.7H20
CoS04.7H20
H3B03
Vitamins
B12
Biotin
Thiamine.HCl
17.5 mg
1.1 mg
2.08 mg
25 ug
61 ug
154 ug
28 ug
6 ug
1425 ug
0.5 ug
0.25 ug
25 ug
7.0 grams
0.44 grams
830 mg
50 mg
24.5 mg
61.5 mg
11.0 mg
2.4 mg
0.57 grams
10 mLe
a Use 2.5 mL per liter for final concentrations
k Original recipe uses sodium glycerophosphate
c From a 1 mg/mL iron stock solution. One g iron powder dissolved
in 10 mL concentrated HC1 and diluted to 1 liter with deionized water.
Original recipe called for Fe(NH4)S04.6H20.
d Original recipe called for MnS04.4H20.
e 200 ug B12, 100 ug biotin, and 10 mg thiamine.HCl per 10 mL of
vitamin stock. Autoclaved, separate from rest of nutrients, only 1
minute (i.e., just bring autoclave up to pressure).
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PAGE 94
Table 4. Recipe for SII trace elements (Provasoli, 1963).
COMPOUND
KBr
KI
NaMo04.2H20
LiCl
SrCl2.6H20
RbCl
CONCENTRATION
FINAL SOLUTION
14.8 mg
13.1 ug
1.3 mg
1.2 mg
6. 1 mg
0.28 mg
PER LITER
CONCENTRATED STOCK3
745 mg
654 ug
63 mg
61 mg
304 mg
14 ag
a Use 10 mL per liter for final concentrations. Add 1 mL
concentrated HC1 to stock solution before autoclaving.
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PAGE 95
Table 5. Comparison of growth and morphology of Champia parvula grown
in artificial seawater (ASP-1) with various nutrient media.
MEDIUM MG DRY WEIGHTa (n=10)
Optimum Natural*3
Seawater Medium (4X) C2.59 + 0.53
PES-M C4.21 + 1.08
Optimum Natural Seawater
Medium + SII Trace Elem. 1.82 + 0.79
PES-M + SII Trace Elements 5.23 + 1.01
a mean + s.d.
b Earlier experiments showed that the 'normal' concentration of
this medium was insufficient.
c No septa and stubby.
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PAGE 96
Table 6. Effect of omitting each of the SII trace elements on growth of
Champia parvula.
TREATMENT MG DRY WEIGHT3 (N=10)
SII Complete 8.60 + 1.84
- Li 10.52 + 1.62
- Rb 8.97 + 1.79
- Sr 7.57 + 1.19
- Mo 11.14 + 2.81
- I • 9.32 + 2.77
- Br b4.22 + 1.79
- all b5.09 + 1.96
a mean + s.d.
b No septa; incorrect morphology.
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PAGE 97
Table 7. Effect of omitting each of the PII trace elements on growth of
Champia parvul-a. Complete S1I trace elements were present in
each treatment.
TREATMENT MG DRY WEIGHTa (N=10)
PII Complete 4.8 +1.2
- Mn 3.9 + 0.6
- Co 4.2 + 1.1
- Zn 4.4 + 1.0
- Fe 4.3 + 0.9
- B 2.6 + 1.2
- all 2.5 + 0.4
a mean + s.d.
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PAGE 98
Table 8. Effect of phenol, toxaphene and isophorone (expressed as % of
control) in both natural and artificial seawaters on growth
and cystocarp formation in Champia parvula. Number underlined
represent lowest concentration that was statistically less than
the control.
COMPOUND
UG/LITER
Phenol
control
7,800
13,000
21,670
36,120
60,200
Toxaphene
control
14
23
39
65
108
180
Isophorone
control
29,500
49,840
83,070
138,450
GROWTH
NATURAL ARTIFICIAL
100
64
58
55
22
dead
100
102
81
68
50
17
4
100
94
75
24
9
aioo
91
75
65
27
9
100
82
37
37
23
6
0
100
83
62
13
13
# CYSTOCARP S
NATURAL ARTIFICIAL
100
50.
4
0
0
dead
100
141
106
60
41
0
0
100
84
60
0
0
100
57
9
0
0
0
100
111
89
44
0
0
0
100
62
46
0
0
a Results are based on the natural seawater control (both of the
phenol artificial seawater controls were unhealthy), therefore these
results can be used only as a rough comparison.
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PAGE 99
Table 9. Chronic values for phenol, toxaphene and isophorone (ug/L) for
Champia parvula in both natural and artificial seawater.
•Numbers represent the lowest concentration tested that was
statistically less than the control (this is the upper limit
for the chronic value, the next lowest concentration can be
found in Table 8).
COMPOUND GROWTH3 REPRODUCTION
NATURAL ARTIFICIAL #CYSTOCARPSb NO CYSTOCARPSC
NATURAL ARTIFICIAL NATURAL ARTIFICIAL
Phenol 7,800 d 7,800 7,800 21,670 21,670
Toxaphene 39 23 39 23 108 65
Isophorone 49,840 49,840 49,840 49,840 83,070 83,070
a Based on statistical difference in dry weight.
b Based on statistical difference in number of cystocarps.
c Based on the concentration that completely inhibits cystocarps.
d Can not be determined because the control was unhealthy.
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PAGE 100
Table 10. Phenol chronic values (ug/L) for three Champia parvula isolates,
Agardhiella subulata, and Grinnellia americana based on growth
(final dry weight) and reproduction of females and tetrasporophytes.
Numbers represent the lowest concentration tested that was
statistically different from the control (this is the upper
limit for the chronic value, the next lowest concentration
was 60% of this value.
Species
CHAMPIA Ia
CHAMPIA II
CHAMPIA III
AGARDHIELLA
GRINNELLIA
GROWTH
FEMALE TETRA.
21,600 13,000
36,120 36,120
7,800 NDe
36,120b ND
36,120° ND
REPRODUCTION
#CYSTO. #TETRASP. +CYSTO. +TETRASP.
7,800 7,800
7,800 7,800
7,800 ND
7,800 ND
Od ND
36,000 60,000
36,120 21,670
13,000 ND
21,670 ND
13,000 ND
a Data from Thursby & Steele [10].
b Growth determined on day 21.
c Based on growth of blade tissue.
d. Immature cystocarps only in control.
e No Data.
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Table 11. Toxaphene chronic values (ug/L) for three Champia parvula
isolates, Agardhiella subulata, and Grinnellia americana
based on growth (final dry weight) and reproduction of females
and tetrasporophytes. Numbers represent the lowest concentration
tested that was statistically different from the control
(this is the upper limit for the chronic value, the next lowest
concentration was 60% of this value.
GROWTH REPRODUCTION
Species FEMALE TETRA. #CYSTO. //TETRASP. +CYSTO. +TETRASP.
CHAMPIA Ia
CHAMPIA II
CHAMPIA III
AGARDHIELLA
GRINNELLIA
23
65
108
108b
>180C
23
65
NDd
ND
ND
39
108
65
180
108
65
65
ND
ND
ND
108
108
108
>180
108
180
180
ND
ND
ND
a Data from Thursby & Steele [10].
b Growth determined on day 21.
c Based on growth of blade tissue.
d No Data.
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PAGE 102
Table 12. Isophorone chronic values (ug/L) for three Champia parvula
isolates, Agardhiella subulata, and Grinnellia americana
based on growth (final dry weight) and reproduction of females
and tetrasporophytes. Numbers represent the lowest concentration
tested that was statistically different from the control
(this is the upper limit for the chronic value, the next lowest
concentration was 60% of this value.
Species
CHAMPIA Ia
CHAMPIA II
CHAMPIA III
AGARDHIELLA
GRINNELLIA
GROWTH
FEMALE TETRA.
83
83
83
49
49
,070
,070
,070
,842b
,842C
49,840
138,450
NDd
ND
ND
REPRODUCTION
//CYSTO. //TETRASP. +CYSTO. +TETRASP.
S3
49
29
49
49
,070 49,840
,842 48,840
,536 ND
,842 ND
,842 ND
83,070 138,450
83,070 138,450
83,070 ND
138,450 ND
49,842 ND
a Data from Thursby & Steele [10].
" Growth determined on day 21.
c Based on growth of blade tissue.
d No Data.
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PAGE 103
REFERENCES
Abbott, I.A. and C.J. Hollenberg. 1976. Marine algae of
California. Stanford University Press, Stanford, p. 565.
ASTM. 1980. (American Society for Testing and Materials).
Standard practice for conducting acute toxicity tests with
fishes, raacroinvertebrates, and amphibians. ASTM E-729-80.
Eide, I. and S. Mykiestad. 1980. Long-term uptake and release
of heavy metals by Ascophyllum nodosum (L.) Le Jolis.
Environmental Pollution 23:19-28.
Lewis, E.J. 1973. The protein, peptide and free amino acid
composition in species of Champia from Saurashtra Coast,
India. Botanica Marina Vol XVI:145-147.
Mann, K.H. 1972. Introductory remarks, Procedings of the
IBP-UNESCO Symposium on detritus and its role in aquatic
ecosystems. Mem. Irt. Ital. Idrobiol. (Suppl.) 29:13-16.
Melhuus, A., K.L. Seip and H.M. Seip. 1978. A preliminary
study of the use of benthic algae as biological indicators of
heavy metal pollution. Environmental Pollution 15:101-109.
Phillips, D.J.H. 1977. Use of biological indicator organisms to
monitor trace metal pollution in marine and estuarine
environments: A review. Environmental Pollution 13:281-317.
Provasoli, L. 1963. Growing marine seaweeds. Proc. Int.
Seaweed Symp. 4:9-17.
Provasoli, L. 1968. Media and prospects for the cultivation of
marine algae. In: Cultures and collections of algae, pp.
63-75, A. Watanabe and A. Hattori (eds.) Japan COnf.
Hakone, September, 1966. Jap. Soc. Plant Physiol.
Reedman, D.J. and H.B.S. Womersley. 1976. Southern Australian
species of Champia and Chylocladia (Rhodymeniales:Rhodophyta).
Trans. R. Soc. S. Aust. 100:75-104.
Steel, R.G. and J.H. Torey. 1960. Principles and procedures
of statistics. McGraw Hill, p. 481.
Steele, R.L. and G.B. Thursby. 1983. A toxicity test using
life stages of Champia parvula (Rhodophyta). In: Aquatic
Toxicology and Hazard Assessment, Sixth Symposium, pp.
73-89, W.C. Bishop, R.D. Cardwell, and B.B. Heidolph
(eds.) ASTM STP 802. American Society for Testing and
Materials, Philadelphia.
-------
PAGE 104
Taylor, W.R. 1957. Marine algae of the Northeastern Coast of
North America. University of Michigan Press, Ann Arbor,
p. 289.
Thursby, G.B. and R.L. Steele. 1984. Toxicity of arsenite and
arsenate to the marine alga Champia parvula (Rhodophyta).
Environ. Toxicol. Chem. 3:391-397.
U.S. EPA. 1974. (U.S. Environmental Protection Agency). Marine
algal assay procedure: Bottle test. National Environmental
Research Center, Corvallis, Ore.
U.S. EPA. 1978. (U.S. Environmental Protection Agency). Bioassay
procedures for the Ocean Disposal Permit Program. EPA report
600/9-78-010.
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PAGE 105
PART VI.
A SHORT-TERM EXPOSURE TEST FOR
CHAMPIA PARVULA FOR USE IN EFFLUENT TESTING
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PAGE 106
INTRODUCTION
A relatively fast, simple and inexpensive toxicity test method has
been developed for the marine red macroalga Champia parvula, to
assess chronic effects of pollutants to marine macroalgae (Steele
and Thursby, 1983). The method has been used to generate data for
water quality criteria. The test method has previously been
evaluated with heavy metals and cyanide (Steele and Thursby, 1983),
arsenite and arsenate (Thursby and Steele, 1984), as well as ten
different organic compounds (see Part I, this, report). This test
method showed that sexual reproduction is the best end point to
use for Champia. If sexual reproduction is used as the sole end
point, then plants should only have to be exposed to toxicants for
a few days (long enough to show any effects on fertilization).
The current toxicity test with Champia last two weeks, however, a
modification is being developed in which females and males are
exposed together to a toxicant for four days (or less).
Champia parvula is a common species in many parts of the world. It has
been reported from Mexico and Southern California on the west
coast of North America, and from the Carribean to Massachusetts on
the east coast. It has also been reported from France, Spain,
Korea, and Southern Australia. Because of its sensitivity and
wide distribution, Champia could be a useful surrogate species for
determining the relative toxicity of a variety of compounds to
marine macroalgae.
The sensitivity of sexual reproduction with the mini-chronic
(short-term exposure) test was compared against existing data from
the two—week test. This comparison was made with single compounds
because effluents can not be tested easily with the two-week test.
Champia must remain uni-algal during the two-week test and
effluent generally introduce microalgal contamination. The
simplified, short-term exposure test was also tested with several
mixed effluents as a part of ERLN's Effluent Testing Program.
MATERIALS AND METHODS
The short-term exposure last no longer than four days (current tests
show promise for reducing this to one or two days) followed by a
seven day recovery period. Test are in either 400 mL of medium in
500 Ellenmeyer, screw-capped flasks or in 80 mL of medium in 100
mL polystyrene cups (with plastic caps). The medium for toxicity
tests consists of 15 mg/L NaN03, 1 mg/L NaH2P04'H20, 15 ug/L iron,
vitamins (0.06 ug Bj, and biotin and 12.5 ug thiamine'HCl) and 150
mg/L NaH2C03. One and a half cm female branches are cut to serve
as inocula for a test. Five branches are placed into each cup.
One male branch (ca 2 cm long), visibly producing spermatia, is
added to each vessel. All tests are performed in duplicate. Room
light is sufficient illumination during the exposure period.
Temperature and salinity are 22-24°C and 30 ppt, respectively. At
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PAGE 107
the end of the exposure period females are rinsed in sterile
seawater to remove excess spermatia and transferred to control
media for the recovery period. This latter step allows time for
cystocarps (evidence of sexual reproduction) to develop. During
the recovery period cultures are aerated (this enhances growth)
and illuminated with 75 uE-m'^-s"1 of cool-white fluorescent light
on a 16h:8h,- light:dark cycle.
The single compounds tested were benzene, isophorone,
pentachlorophenol, and toxaphene. Effluents from GAF Corporation
and Ciba Geigy in New Jersey, as well as three different sources
on the Quinniplac River in Connecticut were tested. The
Connecticut effluents were from Circuit-wise, Upjohn and American
Cyanimid. The endpoint measured for all tests was the number of
cystocarps per plant. From these data the lowest concentration
that results in a statistically significant decrease from the
control was determined. Statistical significance was determined
by ANOVA (alpha = 0.05) followed by Dunnet's multiple range test.
The concentration that completely inhibits sexual reproduction was
also noted. This endpoint is referred to as NSR (No Sexual
Reproduction). Absence of sex is the simplest endpoint to
quantify, and it does not leave any doubt as to whether the
observed effect is biologically significant. Absence of
cystocarps eliminates the next stage in the life history.
RESULTS
Results form the test with organic compounds indicated that the
sensitivity of Champia in the two-week test and the short-term
exposure test are similar (Tables 1-4). Although the dose
response was not always the same, the concentration that
completely inhibited sexual reproduction was.
Results from the short-term exposure tests with mixed effluents are
shown in Tables 5 to 11. The data were generally similar among
replicates with the same effluent.
DISCUSSION
The two-week test measures effects of toxicants on growth and
reproduction. During the test there is approximately three orders
of magnitude change in the dry weight of individual plants. This
test is assumed to be representative of Champia's 'true1
sensitivity to toxicants. The existing data from the two-week
test indicates that Champia's sensitivity is comparable to that of
.the most sensitive marine animala.
The mini-chronic (short-term exposure) test gave the same values for
NSR as the two-week test. The fact that it did not always give
the same dose response is not a major concern. The ultimate goal
is to use NSR as the endpoint for Champia.
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PAGE 108
The repeatability of results for Champia sensitivity to the same
effluent is a definite plus in its favor. The NSR endpoint is
difficult to compare with endpoints of other species, since
chronic values for other species typically refer to statistical
differences from the control. If we examine the data for Champia
for statistical differences from the control, then Champia is
approxiamtely in the middle of the range of results for all of the
species that are being examined for ERLN's Effluent Program.
CONCLUSION
There are still a few details to be examined for field use of the
mini-chronic test with Champia (i.e., storage of plants after
exposure to effluents). However, the current mini-chronic
protocol has been successfully used in the field. The preliminary
protocol is listed in the next section.
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PAGE 109
Table 1. A comparison of the effect of benzene on sexual
reproduction in Champia using the two-week and niini-chronic
procedures. The mini-chronic was a four-day exposure followed by
a seven-day recovery period in control medium.
Concentration # Gystocarps/plant *
(ug/L) Two-Week Test Mini-Chronic
Control 19+8 22+14
34,160 19+8 11+4
56,930 14 + 10 0.8 + 0.4
94,890 0 0
158,150 0 0
* Mean + S.D.
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PAGE 110
Table 2. Comparison of the effect of pentachloroethane on sexual
reproduction in Champia using the two-week and mini-chronic test
procedures. The mini-chronic was a four-day exposure followed by
a seven-day recovery period in control medium. The carrier for
both procedures was triethylene glycol (TEG).
Concentration
(ug/L)
Control
# Cystocarps/Plants *
Two-Week Test Mini-Chronic
43 + 11
Carrier Control 28+15
1,000
1,700
2,850
4,700
7,900
13,100
21,800
43 + 18
37 + 22
25+9
12+9
7+6
0
0
72
97
65
44
41
37
19
0
0
+ 24
+ 22
+ 20
+ 21
+ 23
+ 14
+ 7
* Mean + S.D.
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PAGE 111
Table 3. Comparsion of the effects of isophorone on sexual
reproduction in Champia using the two-week and mini—chronic
procedures. The first mini-chronic (A) was a four-day exposure.
The next two (B and C) were one- and two-day exposures
respectively. All mini-chronics were followed by a seven-day
recovery period in control medium.
Concentration # Cystocarps/Plant *
(ug/L) Two-Week Test Mini-Chronic
A B C
Control
29,500
49,800
83,100
138,450
18 + 4
30 + 9
18 + 10
0.8 + 0.6
0
23 + 6
15 + 7
7 + 4
7 + 6
0
19 + 6
19 + 9
4+3
1 + 1
0
22 + 12
5 + 1
3 + 2
0.7 + 0.8
0
*Mean + S.D.
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Table 4. Comparison of Che effects of toxaphene on sexual
reproduction in Champia using the two-week and mini-chronic
procedures. The two mini-chronics (A and B) were one- and two-day
exposures respectively. Both mini-chronics were followed by a
seven-day recovery period in control medium. The toxaphene was
first dissolved in acetone (1 g in 100 mL) and then diluted (240
uL to ten mL with TEG) to the working stock with triethyleneglycol
(TEG). The carrier was acetone in TEG (240 uL to ten mL with
TEG).
Concentration
(ug/L)
# Cystocarps/Plant *
Two-Week Test Mini-Chronic
A B
Control
Carrier Control
14
23
39
65
108
180
26 +
27 +
34 +
26 +
12 +
1 +
0.1 +
dead
6
12
12
9
3
2
0.3
9 +
-
8 +
6 +
3 +
4 +
3 +
0
4
5
5
2
2
2
15
13
16
15
16
12
5
+ 5
+ 5
± 5
+ 3
+ 7
± 8
± 2
0
* Mean + S.D.
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PAGE 113
Table 5. Number of cystocarps (evidence of sexual reproduction)
produced per plants during a four-day exposure to effluent from
GAF Corporation in New Jersey. The effluent was collected on
May 3. Exposure was followed by a seven-day recovery period in
control medium.
Exposure Concentration # Cystocarps/Plant *
(%) May 21 June 18
Control
0.65
1.1
1.8
2.9
4.8
36 + 12
49 + 12
30 + 13
16 + 7**
0.2 + 0.4
0
58
79
51
26
7
0
+ 15
± 31
± 31
+ 10**
+ 7
* Mean + S.D.
** Significantly less than the control (p > 0.05)
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Table 6. Number of cystocarps (evidence of sexual reproduction)
produced per plant during laboratory exposures to effluent from
Ciba Geigy in New Jersey. Exposure was followed by a seven-day
recovery period in control medium. The pre-trip exposure was
four-days. The post-trip exposures were 1,2 and 4-days (A,B, and
C, respectively).
Exposure Cone
(%)
Control
0.6
1.2
2.4
4.8
7.0
11.0
# Cystocarps/Plant *
Pre-trip Post-trip
ABC
58 + 15 16 + 6 20 + 6 24 + 6
15 + 2 17 + 4 29 + 9
49+12 14+5 21+7 28+8
22 +6** 9 + 5** 12 + 7** 29 + 15
3 + 3 2 + 1 0 19 + 9
0 000**
0 -
*Mean + S.D.
** Significantly less than the control (p < 0.05)
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Table 7. Number of cystocarps (evidence of sexual reproduction)
produced per plant during two- and four-day exposures to effluent
from Ciba Geigy in New Jersey. The exposures were carried out
while on site.
Concentration
(%)
Control
0.1
0.32
1.0
3.2
10.0
32.0
# Cystocarps/Plant *
Two-Day Exp. Four-Day Exp.
52 + 11
67 + 20
66+8
20 + 15**
0
0
0
48 + 34
56 + 22
58 +_ 10
38 + 14
1 + 1**
0
0
*Mean + S.D.
** Significantly less than the control (p < 0.05)
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PAGE 116
Table 8. Number of cystocarps (evidence of sexual reproduction)
produced per plant during a 4-day exposure to effluents from
Circuit-Wise, Upjohn and American Cyanimid. The effluent was
collected prior to the field trip. Exposure was followed by a
7-day recovery period. Salinity was 30 ppt and temperature 22 to
24°C. Ten plants were used per treatment.
Exposure Concentration # Cystocarps per plant
(mean + S.D.)
Circuit-Wise
Control 23+5
0.06 13 + 3*
0.12 8 + 2
0.25 3+2
0.50 1 + 2
1.20 0
Upj ohn
Control 20+5
0.6 15 + 3*
1.2 13+3
2.4 13+4
4.8 13 + 3
7.0 0
11.0 0
American Cyanimid
Control 22+3
0.6 19 + 3
1.2 19+6
2.4 13+3
4.8 18+6
7.0 21+5
11.0 17 + 5
* Statistically less than the control (p < 0.05)
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PAGE 117
Table 9. Number of cystocarps produced per plant during a 3-day
exposure Co effluent from Circuit-Wise. Ten plants were used per
treatment.
Exp.Conc. Field*
(%) A B
Control
0.01
0.032
0.10
0.32
1.0
3.2
10
7
11
dead
3
dead
dead
8
5
7
5
0.8
0.6
0
Aug 15
19 + 9
15 + 4
13+4
15 + 9
9 + 9***
6 + 4
0
Laboratory**
Aug 17 Aug 21
19 + 9
17 + 5
14+4
12 + 12***
10 + 5
2 + 1
0
19 +
11 +
9 +
11 +
1 +
0
0
9
6***
3
6
1
* Both sets of data are from exposures on August 17, 1984.
Plants from 'A' were aerated imediately after the exposure and
plants from 'Bf were stored at room temperature and aeration began
at the main laboratory several days later. No S.D.'s are reported
because plants had severe die-back and had fragmented. Only total
cystocarps per treatment were counted (and then divided by 10).
** Dates represent the days on which the effluent was collected,
Actual testing was started on September 25, 1984.
*** Statistically less than the control (p < 0.05)
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Table 10. Number of cystocarps produced per plant during a 3-day
exposure to effluent from Upjohn. -
Exp.Conc.
(%)
Control
0.032
0.10
0.32
1.0
3.2
10
Field*
9
10
9
7
5
0
dead
Aug 15
14 + 6
20 + 8
26 + 10
23 + 6
12 + 9
o***
0
Laboratory**
Aug 17
13 + 7
20 + 5
24 + 12
15 + 6
12 + 8
o***
0
Aug 21
7 + 4
10 + 3
13 + 2
10 + 2
12+7
Q***
0
* Data from exposures on August 17, 1984. No S.D.'s are
reported since plants experienced severe die-back and had
fragmented Only total cystocarps per treatment were counted
(divided by 10).
** Dates refer to days on which effluent was collected.
Tests were actually started on September 25.
*** Statistically less than the control (p < 0.05)
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Table 11. Number of cystocarps produced per plant during a 3-day
exposure Co effluent from American Cyanimid. Ten plants were used
per treatment.
Exp.Conc.
Control
6.3
12.5
25
50
Field* Laboratory**
Aug 15 Aug 17 Aug 21
12 16 + 5 16 + 5 16 + 5
8 16+4 18+4 10 + 4***
7 2 + 2*** 7 + 3*** 0.5 + 0.5
0000
00 0 0
*Data is from exposure on August 17, 1984. No S.D.'s are
reported since plants experienced severe die-back and fragmentation.
Only total cystocarps per treatment could be counted (divided by
10).
**Dates refer to days only which effluent was collected. Actual
testing started on September 25.
***Statistically less than the control (p < 0.05)
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REFERENCES
Steele, R.L. and G.B. Thursby. 1983. A toxicity test using
Life stages of Champia parvula (Rhodophyta). (in) W.C.
Bishop, R.D. Cardwell, and B.B. Heidolph, eds. , Aquatic
Toxicology and Hazard Assessment, Sixth Symposium. ASTM STP
802. American Society of Testing and Materials,
Philadelphia, pp. 73-89.
Thursby, G.B. and R.L. Steele. 1984. Toxicity of arsenite and
arsenate to the marine red alga Champia parvula. Environ.
Toxicol. Chem. 3:391-397.
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PART VII.
PROTOCOL FOR EFFLUENT FIELD TEST WITH THE
MARINE RED ALGA CHAMPIA PARVULA
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A. MATERIALS:
1. fine point stainless steel forceps
2. 100 mL polystyrene cups with covers
3. 250 mL
4. 100 mL graduated cylinders (2)
5. 1 and 10 mL disposable pipets
6. digital micropipet (200 and 1000 uL maximum)
7. disposable tips for digital micropipets
8. nutrient stock solution (see Appendix I)
9. sodium bicarbonate stock solution (see Appendix I)
10. recovery bottles ("BOD" bottles with 200 mL seawater +
1/2 mL of both the nutrient and bicarbonate stock
solutions). Need one bottle per treatment replicate.
11. air pump
12. air tubing and "gang" valves
13. plastic aeration tubes (1 mL disposable pipets broken
between the 5 and 6 mL mark).
14. thermometer
15. squeeze bottle filled with sterile seawater
16. marking pens and colored tape
B. PLANTS
Cut 1.5 to 2 cm female branch tips, enough for 5 branches per
cup. Cut 2 to 2.5 cm male branch tips, enough for one branch per
cup. Store in aerated, 1 liter flasks with 800 mL of natural
seawater (30 ppt) + nutrients (2.5 mL per liter). Use
screw-capped polycabonate flasks to make transport to field site
easier (no breakage). Keep plants at 50 to 75 uE.m-2.s-l of light
and at 22 to 24°C.
C. EXPERIMENTAL SET UP
1. Set up and label control and treatment cups (two per
treatment).
2. Fill cups with 80 mL of natural seawater.
3. Add 200 uL of both the nutrient and bicarbonate stock solution
to each cup.
4. Add effluent in appropiate amounts with either disposable or
digital pipets.
5. Add 5 female branches and 1 male branch per cup.
6. Place cups in room light away from direct sunlight (to avoid
sharp temperature increases). Place thermometer in a bottle
of water and place near cups.
7. Gently hand-swirl cups twice a day (spermatia are not motile,
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therefore water motion is critical). Record temperature
daily.
8. Day 4.
A. Label recovery bottles ("BOD" bottles)
B. With tweezers, remove females from cups and rinse
briefly with clean seawater (squeeze bottle).
Put females into recovery bottles.
C. Place bottle on counter and aerate. Supplemental
lighting is not necessary, but will speed up
cystocarp development.
9. Return trip home.
A. Remove air tubes and place caps on recovery bottles.
Place bottles back into rack and keep cool (<_ 24 G).
B. When back at the laboratory, aerate bottles again
until cystocarps have developed (at 75 uE'm~^-s~^
of cool-white fluorescent light on a 16hL:8hD
photoperiod the total recovery period is 7 days).
10. Endpoint:
The endpoint measure for all tests is the number of
cystocarps per plant. From these data the lowest effluent
concentration that results in a statistically significant decrease
from the control is determined. Statistical significance is
determined by ANOVA followed by Dunnett's multiple range test
(alpha = 0.05). The concentration that completely inhibits sexual
reproduction is also noted. Absence of sex is the simplest end-
point to quantify. It does not leave any doubt at to whether to
observed effect is biologically significant, since absence of
cystocarps eliminates the next stage in the life history.
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APPENDIX I.
Recipe for optimum nutrient medium for growth of Champia parvula
in natural seatwater. Both EDTA and trace elements have been
omitted.
COMPOUND CONCENTRATION PER LITER
FINAL SOLUTION CONCENTRATED STOCK3
NaN03
NaH2P04'H20
9.35 mg
0.62 mg
3.74 grams
0.25 grams
Ironb
2.6 ug 1.04 mg
Vitamins
B12
Biotin
Thiamine-HCl
0.06 ug
0.06 ug
12.5 ug
10 mLc
a Use 2.5 mL/L (200 uL/80 mL) for final concentrations. Add
150 mg/L sodium bicarbonate when cultures are not aerated.
A stock solution of 60 mg/mL NaH2C03 is prepared by auto-
claving sodium bicarbonate as a dry powder and then
dissolving it in sterile deionized water. Use 2.5 mL/L
(200 uL/80 mL) for the final concentration.
" Iron stock solution prepared by dissolving 1 g iron powder
in 10 mL concentrated HC1 and diluting to 1 liter with
deionized water.
c Vitamin stock solution autoclaved separately in 10 mL
sub-samples. Each 10 mL contains 24 ug 6^2, 24 ug biotin
and 5 mg thiamine.HCl.
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