Development of short-term
exposure tests for marine
macroalgae for use in effluent
testing
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FINAL REPORT FOR COOPERATIVE AGREEMENT f CR812070-01
DEVELOPMENT OF SHORT-TERM EXPOSURE TESTS FOR MARINE
MACROALGAE FOR USE IN EFFLUENT TESTING
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
Noverraber 26, 1986
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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
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. 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,
arsenate and arsenite, as well as ten different organic compounds. This
test method showed that sexual reproduction is the best endpoint to use
for Champia. If sexual reproduction is used as the sole endpoint, then
plants should only have to be exposed to toxicants for a few days (long
enough to show any effects on fertilization). The previous toxicity
test with Champia lasted two weeks, however, a modification has been
developed in which females and males are exposed together to a toxicant
for only two days. The procedure has been used successfully with single
»
compounds and a variety of complex effluents.
Initial steps were also taken in the development of a similar
short-term test with the brown alga, Laminaria saceharina. Laminaria
represents another phylum of algae than Champia, and has the additional
advantage of being both economically and ecologically important. Sexual
reproduction in Laminaria has already been shown to be sensitive to
petroleum products. For the current study, the feasibility of using
Laminaria as a routine toxicity test species was verified. The large
adult sporophyte of Laminaria is difficult to maintain in the labora-
tory. However, the male and female gametophytes are microscopic and are
easily cultured.
The Office of Water's Permits Division of the Environmental
Protection Agency needs toxicity test procedures for marine and
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eatuarine species. The procedures are needed to characterize and
quantify the toxicity of effluent discharges by National Pollutant
Discharge Elimination System permittees. The tests must yield chronic
data in a relatively short time period (7 days or less). The data must
be both scientifically sound and legally defensible. At the same time,
it is necessary for the methods to be simple and cost efficient
(requiring standard hardware and laboratory facilities). The test
species should be readily available, and testing should be practical for
both on-site and off-site. All of these requirments are met by the test
methods developed for the marine algae, Champia parvula and Laminaria
saccharina.
This report covers work completed during the time period from
November 1, 1984 through October 31, 1986. The specific objectives of
the cooperative agreement were:
I. Champia parvula
A. Write final guidance manual for conducting short-term chronic
tests.
B. Compare data from above test procedure with that from the
existing two-week test.
C. Test short-term test in field and laboratory with complex
effluents.
II. Laminaria saccharina
A. Test feasibility of Laminaria saccharina as a routine toxicity
test species for short-term tests.
B. Compare the sensitivity of Laminaria with that of Champia.
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The techniques developed during this cooperative agreement were
presented at three effluent monitoring workshops. The Champia technique
was presented at EPA1s Narraganaett, Rhode Island laboratory in October,
1985, and at EPA'3 Gulf Breeze, Florida laboratory in February, 1986.
The Laminaria technique was presented at the workshop held in Newport,
Oregon, in October, 1986.
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TABLE OF CONTENTS
PART I. GUIDANCE MANUAL FOR CONDUCTING SEXUAL REPRODUC-
TION TESTS WITH THE MARINE MACROALGA CHAMPIA PARVULA
FOR USE IN TESTING COMPLEX EFFLUENTS
PART II. COMPARISON OF SHORT- AND LONG TERM SEXUAL
REPRODUCTION TESTS WITH THE MARINE RED ALGA CHAMPIA
PARUVLA
THAT OF CHAMPIA
PAGE
1-1
II-l
PART III. SUMMARY OF RESULTS FROM TESTING COMPLEX EFFLUENTS .....
PART IV. PRELIMINARY GUIDANCE MANUAL FOR CONDUCTING
SEXUAL REPRODUCTION TESTS WITH THE MARINE MACROALGA,
LAMINARIA SACCHARINA, FOR USE IN TESTING COMPLEX
EFFLUENTS ............. . ...................................... IV_1
PART V. COMPARISON OF THE SENSITIVITY OF LAMINARIA WITH
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PART I
GUIDANCE MANUAL FOR CONDUCTING SEXUAL REPRODUCTION TEST WITH
THE MARINE MACROALGA CHAMPIA PARVULA
FOR USE IN TESTING COMPLEX EFFLUENTS
1-1
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TABLE OF CONTENTS
page
INTRODUCTION 3
BACKGROUND 5
MAINTENANCE OF STOCK CULTURES 8
ARTIFICIAL SEAWATER 12
SALINITY ADJUSTMENTS 14
PREPARATION OF PLANTS FOR A TEST 15
TEST CHAMBERS 18
TEST CONDITIONS 19
PROTOCOL 22
STATISTICAL TREATMENT OF DATA 25
CRITERIA FOR ACCEPTABILITY 26
REFERENCES 27
ADDENDA 29
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INTRODUCTION
Seaweeds have been considered less useful for toxicity testing than
microalgae (Jensen, 1984), and microalgae are often considered less
sensitive than aquatic animals (Kenaga and Moolenaar, 1976; Kenaga,
1982). Therefore, one could easily come to the erroneous conclusion
that toxicity testing with seaweeds is not necessary. As recently as
1983 the statement was made that "seaweeds seem to be rather insensitive
to many chemicals and will probably survive pollution better than many
other organisms in the marine environment" (Jensen, 1984).
A two-week toxicity test method has already been developed for the
maeroalga, Champia parvula, to assess chronic effects of pollutants to
marine seaweeds (Steele and Thursby, 1983). The test has previously
been evaluated with heavy metals (Steele and Thursby, 1983), arsenite
and arsenate (Thursby and Steele, 1984), and ten different organic
compounds (Thursby, et al., 1985). This test method shows that sexual
reproduction is generally the most sensitive and practical endpoint to
use for Champia.
Pollution assessments with macroalgae must take reproduction into
consideration if an accurate picture of the potential harm is to be
drawn. Previous conclusions about seaweed sensitivity were based large-
ly on growth as the endpoint. The ability to measure the sensitivity of
seaweeds to toxicants increases when sexual reproduction is used as an
endpoint; and can be greater than many aquatic animals that have been
tested. This has been shown for Champia parvula (see above references)
as well as for the brown macroalgae Fucus edentatus and Laminar!a
saccharina (Steele and Hanisak, 1978).
The above toxicity test with Champia parvula is a two-week growth
and reproduction study and requires that the cultures remain unialgal.
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This time period makes the test unaccepatable for testing complex ef-
fluents and their receiving waters. Fine-filtering, which would be
necessary to remove unwanted microalgae, could change the character of
the effluent or receiving water. Any microalgae introduced with the
effluent would compete with Champia for light and nutrients, thus in-
fluencing Champia's growth rate. However, if sexual reproduction is
used as the sole endpoint, then plants only need to be exposed for a few
days (long enough to show effects on fertilization). Any effect of
other organisms on the growth rate of Champia would not be serious since
interest would only be in whether sexual reproduction had taken place.
Sexual reproduction was selected as the endpoint for effluent.
testing for several reasons. It had previously proven to be a sensitive
endpoint from the two-week toxidty test procedure using single com-
pounds. .A sexual reproduction test for toxicity could be short enough
to fit the tine-constraints for tests used in the effluent program.
Finally, Champia is an annual plant and inhibition or absence of sexual
reproduction reduces or eliminates the next stage in its life history.
Total absence of cystocarp formation is the easiest endpoint to inter-
pret as far as field populations are concerned. In most of the red
algae each fertilization results in the formation of a new life history
stage, the carposporophyte, "parasitic" on the female and housed within
the cystocarp. Each carposporophyte is capable of producing many spores
(perhaps a hundred or more in the case of Champia). This characteristic
makes it difficult to interpret the biological significance of a statis-
tical decrease in the number of cystocarps or an arbitrary percent
decrease such as 507.. Absence of reproduction leaves no doubt about its
biological significance.
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BACKGROUND
This paper describes a method which uses sexual reproduction, after
a short-term exposure to effluents, to estimate chronic toxicity. In
brief, the method consists of exposing males and females to effluents or
receiving waters for two days, followed by a 5- to 7-day recovery period
in control medium. The recovery period allows time for any cystocarps
to mature. At the end of the recovery period the number of cystocarps
per plant are counted. The goal for Champia within the effluent program
is to use absence of sexual reproduction as the endpoint. Statistical
differences (or other "cut-offs"), although more difficult to interpret
ecologically, can also be included to more easily make comparisons with
other marine species in the program. In addition, the reporting of
concentrations that cause the total absence of sexual reproduction and
statistical differences will give some idea of the steepness of the dose
response curve.
r
The method described here has been used for both single compounds
and complex effluents. Tests have been conducted on-site in a mobile
laboratory and at the EPA's Environment1 Research Laboratory, Narragan-
sett, HI. Several different types of effluents have been tested. These
included one from a pulp mill; two industrial sites that discharge
effluents containing heavy metals; five industrial sites discharging
organically contaminated effluents, including pesticides and dyes; and
17 different sewage effluents. In addition several receiving waters
have also been tested. In all, more than 30 tests have been performed.
When basing the test endpoint on the absence of reproduction, the pulp
mill effluent had its effect between 1.0 and 2.5 7. effluent; the heavy
metal effluents had a range of effect of 0.054 to 0.50 7. effluent;
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organic effluents had a range of 3 to 20 7. effluent (generally <5 7.};
and the sewage effluents ranged from 2.5 to 25 7. effluent (generally <
10 7.). Receiving water effects have been detected and ranged from
little or no effect on sexual reproduction to total elimination of
sexual reproduction.
Nine single compounds have been used to compare effects on sexual
reproduction using the two-week test and the two-day exposure. Several
different cut-off points for the endpoint of sexual reproduction from
the two-day exposure were compared against the no sexual reproduction
(HSR) endpoint from the two-week test (Thursby and Steele, 1986). From
among these comparisons 37.-of-the-control (95% or greater decrease) gave
the best estimate of the NSR effect for the two-week test (Table 1).
Therefore, cystocarp counts at < 5% of the control are considered zero
for the short-term exposure test.
The sexual reproduction, two-day exposure test has been developed
as a static, non-renewal test (although daily media changes are possi-
ble) for effluents and receiving waters. The method is easy and cost
efficient to perform. Stock cultures are maintained in the laboratory
with standard laboratory equipment, therefore, plant material can be
available year-round. The test procedure is intended to be used to
estimate chronic effects of complex effluents on marine oacroalgae.
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Table 1. Comparison of the short-term exposure test and the two-week
test using single compounds. Values listed are the geometric means of
the effect and no-effect concentrations in ug/L. Effect is defined as
either £ 57. of the control (short-term exposure test) or no sexual
reproduction (two-week test).
COMPOUND
Arsenite
Cadmium
Copper
Silver
Benzene
Isophorone
Pentachloro-
SHORT-TERM (STE)
232
>iooa
7.7
0.92
73,600b
107,050
10,170
TWO- WEEK (TWT)
139
77
7.7
1.50
73,600
107,050
10,170
RATIO (STE/TWT)
1.67
>1.30
1.00
0.61
1.00
1.00
1.00
ethane
Pentachloro- 465 465 1.00
phenol
Toxaphene .140 140 1.00
aSTE was run in polystrene cups, in 125 Erlenmeyer flasks and in 500 mL,
screw-capped flasks with 400 mL of medium. The results were always the
same. The STE may not work for cadmium because it is a slow toxicant
and two days is not enough time to see its full effect.
STE was run in 400 mL of medium in 500 mL, screw-capped flasks. The
STE did not compare well with the TWT when the cups were used. A
larger volume may be necessary when working with highly volatile com-
pounds .
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MAINTENANCE OF STOCK CULTURES OF CHAMPIA PARVULA
Plants of Champia parvula (C. Agardh) Harvey (Rhodophyta) are bushy
and 5 to 10 ca tall in the field. The main axis and branches are
cylindrical, hollow and septate. Champia's life history is an alterna-
tion of isomorphic generations (Fig. 1). The clone presently used was
isolated from Rhode Island waters in 1979. It is probably not essential
to have a standard clone, however, some experience is required to iso-
late new clones from the field into unialgal culture. Unialgal stock
cultures are necessary to maintain healthy, actively growing plants for
use in testing. 7egetatively propagated plant material from the 1979
clone is available from the U.S. Environmental Protection Agency, South
Ferry Road, Narragansett, RI 02882.
tetraaporonqia—?
MALE
spermatic
a
TETRASPOROPHYTE
FEMALE
5mm
fertilization
^cystocarp
Figure 1. Life history of the marine red alga Champia parvula.
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Male and female plants of Champia can be maintained easily in
unialgal culture in the laboratory. New cultures can be started from
excised branches, making it possible to maintain clonal material indef-
initely. No special preconditioning is required to induce reproduction.
Under the conditions listed below, male gaoetophytes produce spermatia
continuously and females are always receptive. Thus, plant material can
be available at any time for testing.
Laboratory cultures of Ghanpia provide test plants of similar
preconditioning. Unialgal stock cultures of both males and females are
maintained in separate, aerated 1000 mL Erlenmeyer flasks containing 300
mL of the culture medium. The choice of this flask is one of prefer-
ence rather than necessity. All culture glassware should be acid-strip-
ped in 10 to 12 7. HC1 and rinsed in deionized water after washing. This
is necessary since many detergents can leave a residue that is toxic to
Champia. The culture medium is made from natural seawater to which
additional nutrients are added (Table 2). The nutrients used with
artificial seawater can also b« used (see ARTIFICIAL SEAWATER). The
seawater is autoclaved for 30 min at 15 psi. The culture flasks are
capped with aluminum foil and autoclaved dry, for 10 min. Culture
medium is made up by dispensing seawater into the sterile flasks and
adding the appropriate nutrients from a sterile stock solution. Alter-
nately, liter flasks could be autoclaved with the seawater already in
them. Sterilization is used to prevent microalgal contamination, and
not to keep cultures bacteria-free.
We recommend that several cultures of both males and females be
maintained simultaneously to keep a constant supply of plant material
available. Some cultures should be at different stages of development
(i.e., with different amounts of tissue per flask). Initial stock
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cultures should be started weekly with about twenty 1.0 cm branch tips.
Cultures are gently aerated through sterile, cotton-plugged, disposable,
polystyrene 1 mL pipets. Cultures are capped with foam plugs and alumi-
num foil and illuminated from the side with 75 uE m" s" of cool-white
fluorescent light on a 16:3, light:dark cycle. The temperature is 22 to
24 'C and the salinity 28 to 30 °/,a. Media are changed once a week.
Table 2. Recipe for additional nutrients to be added to natural sea-
water for stock cultures and test medium. Both EDTA and trace metals
have been omitted. The concentrated stock solution is autoclaved at
standard temperature and pressure for 15 minutes (the pH is adjusted to
2.0 prior to autoclaving to prevent precipitation).
COMPOUND AMOUNT/LITER
TEST MEDIUM CONC. CONCENTRATED STOCK*
Na»03
N«H2P04«H20
Iron
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 mL6"
use 0.25 mL/100mL (2.5 mL/L) for test medium concentrations and 10 mL/L
for stock cultures. For test medium only, add 0.25 mL/100mL of a
sodium bicarbonate solution. A stock solution of 60 mg/mL sodium
bicarbonate is prepared by autocalving it as a dry powder and then
dissolving it in sterile deionized water.
Iron stock solution prepared by dissolving 1 g iron powder in 10 mL
concentrated HC1 and diluting to 1 liter with deionized water. Accept-
able stock soultions could also be made with ferric or ferrous
chloride.
Vitamin stock solution autoclaved separately in 10 mL sub-samples.
Each 10 mL contains 24 ug B12, 24 ug biotin and 5 mg thiamine-HCl.
Adjust pH to ca 4.0 before autoclaving for 2 min.
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About half of the plants should be discarded (or placed into anoth-
er culture vessel) with each weekly medium change to reduce the amount
of biomass as the plants grow. At the end of three weeks plants will be
ready to use for testing. Readiness is defined as having enough plant
material to perform at least one test. With this procedure, actively
growing plants will be continuously available. The total number of
cultures maintained will depend on the expected frequency of testing.
A stock culture should not be used as a source of test material if
the plants appear to be stressed or undernourished. Under conditions
of stress the tips of the branches will turn "pink" and the older tissue
will generally be much paler. In addition, the sterile hairs of stressed
plants will appear stubby, especially near the branch tips. The tricho-
gynes of stressed female plants will also be stunted or absent. This can
be evident even in plants that do not have any apparent color change.
Under conditions of nutrient deficiency (resulting usually from too much
plant 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 necrbtic tissue). If cystocarps are pres-
ent on females in the stock cultures, the plants are not suitable for
testing (this usually happens as a result of contamination by males or
water from male cultures).
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ARTIFICIAL SZAWATER
Because salinity adjustments will be necessary in many complex
effluent tests, an artificial seawater recipe that yields good growth of
Champia is desirable. An artificial seawater would also make the test
method more readily available to laboratories that do not have access to
clean natural seawater. Some commercial preparations are toxic to
Champia (presumably due to the presence of high concentrations of trace
metals that are in the commercial grade of salts used in their prepara-
tion). We have had success with artificial seawater using GP2 (Spotte,
et al., 1984). Plants grow and reproduce well and have the correct
morphology in GP2, although they may be slightly smaller in diameter
than plants grown in natural seawater. Plants require approximately two
weeks to acclimate to the artificial seawater. The recipe for arti-
ficial seawater with GP2 is listed in Table 3.
A comparison between the sensitivity to toxicants in the GP2 medium
with that in natural seawater has only been made for copper. Results
suggest that plants grown and tested in GP2 are slightly less sensitive
to copper than plants from natural seawater. This may be due to an
acclimation to higher levels of heavy metals in the GP2 medium (from the
reagent grade salts). However, comparisons with more compounds are
needed before we can conclude that plants grown in GP2 are always Less
sensitive.
During the months of June, July and August the quality of the
seawater that we normally use to culture and test Champia parvula is
often poor. We have, however, been able to obtain excellent growth and
reproduction from Champia during this period of time by mixing GP2 and
natural seawater in a 50:50 ratio. The plants required no acclimation
period to this mixture.
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Table 3. Recipe for artificial seawater using GP2. The concentrations of
the salts have been adjusted to give a final salinity of 30a/«o. The
original recipe calls for autoclaving anhydrous and hydrated salts
separately to avoid precipitation. However, if the sodium bicarbonate
is autoclaved separately (dry), then all of the salts can be autoclaved
together. Since no nutrients are added until needed, autoclaving is
not critical for effluent testing. To minimize microalgal contamination
the artificial seawater should be autoclaved when used for stock cul-
tures. Autoclaving should be for at Least 10 min for 1 liter batches
and 20 min for 10 to 20 liter volumes (at standard temperature and
pressure).
COMPOUND GRAMS/LITER*
NaCI
Na2S04
KC1
KBr
Na2B40?.10H20
MgCl2-6H20
CaCl2.2H20
SrCl2-6H20
NaHC03b
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
Generally made in 10 to 20L batches.
A stock solution of 68 mg/mL sodium bicarbonate is prepared by auto-
claving it as a dry powder and then dissolving it in sterile deionized
water. For each liter of GP2 use 2.5 oL of this stock solution.
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SALINITY ADJUSTMENTS
We have used natural seawater brine (made by evaporation to ca. 100
"/oo), GP2 brine (GP2 made to 3x strength), and concentrated GP2 salts
added separately, to adjust brackish receiving waters to 30 */««•
Salinity adjustments with dry salts are usually too cumbersome, espe-
cially for use in a mobile laboratory. If the initial salinity of the
receiving water is >15 °/a«, then plants grown in natural seawater can
b« used with any of the above methods of salinity adjustment. However,
if the initial salinity of the receiving water is <15 '/„<>» then plants
previously acclimated to artificial seawater are recommended. In either
case, a control using clean natural seawater diluted with deionized
water to the lowest salinity to be tested (then adjusted upwarded to 30
*/ao with one of the techniques above) should be used. If the salinity
range of the receiving waters to be tested from a given location is
great, then it is advisable to make up diluted controls at several
»
salinities.
Plants grown in artificial seawater have always done well when
placed into natural seawater (i.e., no acclimation period required).
However, plants grown in natural seawater do not always do well when
placed into artificial seawater.. This is true even for the two day
exposure period.
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PREPARATION OF PLANTS FOR A TEST
Stock cultures should be checked for their readiness for use in
toxicity tests. Females can be checked by examining a few branch tips
under a compound microscope (100 X or greater). Several trichogynes
(reproductive hairs to which the spermatia attach) should be easily seen
near the apex (Fig. 2). Male plants should be visibly producing sperma-
tia. This can be checked by placing some male tissue in a petri dish,
holding it against a dark background and looking for the presence of
spermatial sori (Fig. 3). Another way is to examine the males under a
compound microscope. A mature sorus can be easily identified by looking
at the edge of the thallus (Fig. 4). A final, quick way to determine
the relative "health" of the male stock culture is to place a portion of
a female plant into some of the water from the male culture for a few
seconds. Under a compound microscope numerous spermatia should be
attached to both the sterile hairs and the trichogynes (Fig. 5).
Once cultures are determined to be usable for toxicity testing,
branch tips should be cut into their final size. For females, cut 7 to
10 mm branch tips, enough for 5 per treatment chamber (try to be consis-
tent in the degree of branching; see Fig. 6). The cutting can be easily
done with fine-point forceps with the plants in a little seawater in a
petri dish. Repeat for males, except cut 1.5 to 2 cm branches and only
one per treatment chamber. The males should visibly be producing sper-
matia (i.e. two or more spermatial sori present). Cut the females first
to minimize the chances of contaminating them with water from the male
stock cultures.
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sterile hairs
-^trichogynes
1 mm
Figure 2. Apical tip of female branch of Champia parvula showing
sterile hairs and trichogynes (reproductive hairs). Sterile hairs
aze wider and generally ouch longer than trichogynes. They also
appear to be hollow except at their apex. Both types of hairs
occur around the entire circumference of the thallus but are seen
easiest at the "edges". Receptive trichogynes occur only near the
branch tips.
1 cm
spermatial
sorus
Figure 3. A portion of a male thallus of Champia parvula showing sper-
matial sori. The sorus areas are generally slightly thicker in
diameter and a little lighter in color.
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cuticle
ICO^m
0 ^
^ 0 spermatia
«
thallus surface
Figure 4. A close-up of a portion of a spermatial sons, note the rows
of cells that protrude from the thallus surface.
spermatic
Figure 5. Apical tip of a female of Champia parvula that had been
"dipped" in water from a mala culture. The sterile and reproduc-
tive hairs are covered with spermatia. Note that older hairs
(those more than about one mm back from the apex) have few to no
spermatia attached to them.
1 cm
Figure 6. The size and degree of branching that is generally used for
the female starting plants. Occasionally the branches will be
longer near the tip, try to be consistent in the degree of branch-
ing since the receptive trichogynes are at the branch tips. There-
fore, the more variation in the degree of branching, the more
variation in the potential number of cystocarps per plant.
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TEST CHAMBERS
Most testing to date has been performed with 100 mL in ca. 110 mL
polypropylene cups with fitted polyethylene caps (Falcon*). These cups
offer the advantage of being disposable, and their wide opening allows
easy access to plants for transferring to recovery bottles. The use of
these cups is out of preference rather than necessity. Successful tests
have also been run in 125 mL Erlenaeyer, Pyrex* flasks and in 100 oL
polystryene cups with plastic caps.
If glass test chambers are used, then they should be acid-stripped
for ca. 10 tain, in 10 to 12 7, HC1 and rinsed in deionized water after
washing. This removes potentially toxic residues left by the detergent.
If organic compounds have been previously tested in the glassware, then
a rinse with acetone prior to washing is recommended.
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TEST CONDITIONS
Temperature. Salinity, Light and Aeration
The test exposure duration is 2 days followed by a 5 to 7 day
recovery period for the development of. cystocarps. The exposure temper-
ature should be between 22 and 24'C, and the test salinity should be
between 28 and 32 4/«0> For testing receiving waters, salinity will
often be below the desired range and oust be adjusted with artificial
sea salts (see SALINITY ADJUSTMENTS). The photoperiod should be a
16h:8h, light:dark cycle of ca. 75 uE m~ s" of cool-white fluorescent
light. It is not necessary for the recovery conditions to be the same
as the exposure conditions. However, the conditions listed are optimal
and will result in the fastest cystocarp development.
Plants are not aerated during the exposure period. Chambers are
either shaken at 100 rpm on a rotary shaker or briefly hand-swirled
twice a day. Spermatia are not motile, therefore some water notion is
critical. Aeration will inhance the growth rate of plants in the recov-
ery bottles, although, adequate growth will occur using a shaker.
Nutrients
The enrichment for natural seawater is listed in Table 2. Both
EDTA and trace metals have been omitted. This recipe should be used for
the 2-day exposure period, however, it is not critical for the recovery
period. Since natural seawater quality can vary among laboratories, a
more complete nutrient medium (e.g. + EDTA) may result in faster growth
(and therefore faster cystocarp development) during the recovery period.
The nutrients recomended for natural seawater are not sufficient
for healthy plants in artificial seawater. The nutrients for the GP2
artificial seawater are listed in Table 4. EDTA has act been elimi-
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nated, but has been reduced to 20 ug/L (<17. of the original recipe).
One of the reasons natural seawater is better than artificial seawater
is probably due to the variety of natural organic chelators in the
former. Therefore, elimination of all organic chelators from artificial
seawater should not be necessary. In fact, total elimination of EDTA
from artificial seawater can result in a greater sensitivity to toxi-
cants such as copper when compared to results with natural seawater.
Effluent Concentrations
For end-of-the-pipe samples, the concentrations that are currently
being used for Champia are 0.63, 1.22, 2.5, 5 and 10 7. effluent plus a
control. The concentrations recommended in the effluent handling adden-
dum of this Guidance Manual are also acceptable.
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Table 4. Recipe of nutrients to be added to GP2 artificial seawater for
stock cultures and test medium. See footnotes for differences between
test medium and culture medium. The concentrated stock solution is
autoclaved at standard temperature and pressure for 15 min (the pH is
adjusted to 2.0 with HC1 prior to autoclaving to prevent precipitation).
COMPOUND
NaH03
N«H2P04.H20
Na2EDTA.2H20
NaC6H507 (citrate)
Ironc
Trace Elements
tU2«o04.2H20
KI
ZnS04.7H20
N«V03
MnS04.H20
Vitamins
Thiamine-HCl
Biotin
B12
AMOUNT/LITER
STOCK CULTURES CONCENTRATED STOCK
127 mg
12.3 mg
2.66 mgb
1.03 mg
195 ug
24.2 ug
33 ug
21.8 ug
6.1 ug
0.61 ug
1.95 mg
1.0 ug
1.0 ug
12.7 g
1.28 g
266 mg
103 mg
19.5 mg
2.42 mg
3.3 mg
2.18 mg
0.61 mg
61.0 ug
10 mL6
*Use 1.25 mL/L for test medium (with the adjusted EDTA concentration)
and 5 mL/L for stock cultures. For test media an additional 2.5 mL/L
of the sodium bicarbonate stock solution is added (see Table 3).
concentration is 20 ug/L for final solution of test media in
artificial seawater; EDTA is omitted entirely if the test medium is
natural seawater.
cThe same stock solution as for Table 2.
^Trace elements are omitted for toxicity test medium (they are also
generally omitted when this medium is used with natural seawater) .
Vitamin stock solution autoclaved separately in 10 mL sub-samples.
Each 10 mL contains 195 mg of thiamine«HCl, 100 ug biotin, and 100 ug
B«2. Adjust pH to ca 4.0 before autoclaving for 2 min.
1-21
-------
PROTOCOL
1. Set up and label control and treatment chambers; three per treatment
and controls.
2. Fill chambers with 100 oL of control or treatment water (28 to 30
*/«0). Alternately, all chambers can be filled with control water
and the toxicant added with micropipets. For toxicant volumes
exceeding 1 mL, adjust amount of dilution water to give a final
volume of 100 mL.
3. Add the appropriate nutrients and bicarbonate to each chamber (see
Table 2 or 4).
4. Add five female branch tips and one male branch to each chamber.
Hake sure the toxicant is present before the male is added.
5. Place chambers under cool-white light (ca 75 uE m s" ) at 22 to 24
*C. Place a thermometer in a flask of water among the chambers.
6. Gently hand-swirl chambers twice a day. Alternately, shake con-
tinuously at 100 rpm on a rotary shaker. Record temperature daily.
7. If desired, media can be changed after one day (24 hr).
3. After 2 days (43 hr):
A. Label recovery bottles (these can be almost any type of
container or flasks with 200 to 400 oL of natural seawater plus
the additional nutrients (see Table 2 or 4). Smaller volumes can
be used, but should be checked to make sure that adequate growth
will occur without having to change the medium. As with culture
vessels, all glassware should be acid-stripped with 10 to 15 7.
HC1.
3. With forceps, gently remove females from test chambers and place
into recovery bottles. Add aeration tubes and foam stoppers.
I-
22
-------
C. Place bottles under cool-white light (at the same irradiance as
the stock cultures) and aerate for the 5- to 7-day recovery
period. If recovery is on a shaker, then eliminate aeration
tubes and reduce the volume of seawater to approximately one-half
of the vessel volume (this will enhance the water motion).
9. At the end of the recovery period count the number of cystocarps per
female and record the data (Addendum C-V). Cystocarps are counted
by placing females between the inverted halves of a polystyrene
petri dish with a small amount of seawater (to hold the entire plant
in one focal plane). Using a stereo-microscope, the emergent
cystocazps can be easily counted* Cystocarps are distinguished from
young branches because they possess an apical ostiole (opening for
spore release) and darkly pigmented spores (see Figs. 7,8). One of
the advantages to this test procedure is that if there is uncertain-
ty about the identification of an immature cystocarp, then the
plants can just be aerated for a little longer in the recovery
bottles. Within 24 to 48 hr the structure in question will either
look more like a mature cystocarp; look more like a young branch; or
have changed very little, if at all (i.e., it is an aborted
cystocarp). No new cystocarps will form since the males have been
removed, the plants will only get bigger. Occasionally, cystocarps
will abort, and these should not be included in the cystocarp
counts. Aborted cystocarps are easily identified by their dark
pigmentation (Fig. 9). They also often begin to form a new branch
at their apex.
1-23
-------
1mm
Figure 7. A mature cystocarp of Champla parvula.
the lower effluent concentrations, cystocarps
in groups of as many as 10 to 12.
In the controls and
are often clustered
cells
young branch
immature
cystocarp
Figure 3. Comparison of a very young branch with an immature cysto-
carp. Both can have sterile hairs and the young branch may or may
not have trichogynes. However, the immature cystocarps will never
have trichogynes. Young branches are usually more pointed at the
apex and will not form an ostiole. The cell dimensions of young
branches are larger than those of the cystocarp.
1mm
Figure 9. An aborted cystocarp of Champia parvula.
eventually develop at the apex.
A new branch will
I-
-------
STATISTICAL TREATMENT OF DATA
Chronic values are calculated using two different criteria for
determining significant differences. One criterion for difference from
controls is absence of cystocarps; >_ 957. decrease from the control
(Thursby and Steele, 1986). The other criterion is based on statistical
differences. Statistically significant decreases in the number of
cystocarps are determined by one-way analysis of variance (ANOVA) fol-
lowed by Dunnett's mean separation test (alpha * 0.05) for comparison of
treatments with a control (Steel and Torrey, 1960). The results from
each replicate chamber are reported as the mean number of cystocarps per
plant (n«3 for each treatment).
Chronic values are expressed either as the no-effect range, the
lowest concentration that results in a significant difference from the
control and the next lowest concentration tested, or as the geometric
mean of these two values (Buikema, et als j—1982). If the lowest coneen-
f
tration that results in a significant difference is the lowest concen-
tration tested, then the geometric mean is not calculated. One can only
report the lowest concentration that gave the significant difference
(one should also consider repeating this test). In practice, the
"absence of cystocarps" endpoint is generally used for determining the
effect concentration from an effluent diluton series. However, if the
concentration that causes a statistical decrease from the control is
also reported, then some idea of the steepness of the dose response
curve can be inferred. The statistical difference is used primarily
when testing receiving waters where dilutions are usually not made.
I-
25
-------
CRITERIA FOR ACCEPTABILITY
1. A test is not acceptable if control mortality exceeds 207. (generally
there is no control mortality).
2. If plants fragment in either the controls or the lowest exposure
concentration so that individual plants can not be identified, then
the test is not acceptable. This is aot critical if absence of sex
is the only endpoint of interest. However, the fact that the plants
fragmented indicates they are not at their best and the data may be
biased toward the lower concentrations.
3. A test should not be considered definitive if the controls average
fewer than 10 cystocarps per plant. If no sexual reproduction
occurs in the controls, then this test can not be considered accept-
able.
4. If the plants in the two control chambers are suspected of
responding differently (this can be checked with a t-test), then the
test should not be considered acceptable.
5. The data from all replicates at the effluent concentration deter-
mined to be the effect concentration should be statistically equal.
That is, all replicates should show the effect.
1-26
-------
REFERENCES
Buikema A.L., Niederlehner B.R., Cairns J. 1982. Biological monitor-
ing, Part IV, Toxicity Testing. Water Res. 16:239-262.
Jensen A. 1984a. Marine ecotoxicological tests with seaweeds, pp. 181-
193. In: Ecotoxicological Testing for the Marine Environment.
Persoone G., Jaspers E., Claus C. (eds). State Oniv. Ghent and
Inst. Mar. Sclent. Res., Bredene, Belgium. 7ol 1.
Jensen A. 1984b. Marine Ecotoxicological Tests with Phytoplankton. pp.
195-213. ibid.
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., Moolenaar R.J. 1976. Fish and Daphnia toxicity as
surrogates for aquatic vascular plants and algae. Environ. Sci.
Techno1. 13:1479-1480.
Spotte S., Adams 6., Bubucis P.M. 1984. GP2 medium is an artificial
seawater for culture or maintenance of marine organisms. Zoo.
Biol. 3:229-240.
Steel R.G., Torrey J.H. 1960. Princilples and procedures of statis-
tics. McGraw Hill. 481 pp.
Steele R.L., Hanisak M.O. 1978. Sensitivity of some brown algal
reproductive stages to oil pollution. In: Proceedings of the Ninth
International Seaweed Symposium, pp. 181-190. Jensen A., Stein J.R.
(eds). Science Press.
Steele R.L., Thursby G.B. 1983. A toxicity test using life stages of
Champia parvula (Rhodophyta). In: Aquatic Toxicology and Hazard
Assessment, Sixth Symposium, pp. 73-89. Bishop WC, Cardwell RD,
Heidolph BB (eds). ASTM STP 802. American Society of Testing and
1-27
-------
Materials, Philadelphia.
Thuzsby G.B., Steele R.L. 1984. Toxicity of arsenite and arsenate to
the marine macroalga Champia pannila (Rhodophyta). Environ.
Toxicol. Chen. 3:391-397.
Thursby G.B., Steele R.L., Kane M.E. 1985. Effects of organic chemicals
on growth and reproduction in the marine red alga Champia parvula.
Environ. Toxicol. Chem. 4:797-805.
Thursby, G.B., Steele, R.L. 1986. Comparison of short- and long-term
sexual reproduction tests using Champia parvula. Environ. Toxicol.
Chem. (in press).
1-28
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ADDENDUM C-I
List of materials for performing toxicity test3 with Champia parvula for
effluent program.
1. Fine-point stainless steel forceps.
2. 100 mL polypropylene cups with covers (or 125 mL Erlenmeyer flasks)
to be used as exposure chamabers.
3. Polystrene petri dishes to hold plants for counting cystocarps and
for cutting branch tips.
4. 100 mL graduated cylinder to measure control and treatment volumes.
5. 1 and 10 mL disposable pipets.
6. Digital micropipets (200 and 1000 uL maxima) if dilutions are made
directly in test chambers.
7. Disposable tips for micropipets.
3. Nutrient and sodium bicarbonate stock solutions.
9. Recovery bottles or flasks, need one per treatment or control
chamber.
10. Aquarium pump(s) and air tubing.
11. Plastic aeration tubes (1 mL disposable pipets work fine) and foam
plugs.
12. Thermometer and flask or bottle to hold it.
13. Marking pens and colored tape.
14. Cool-white fluorescent, lighting, sufficient to give 75 uE m~ s"
(ca 500 foot-candles).
15. Rotary shaker for exposure chambers (hand-swirling twice a day can
be substituted).
16. Stereomicroscope for counting cystocarps.
17. Refractometer for salinity measurements.
18. Data sheets (one per test).
19. Protocol.
I- 29
-------
ADDENDUM Oil
Summary of test conditions for Champia parvula sexual reproduction test,
1. Test type:
2. Salinity:
3. Temperature:
4. Photoperiod:
5. Light source:
6. Irradiance:
7. Test solution volume:
3. Test chamber size:
9. Number of test organisms
per test chamber:
10. Number of replicate
chambers per treatment:
11. Aeration:
12. Dilution water:
13. Test duration:
14. Effect measured:
Static, non-renewal
30V,,
22 to 24 «C
16h light:8h dark
cool-white fluorescent
ca 75 uE m"2 s"1
100 oL
110 mL polypropylene cups (with
covers) or 125 mL Erlenmeyer flasks
5 female branch tips and one male
None; chambers are either shaken at
100 rpm on a rotary shaker or hand-
swirled twice a day
30 Va, natural or artificial
seawater with additional nutrients
added
2 day exposure followed by a 5- to
7-day recovery period for cystocarp
development
Sexual reproduction (number of
cystocarps per female)
1-30
-------
ADDENDUM C-III
Equations for making salinity adjustments.
A. To dilute to a desired salinity with deionized water.
Six7i-Sfx7f
where:
S » initial salinity (measured)
Sf • final salinity (selected)
V * initial volume (unknown)
7. - final volume (selected)
Solve for V., then dilute to the final (selected) volume with deionized
water.
B. To mix two different salinities to get a third salinity.
si(vf - V * sn * 7n - sf x 7f
and
7 » 7 +
Vf VI
where:
S_ * salinity of the first solution (measured)
S__ • salinity of the second solution (measured)
S. • salinity of the final solution (selected)
7_ - volume of the first solution (unknown)
7_T » volume of the second solution (unknown)
V, * volume of the final solution (selected)
Solve the first equation for 7 , then solve the second equation for
Example: Solution I salinity » 10 a/ao;
solution II salinity - 90 °/«a;
final solution salinity desired « 30- a/00;
final volume wanted « 1000 mL.
1-31
-------
First equation becomes: 10 (1000 - 7 ) + 90 7 _ » 30 x 1000
10,000 - 10 V + 90 VTI - 30,000
80 Vjj - 20,000
7 - 250 mL
Second equation becomes: 7. > 1000 - 250
7 - 750 mL
Any of the three volumes can be selected as the constant, solving for
the other two volumes. For example you may have 500 mL of solution II
and wish to know how much of solution I to add to get 30 */a«. In this
case you would just solve the first equation for 7. and continue from
there .
I- 32
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ADDENDUM C-IV
Precision testing for Champia parvula short-term.
Intralaboratory precision testing of the short-term exposure repro-
ductive test with Champia parvula has not been conducted yet. However,
we have several repeat tests with effluents and one single compound
(copper). All tests concentrations were unmeasured. The concentration
given is the geometric mean of the effect/no-effect level. Effect is
defined as having any number of cystocarps/plant £ 51. of the control. In
general, the agreement among the repeat tests was very good. For the
heavy metal effluent, the variation was by a factor of 3, but the two
tests (on the same sample) were run approximately 2 months apart.
COMPOUND
COLLECTION DATE
TEST DATE
Heavy Metal
Effluent
Organic Effluent
8/17/84
8/17/84
5/8/85
5/8/85
EFFECT LEVEL
Copper
2/18/85
2/18/85
4/3/85
4/3/85
6/7/85
~ 8.8 ug/L
8.8
8.8
8.8
8.8
9/25/84 1.76 I
11/27/84 0.56
5/16-shaken 14.1 7.
5/16-not shaken 14.1
1-33
-------
ADDENDUM C-V
1-34
-------
CHAMP/A PARVULA
CYSTOCARP DATA SHEET
COLLECTION DATE
EXPOSURE BEGAN (date).
RECOVERY BEGAN (date),
COUNTED (date)
EFFLUENT OR TOXICANT
TREATMENTS (% EFFLUENT, ^/G/L, or REC. WATER SITES)
REPLICATES
A 1
2
3
4
5
CONTROL
MEAN
9 1
2
3
4
3
- -:
MEAN
c ^
2
3
4
5
MEAN
OVERALL
MEAN
Temperature.
Salinity ——
Light
Source of Dilution Water.
-------
RECEIVING WATER SUMMARY SHEET
SITE
COLLECTION DATE
TEST DATE
LOCATION
INITIAL
SALINITY
FINAL
SALINITY
SOURCE OF SALTS FOR
SALINITY ADJUSTMENT *
* .
i.e. natural seawoter brine, GP2 brine, GP2 salts , etc.
(include some indication of amount)
COMMENTS:
-------
PART II
COMPARISON OF SHORT- AND LONG-TERM SEXUAL REPRODUCTION TESTS
WITH THE MARINE RED ALGA CHAMPIA PARVULA
II-l
-------
Abatract--A two-day exposure test using the marine red alga Champia
parvula has been developed for assessing the toxic effects of complex
effluents entering the marine and estuarine environments. The initial
exposure was followed by a 5- to 7-day recovery period to allow the
development of any cystocarps (evidence of sexual reproduction—the
endpoint measured). The two-day exposure test was validated by compari-
son with a previously developed two-week test in which "no sexual repro-
duction" (NSR) was used as the reproductive endpoint measured. Single
compounds can be more accurately tested with the two-week test procedure
than effluents, therefore, they were used to compare the two-day expo-
sure test with the two-week exposure. A total of nine single compounds
were tested using the two testing procedures. Concentrations that
resulted in a 95H or greater decrease from the control response with the
two-day exposure were considered the best estimate of the NSR response
from the two-week test. All of the single compounds tested, except
cadmium, yielded essentially the same results with both tests.
Keywords--Champia, effluents, sexual reproduction
II-2
-------
DTTBODUCnOM
The Permits Division of the U.S. Environmental Protection Agency's
(EPA) Office of Water needs toxicity test methods for marine and estua-
rine species. The U.S. EPA'a Environmental Research Laboratory at
Narragansett, Rhode Island has developed or modified four toxicity tests
to begin to address the above need. The four species used are: the red
macroalga, Champia parvula; the sea urchin, Arbacia punctulata; the
my3id, Myaidopsis bahia; and the sheepshead minnow, Cyprinodon
veriegatus. The methods are needed to characterize the toxicity of
effluent discharges within the National Pollutant Discharge Elimination
System (NPDES). Since March 9, 1984, EPA has had the authority to
require biological testing as a condition for issuing HPDES permits [!]•
These tests should yield chronic data in a relatively short time period
(7 days or less). It is also necessary that the methods be simple and
cost efficient (requiring standard hardware and laboratory facilities).
•>
The species used should be readily available, and should also be practi-
cal for both on-site and off-site testing. All of the requirements are
met by the test method for the marine red macroalga, Champia parvula.
Seaweeds have been considered-less useful for toxicity testing than
microalgae [2], and microalgae are often considered less sensitive than
aquatic animals [3,4]. Therefore, one could easily come to the erron-
eous conclusion that toxicity testing with seaweeds is not necessary.
Recently the statement was made that "seaweeds seem to be rather insen-
sitive to many chemicals and will probably survive pollution better that
many other organisms in the marine environment" [2].
Previous conclusions about seaweed sensitivity were based on only a
few species, and generally considered only vegetative growth of the
II-3
-------
macroscopic life history stage as the endpoint. The sensitivity of
seaweeds can increase when sexual reproduction is used as an endpoint,
and can be greater than many aquatic animals that have been tested.
This has been shown for the red alga, Champia parvula [5-7], as well as
with the brown algae Fucus edentatus and Laninaria saccharina [8]. In
addition, using growth of young sporophytes, ^ saccharina has been
placed among the most sensitive marine organisms for toxicity of copper,
zinc and mercury [9].
A two-week toxicity test has already been developed for the macro-
alga, Champia parvula, to assess chronic effects of pollutants to marine
seaweeds [5]. The test has been evaluated with heavy metals [5],
arsenite and arsenate [6] and ten different organic compounds [7].
Sexual reproduction was generally the most sensitive and practical
endpoint to use for C. parvula. The two-week exposure, however, made
this test procedure unacceptable for testing complex effluents and their
receiving waters.'
Effluents can not be easily tested using the two-week test
procedure. The two-week toxicty test with Champia parvula requires that
the cultures remain unialgal during the test period. Fine-filtering or
autoelaving, which would be necessary to eliminate unwanted microalgae,
could change the character of the effluent or receiving water. Any
microalgae introduced with the effluent would compete with C._ parvula
for light and nutrients, thus influencing^ parvula's growth rate.
However, if sexual reproduction is used as the sole endpoint, then
plants only need to be exposed for a few days (long enough to show
effects on fertilization). Any effect of other organisms on the growth
rate of C. parvula should not be serious since interest would only be in
whether sexual reproduction had taken place. The two-week procedure
II-4
-------
also requires that the media be changed during the test. Therefore,
either the effluent sample would have to be stored (i.e. refrigerated)
or additional samples would have to be collected during the test period.
Both of these alternatives would result in variable toxicity. Single
compounds can be more accurately tested with the two-week test procedure
than effluents* therefore, they were used to compare the two-day
exposure test with the two-week exposure.
This paper describes a method which uses sexual reproduction to
estimate chronic toxicity after a short-term exposure to toxicants. In
brief, the method consists of exposing males and females to effluents or
receiving waters for two days, followed by a 5- to 7-day recovery period
in control medium. The recovery period allows time for any cystocarps
to mature. At the end of the recovery period the number of cystocarps
per plant are counted.
II-5
-------
MATERIALS AHD METHODS
Maintenance of stock cultures
Unialgal stock cultures of both male and female gametophytes of
Champia parvula (C. Agardh) Harvey were maintained in separate, aerated
1000 mL Erlenmeyer flasks containing 800 mL of culture medium. All
culture glassware was acid-stripped in 10 to 15 % HC1 and rinsed in
deionized water after washing. The culture medium was made from natural
seawater (from lover Narragansett Bay, RI) to which additional nutrients
were added (Table 1). The seawater was filtered through a 15-urn char-
coal filter and a 0.3-um Balston filter, then autoelaved for 30 min at
15 pai in 20 L carboys. The culture flasks were capped with aluminum
foil and autoelaved dry for 10 min. Culture medium was formulated by
dispensing seawater into the sterile flasks and adding the appropriate
nutrients from a sterile stock solution.
Initial stock cultures were started weekly with about twenty 0.5 to
»
1.0 cm branch tips. Cultures were gently aerated through sterile,
cotton-plugged, disposable, polystyrene 1 mL pipets. Cultures were
capped with foam plugs and aluminum foil and illuminated from the aide
•2 -1
with 75 uE m s of cool-white fluorescent light on a 16h:8h,
light:dark cycle. The temperature was 22 to 24 °C and the salinity 28
to 30 °/«0* Media were changed once each week.
About half of the plants were discarded (or placed into another
culture vessel) with each weekly medium change to reduce the amount of
biomass as the plants grew. At the end of three weeks plants were ready
to use for testing. Readiness was defined as having enough plant
material to perform at least one test. With this procedure, actively
growing plants were continuously available.
II-6
-------
A stock culture was not used as a source of test material if the
plants appeared to be stressed or undernourished. Under conditions of
stress the tips of the branches turned "pink" and the older tissue was
generally much paler. Under conditions of nutrient deficiency
(resulting usually from too much plant material in the culture flask or
too long since the last medium change) the entire plant turned pale
yellow. If the stress was severe enough the older tissues (main axes)
or occasionally the branch tips turned white (evidence of necrotic
tissue).
Two-week test
All procedures for the two-week test followed those previously
described [5], except EDTA was omitted from the medium and vitamins were
added. The test medium was then identical to that used for the two-day
exposure test. Toxicity test duration was 14 days. Tests were per-
formed with 400 oL volumes in 500 mL, screw-capped Erlenmeyer flasks.
The medium was fc strength of that used for the stock cultures. In
addition 150 mg/L sodium bicarbonate was added rather than 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 cultures were rinsed in sterile seawater to remove
traces of old medium, 2- to 3-mm branch tips were cut from females to
serve as inocula for toxicity tests. Five branch tips 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 each toxicity test,
females were examined for the presence of cystocarps (evidence of sexual
II-7
-------
reproduction). The concentration which resulted in no sexual reproduc-
tion (HSR) was determined.
Two-day exposure
Stock cultures were rinsed in sterile seawater to remove traces of
old medium. Female branch tips, 7 to 10 mm in length, were cut to serve
as inocula. Five tips were placed into each treatment cup, along with
one male branch (1.5 to 2.0 cm long), visibly producing spermatia.
Tests were performed in replicate 100 mL polystyrene cups, with plastic
caps, containing 80 mL of medium. The nutrient medium was the same as
for the two-week tests.
The two day exposure was followed by a5- to 7- day recovery period
(for females only) in control medium for cystocarp development.
Temperature, salinity and light conditions were the same as for stock
cultures, except that light was from above. Plants were not aerated
during the exposure period. Exposure chambers were shaken at 100 rpm on
a rotary shaker. Recovery bottles were aerated, since this enhanced the
growth rate of plants and therefore the rate of development of
cystocarps.
At the end of the recovery period the number of cystocarps per
female were counted. The results from each replicate cup were reported
as the mean number of cystocarps per plant (n » 2 for the treatment).
The data were examined for HSR, 95 and 507. decreases from the control,
and statistical differences from the control. Statistical decreases in
the number of cystocarps were determined by one-way analysis of variance
(ANOVA) followed by Dunnett's means separation test (alpha - 0.05) for
comparisons of treatments with a control [10].
One advantage of this test procedure is if there is uncertainty
II-8
-------
about the identification of an immature cystocarp, then the plants can
Just be aerated a little longer in the recovery bottles. No new cysto-
carps will form since the male gametophytes have been removed; the
plants will only get bigger.
Toxicant concentrations
The highest concentration tested was based on preliminary
experiments and was chosen to cause death or a near death response after
a two-week exposure. The dilution factor for all test runs was 0.6.
Only one stock solution was prepared for each toxicant. All
concentrations were obtained by dispensing from these stock solutions
with adjustable micropipets.
Stock solutions of sodium arsenite, copper sulfate, cadmium
chloride and silver nitrate were prepared in deionized water. Toxaphene
was dissolved first in acetone and then diluted with triethylene glycol
(TEG) to give a final acetone to TEG ratio of 1 to 20. Pentachloro-
ethane and pentachlorophenol were prepared in TEG alone. Benzene and
isophorone were dispensed directly. For benzene, isophorone and penta-
chloroethane (all liquids), density was used to calculate the weight of
the compound in solution. A carrier control (at the highest concentra-
tion of carrier used with a toxicant) was used with those toxicants that
required a carrier for solubility. Water samples from the test chambers
were not chemically analyzed for toxicant concentrations, therefore all
concentrations reported are nominal concentrations added.
II-9
-------
RESULTS AMD DISCUSSION
The results of the comparison of the two-day exposure test with the
two-week test using single compounds are shown in Table 2. Only data
for concentrations causing no sexual reproduction (HSR) are reported for
the two-week test. Several values are listed for the two-day exposure
test. The object of the two-day exposure was to estimate the chronic
effect from the two-week test. The endpoint of choice for the two-week
test was NSR, therefore all two-day results are compared only to this
number. The same concentrations were tested in both test procedures,
and for four of the nine compounds the NSR endpoint was not achieved for
the two-day test.
The best two-day values for estimating the two-week NSR results
were those from the concentration that resulted in a 95% or greater
decrease in the number of cystocarps (0 to 5% of control) when compared
to the controls. This is seen more clearly in Table 3 which shows the
ratios of the values in Table 2 to the two-week values. Cadmium was the
only compound tested that did not give a good relationship between the
two-day and the two-week results. Cadmium is generally a slow acting
toxicant [11, 12] and two days may not have been enough time to elicit
its effect. The ratios for arsenite and silver are close to one, while
the remaining are one. Arsenite and silver were the only two compounds
for which the two test procedures were not started on the same date. It
should be noted that the two-day benzene test was performed in 400 mL,
screw-capped Erlenmeyer flasks instead of the polystyrene cups. When
plants were exposed in 80 mL test solution the toxicity was much less,
probably due to the high volatility of benzene. If volatile compounds
are being tested, then larger, air-tight exposure chambers should prob-
ably be used. However, the two day test procedure is designed for
11-10
-------
testing effluents and receiving waters. Highly volatile compounds are
significantly reduced in pretreatment before effluents are discharged.
Therefore the failure of the test with benzene when using smaller, non-
air-tight vessels is not expected to be a problem when testing ef-
fluents .
A sample data sat is graphed for pentachloroethane in Figure 1,
illustrating that a 95% decrease from the control is a good value for
estimating the two-week results. The dose-response curves for both the
two-week test and the short-term exposure tests are similar. However,
no cystocarps were produced during the two-week test at 13,000 or 22,000
jug L* , whereas a few were produced at both concentrations during the
short-term test. A similar relationship between the two-day an the two-
week results was seen for most of the compounds tested.
Sexual reproduction was selected as the endpoint for two-day test-
ing for several reasons. It was proven previously to be a sensitive and
»
practical endpoint from the two-week toxicity test procedure [5-7]. A
sexual reproduction test for toxicity could be short enough to fit the
time constraints for tests used in the effluent program (ca. 7 days).
Finally, Champia parvula is an annual plant and inhibition or absence of
sexual reproduction reduces or eliminates the next stage in the life
history. Total absence of cystocarp formation is the easiest endpoint
to interpret as far as field populations are concerned. In most of the
red algae, each fertilization results in the formation of a new life
history stage, the earposporophyte, "parasitic" on the female and housed
within the cystocarp. Each carposporophyte is capable of producing many
spores (perhaps a hundred or more in the case of C. parvula). This
characteristic makes it difficult to interpret the biological signif-
11-11
-------
Icance of a statistical decrease in the number of cystocarps or an
arbitrary percent decrease such as 501. Absence of reproduction leaves
no doubt about its biological significance.
The two-day exposure test has been used successfully in both a
mobile laboratory and in the main laboratory (EPA, Environmental
Research Laboratory, Narragansett, RI, USA). The method is easy and
coat-efficient to perform. Stock cultures are maintained in the labora-
tory with standard laboratory equipment, therefore, plant material can
be available throughout the year. The test procedure la intended to be
used to estimate chronic effects of complex effluents on marine macro-
algae, although it can obviously be used for single compounds. The
procedure has already been included in a draft guidance manual for
tasting marine and estuarine effluents. Comparing this test with a two-
week chronic teat using single compounds has shown that the test can be
uaed for determining adverse effects on sexual reproduction in the
marine alga, Champia parvula.
Acknowledgements—The technical assistance of Raymond Palmquist is
greatly appreciated.
11-12
-------
UFfiUKBO
1. U.S. Bmjjiuuaeutal Protection Agency. 1984. Development of water
quality-baaed permit limitations for toxic pollutants: National
policy. Fed. Reg. Vol. 49 No. 48. Friday, March 9, 1984.
2. Jensen, A. 1984. Marine ecotoxicological tests with seaweeds,
In G.Persoone, E. Jaspers and C. Glaus, eds., Ecotoxieologieal
Testing for the Marine Environment, Vol. 1. State Univ. Ghent and
Znst. Mar. Scient. Res., Bredene, Belgium, pp. 181-193.
3. g*"*tr, K.K. and R.J* Moolenaax. 1976. Fish and Daphnia toxicity
as surrogates for aquatic vascular plants and algae. Environ. Sci.
Techno1. 13*1479-1480.
4. g^"*tTi K«Ea 1982. The use of environmental toxicology and
chemistry data in hazard assessment: Progress, needs, challenges.
Environ. Toxieol. Chem. 1:69-79.
5. Stewla, R.L. and G.B. Thnraby. 1983. A toxicity test using life
stages of Champia parvula (Rhodophyta). In V.E. Bishop, R.D.
Cardwell and B.B. Heidolph, eds., Aquatic Toxicology and Hazard
Assessment: Sixth Symposium. ASTM STP 802. American Society for
Testing and Materials, Philadelphia, PA, pp. 73-89.
6. Tharsbr, G.B. and R.L. Steele. 1984. Toxicity of arsenite and
arsenate to the marine macrolaga Champia parvula (Rhodophyta).
Environ. Toxieol. Chem. 3:391-397.
7. Tharabr, G.B., R.L. Steole and H.E. Kane. 1985. Effect of organic
chemicals on growth and reproduction in the marine red alga Champia
oarvula. Environ. Toxieol. Chem. 4:797-805.
II
-13
-------
8. Steele, R.L. and M.D. Hanisak. 1978. Sensitivity of some brown
algal reproductive stages to oil pollution. In A. Jensen and J.R.
Stein, «ds., Proceedings £f_ the Ninth International Seaweed
Symposium, Vol. 9, pp. 181-190.
9. Tbo«B«o», R.S. and S.M. BOTTOM. 1984. The toxicity of copper,
zinc, and mercury to the brown macroalga Laminaria saccharine, In
G.Persoone, E. Jaspers and C. Glaus, ads., Ecotoxicological Testing
for the Marine Environment, Vol. 2. State Univ. Ghent and last.
Mar. Sclent. Res., Bredene, Belgium, pp. 259-269.
10. Steel, R.6* and J.H. Terries. 1960. Principles and Procedures £f_
Statistics. McGraw-Hill, New York, NT
11. lagexaoil, C.G« and R.L. Winner. 1982. The effect on Daphnia pulex
of daily pulse exposure to copper or cadmium. Environ. Toxicol.
Chem. 1)321-327.
12. Arillo, A., D. CalasMxi, C. Margiocco, P. Melodla and P. Meaai.
1984. Biochemical effects of long-term exposure to cadmium and
copper on rainbow trout (Salmo gairdneri): Validation of water
quality criteria. Ecotoxico1. Environ. Safety 8>106-117.
11-14
-------
Table 1. Recipe fox additional nutrients to be added to natural sea-
water for Champia parvula stock cultures and test medium. Both EDTA
and trace metals have been omitted. The concentrated stock solution is
autoclaved at standard temperature and pressure for 15 minutes.
COMPOUND
NaN03
NaHjPO^HjO
Ironb
Vitamins
B12
Biotin
Thiaaln««HCl
TEST MEDIUM C
9.35 mg
0.62 mg
2.6 ug
0.06 ug
0.06 ug
12.5 ug
AMOUNT/LITER
ONC. CONCENTRATED STOCK*
3.74 grams
0.25 grams
1.04 mg
10 mLe
*Use 0.2 oL/SOmL (2.5 mL/L) for test medium concentrations and 10 mL/L
for stock cultures. For test medium only, add 0.2 mL/80mL of a sodium
bicarbonate solution. A stock solution of 60 mg/mL sodium bicarbonate
is prepared by autoclaving it as a dry powder and then dissolving it in
sterile deionized water.
Iron stock solution prepared by dissolving 1 g iron powder in 10 mL
concentrated HC1 plus ca. 1 mL of deionized water. This is diluted to
1 liter with deionized water. Acceptable stock solutions can also be
made with ferric or ferrous chloride.
Vitamin stock solution autoclaved (2 min) separately in 10 mL sub-
samples. Each 10 mL contains 24 ug B12, 24 ug biotin and 5 mg
thiamine*HCl. Adjust pH to ca. 4.0 before autocalving.
11-15
-------
Table 2. Test results for Champla parvula comparing the two-day exposure
test and the two-week test using single compounds. Values listed are
the geometric means of the effect and the no-effect concentrations in
ug/L. HSR refers to no sexual reproduction, i.e. no cystocarps were
formed at these concentrations. The other column headings refer to
concentrations that resulted in 95 and 501 decreases from controls, and
concentrations that resulted in a number of cystocarps statistically
less than controls.
COMPOUND
Arsenite*
Copper
Q4HJm^ni»
Silver*
b
Benzene
Pentachloro-
e thane
Pentachloro-
phenol
Toxaphene
Isophorone
NSR
>300
7.7
>100
1.5
7 3 ,.600
> 21,800
465
140
> 138, 500
TWO-DAT
951
230
4.6
>100
0.9
73,600
10,200
465
140
107,300
EXPOSURE
501
84
1.0
>100
0.9
< 34, 3 00
2,200
280
84
38,300
Stat. Dlf.
84
<0.8
17
0.5
44,250
<1,700
280
84
38,300
TWO-WEEK
NSR
140
4.6
77
1.5
73,600
10,200
465
140
107,300
aShort-term exposure and two-week teat not run silmultaneously.
Both short-term exposure and two-week test run in 400 mL volume.
11-16
-------
Table 3. Ratio of two-day exposure results and two-week test results
from Table 2 for Champia parvula. See Table 2 for explanation of column
headings.
COMPOUND HSR 57. 501 Stat. Dif.
Arsenite >2.1 1.6 0.6 0.6
Copper 1.8 1.0 0.2 <0.2
Cadmium >1.3 >1.3 >1.3 0.2
Silver 1.0 0.6 0.6 0.3
Benzene 1.0 1.0 <0.5 0.6
Pentachloroethane >2.1 1.0 0.2 <0.2
Pentaehlorophenol 1.0 1.0 0.6 0.6
Toxaphene 1.0 1.0 0.6 0.6
Isophorone >1.3 1.0 0.4 0.4
11-17
-------
Figure 1. Comparison of the two-day exposure test results and the two-
week teat results for Champia parvula using pentachloroethane. The
graph illustrates the use of the 95% decrease as the cut off point for
the two-day exposure test. Note that some cystocarps were produced at
the two highest concentrations during the two-day exposure, but not
during the two-week test.
11-18
-------
IOOC
O TWO WEEK TEST
• TWO DAY EXPOSURE
O
o:
O 50
O
vP
0
PENTACHLOROETHANE (x!0//g/L)
-------
PART III
SUMMARY OF RESULTS FROM TESTING COMPLEX EFFLUENTS
-------
INTRODUCTION
Tests have been conducted on-site in a mobile laboratory and at
the EPA1s Environmental Research Laboratory, Narragansett, RI. Several
different types of complex effluents have been tested over the past two
years. These include one from a pulp mill; two industrial sites
that discharge effluents containing heavy metals; five industrial sites
discharging organically contaminated effluents, including pestcides and
dyes; and 17 different sewage effluents* In all, over 100 tests were
conducted.
More complete data is included for the pulp mill effluent (Tables 2
and 3, and Figures 1 and 2) from ITT Rayonier in Fernadina Beach,
Florida. These results may be incorporated into the first marine
toxicity-based NPDES permit.
MATERIALS AND METHODS
The test procedure used is described in the guidance manual (Part I
of this report). In brief, the method consists of exposing males and
females to effluents or receiving waters for two days, followed by a
five- to seven-day recovery period in control medium. The recovery
period allows time for any cystocarps to mature. At the end of the
recovery period the number of cystocarps per plant are counted. Some of
the earliest tests differed in that the exposure period was four days
instead of the now standard two days. However, our earlier work also
showed that two and four day exposures yielded essentially the same
results.
Chronic values can be calculated using two different criteria for
determining significant differences. One criterion for difference from
controls is absence of cystocarps;> 957. decrease from the control. The
III-2
-------
other criterion is based on statistical differences. See Parts I and II
of this report for a further explanation.
RESULTS
Table 1 summarizes all of the effluent test results to date. The
summary is based on absence of reproduction as the measurement of the
effect. In general the effects could be separated based on effluent
type. Heavy metal containing effluents were by far the most toxic,
followed by oranic and pulp mill effluents, and then sewage effluents.
Tests on the pulp mill effluent from ITT Rayonier were performed on
four separate effluent and receiving water collections (May 15, 17, 18
and 19, 1986). The tests set up on May 15th and 19th are not included
in these results. The control values from these two runs were unaccept-
able (an average of <10 cystocarps per plant). The May 15th run used
plants cultured in artificial seawater, these plants are more difficult
to judge as ready-to-use in the absence of a compound microscope. The
*
test runs on the other three days all used plants cultured in natural
seawater (where color of the tissue is a good indicator of "readiness").
However, the test run set up on the 19th lasted only one day instead of
the usual two.
Tables 3 and 4 list the data from the May 17th and 18th collections
for the effluent and receiving waters respectively. Figure 1 is a graph
of the May 18th data against percent effluent, based on the previous
week's dye study. These results indicated that the effect
concentrations in the receiving water was consistent with that
determined in the effluent test. Figure 2 is a graph of the May 17th
and 18th effluent data and data from an ammonium chloride toxicity test
run started on June 11, 1986. The similarity between the two curves
III-3
-------
suggests that ammonia may be the main toxicant in the ITT Rayonier
effluent that inhibits sexual reproduction in Champia. This conclusion
supports the chemical fractionation studies conducted on-site by EPA's
Duluth laboratory inwhich unionized ammonia was the primary toxic
comnponent of the effluent.
III-4
-------
Table 1. Summary of the results for all the effluents tested. The
values represent the range of response for the No Sexual Reproduction
effect (> 957. decrease from the control). The number in parenthesis
after the~"effluent type is the number of effluents of that type tested.
EFFLUENT TYPE RANGE OF RESPONSE (7.)
Heavy Metals (2) 0.05-0.5
Pulp Mill (1) 1.8
Organics (5)
(pesticides, dyes, etc.) 3-20 (generally <5)
Sewage (17) 2.5-25 (generally 5-10)
III-5
-------
Table 2L. The effect of effluent from ITT Rayonier on formation of
cystocarps by Champia parvula. Temperature was 23.to 25 °C, salinity
was 30°/oe, and light density was ca 100 uE m" 3" of daylight
fluorescent light on a 16h:8h, light:dark cycle. The concentration
resulting in no sexual reproduction (_>957. decrease from control) was 2.5
7. effluent with both test runs. The data from the May 15th and 19th
test runs also had 2.5 7. as the cut off point even though these data
were considered unacceptable. Mo necrotic tissue was observed at any of
the concentrations tested.
Effluent Number of Cystocarps per Plant (n-3)
May 17 May 18
Control
0.5
1.0
2.5
5.0
10.0
11 ± 2
12 ± 3
13 ± 1
0.2
0
0
14 ± 5
13 ± 3
9 ± 3
0
0
0
III-6
-------
Table 3. The effect of receiving waters from the Amelia River on the
formation of cystocarps by Champia parvula. Temperature, salinity and
light conditions were the same as for the tests in Table 1. Effluent
percents were calculated based on the dye study. No necrotic tissue was
observed in any of the treatments. A blank space means missing data.
Sta. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
% Eff .
2.0
b
1.5
0.05
1.4
0.08
1.4
0.5
0.2
0.9
0.7
0.5
0.5
0.6
0.5
0.4
0.4
0.5
Number of Cystocarps per Plant
May 17 May 18
Replicate3 Replicate
ABC Mean A B
0.4 0.8 0.2 0.5* 0.0
17.2 12.2 14.6 14.7 20.6
1.0 1.6 0.4 1.0* 2.0
11.8
4.6 1.6 0.6 2.3* 0.0
1.6
2.4
1.6 11.6 11.6 8.3 23.6
1.2
14.4 15.4 15.8 15.2 10.4
10.8 13.2 14.4 12.8 26.6
8.4
2.0
5.8
6.0
12.0
7.8
15.0
10.8
9.2
1.6
17.4
4.0
9.0
1.2
0.6
4.4
18.4
1.0
16.6
13.3
6.0
8.6
4.6
9.8
11.4
11.6
5.8
9.6
10.0
Mean
0.8*
19.0
3.0*
10.4
0.6*
1.1*
3.4*
21.0
1.1*
13.5
19.9
7.2
5.3*
5.2*
7.9
11.7
9.7
10.4
10.2
9.6
a Mean of five plants per replicate.
Control station, assume effluent 7. to be close to zero.
* Statistically less than station 2 (ANOVA followed by Dunnett's mean
separation test).
III-7
-------
25
20
• Ambient
O Effluent Dilution
6 I 5
5 I5o
Q.
cr
i 10
h-
Cfl
o
•
•Sto.4
\
\
\
\
X
Sta.6
0.5
1.0 1.5
% EFFLUENT
2.0
2.5
Figure 1. Number of cystocarps plotted againsts 7. effluent for data
from May 18th. The 7. effluent was based on the dye study of the
previous week. Plants treated with receiving water from station 4 had
an unusually large number of cystocarps for the effluent 7.. Plants
treated with water from station 6 had an unusually low number of
cystocarps. This may be due to the time difference between the dye
study and the toxicity testing. However, considering this time
difference, the rest of the data show a good correlation.
-------
20 r
A Effluent Dilution
O NH4CI
0 L
Figure 2. Number of cystocarps (as 7. of control) plotted against 7.
effluent. The effluent data are averages from May 17th and 18th. The
ammonium chloride data are based on 70 mg NH,-N/L in the effluent. A
stock solution of 26.7 mg NH.C1/100 mL was used.
-------
PART IV
PRELIMINARY GUIDANCE MANUAL FOR CONDUCTING SEXUAL REPRODUCTION
TESTS WITH THE MARINE MACROALGA, LAMINARIA SACCHARINA,
FOR USE IN TESTING COMPLEX EFFLUENTS
IV-1
-------
INTRODUCTION
A test was developed to use kelp, Laminaria saccharine, as a
toxicity test species for short-term exposure to toxicants. A test has
also been developed using the red alga Champia parvula, however, this
alga is primarily a warm water species. The kelps are more normally
found in colder waters, and thus would make a good complimentary test
organism. As with Champia, sexual reproduction was used as the endpoint
for accessing the effects of toxicants with Laminaria. The test differs
from that of Champia in that it requires preconditioning in order to
perform tests.
The current Laminaria test is based on techniques that were first
used with oil studies several years ago (Steele and Hanisak, 1979).
Those tests used material derived from nature for each experiment,
hereas the present test arelies on cultured gametophytic material.
IV-2
-------
MAIHTEHABCE OF STOOL COLTDBES OF T-AMTHARTA
Laminar ia* 3 life history is an alternation of microscopic
gametophytes with a. large diploid blade. The clones presently being
used were isolated from Rhode Island in 1985. It is probably not
essential to have a standard clone, however, some experience is required
to isolate new clones from the field into unialgal culture. Unialgal
stock cultures are necessary to maintain healthy, actively growing
plants for use in testing.
Male and female plants of Laminaria can be maintained easily in
unialgal culture in the laboratory. New cultures can be started by
blending old cultures, and splitting into several new culture vessels.
Blending is accomplished using a food blender at its fastest speed (for
approximately 1 min). For maintenance cultures, a nutrient medium
without added iron is used to inhibit gametogenesis; allowing greater
vegetative growth. Some preconditioning is required to induce reproduc-
tion. Under the conditons listed below, male and female gametophytes
will produce gametes. Thus, plant material can be available at any time
for testing.
Unialgal stock cultures of both males and females are maintained in
separate, aerated 500 mL Erlenmeyer flasks containing 400 mL of the
culture medium. The choice of these flasks is one of preference rather
than necessity. The maintenance culture medium is artificial or natural
seawater to which additional nutrients are added (Table 1). Seawater is
autoclaved for 30 min at 15 psi. The culture flasks are capped with
aluminum foil and autoclaved dry, for 10 min. Culture medium is made up
by dispensing seawater into the sterile flasks and adding the appro-
priate nutrients from a sterile stock solution. Alternately, 500 mL
IV-3
-------
flasks could be autoelaved with the seawater already in them. Sterili-
zation is used to prevent microalgal contamination, and not to keep
cultures bacteria-free.
We recommend that several cultures of both males and females be
maintained simultaneously to keep a constant supply of plant material
available. Initial stock cultures should be started weekly or biweekly
with freshly blended material. Cultures are gently aerated through
sterile, cotton-plugged, disposable, polystyrene 1 mL pipets. Cultures
are capped with foam plugs and aluminum foil and illuminated with ca 75
-2 -1
uE m s of cool-white fluorescent light on a 16:8, light:dark cycle.
The temperature is 12 to 15 °C and the salinity 28 to 30°/ao. Media are
changed biweekly. About one-half to one-third of the plant material
should be placed into another culture vessel with each medium change to
reduce the amount of biomass as the plants grow. With the above proce-
dure, actively growing plants will be continuously available. The total
number of cultures^maintained will depend on the expected frequency of
testing. We keep 7 actively growing cultures of each sex. In this way
we can use a different culture each day and not reuse it for at least a
week; allowing ample time for regrowth of the blended material.
ARTIFICIAL
Because salinity adjustments will be necessary in testing most
complex effluents (particularly their receiving waters), an artificial
seawater recipe that yields good growth of Laminaria is desirable. An
artificial seawater would also make the test method more readily avail-
able to laboratories that do not have access to clean natural seawater.
The recipe for the artificial seawater that we use is listed in Table 2.
Comparisons between the sensitivity to effluents in this medium with
IV-4
-------
that in natural seawater have not yet been conducted.
PREPARATION OF PLABTS FOR A TEST
Stock cultures should be checked for their readiness for use in
toxicity tests. Plants can be checked by examination under a compound
microscope (50-100X). Healthy gametophytes of both sexes are highly
branched and the condition of individual cells can be accessed by obser-
ving the chroma tophores. These should appear as discrete pale brown
discoid objects evenly dispersed in the cell. Senescent cells will
appear much darker and the chroma tophores will not be discrete. Once
cultures are determined to be usable for toxicity testing, plants should
be blended using a commercial food blender at the highest speed. The
resulting suspension should be filtered through a 30-60 urn nylon
screening, and the portion that passes through used for testing. These
cells can be diluted and pipetted into the test chambers.
Host testing to date has been performed in ca. 30 mL (using 60x25mm
petri dishes). These dishes offer the advantage of being disposable, as
well as being deeper than the standard petri dish (Lab Tek #4036).
Recovery chambers can 'be either these dishes or scintillation vials.
TEST CUHDITOBS
Temperature, Salinity, Light and Aeration
The test exposure duration is 2 days followed by a 3-7 day recovery
period for females for the development of sporophytes. The exposure
temperature should be between 10-12°C, and the salinity should be be-
tween 28-32°/oe* For receiving waters, salinity will often be below the
desired range and must be adjusted with artificial sea salts. The
IV-5
-------
•2 -1
photoperiod should be a 16h:8h, light:dark cycle of ca. 75 uE m s of
cool-white fluorescent light. Plants are recovered under the same
salinity and light conditions, but the temperature is raised to 16°C to
slow the growth of the females while the sporophytes develop (this is
not essential, but it makes the counting of the sporophytes easier).
Nutrient
The nutrient recipe is listed in Table 1. Trace metals and EDTA
are omitted from the medium during test exposures (except in artificial
seawater, where EDTA is added at 20 ug/L).
FB0TOGOL
1. At least one week prior to testing, blend males, filter and dilute
and allow to settle onto cover slips (100-200/cover slip). The minus
iron medium is used at this point. This allows the males to attach
better to the cover slip before gametogenesis begins.
2. After 2-3 days replace medium for males with one that contains
double strength iron/EDTA.
3. On the same day as #2, blend the females, filter and dilute and
allow to settle onto cover slips or small pieces of glass slides (100-
200/slide). The complete medium containing double strength iron is used.
4. After an additional 4-5 days check males and females for the
presence of gametes. If gametes are seen then testing can begin.
5. Set up and label control and treatment dishes; three per treatment
and controls.
6. Fill dishes with 30 mL of control or treatment water. Use the 1/8
strength nutrient medium minus trace elements and with the adjusted
EDTA.
7. Add one cover slip (or glass slide) each of males and females.
IV-6
-------
Rinse males briefly in seawater to remove loosely attached plants. This
will minimize the transfer of males to the slide containing the females.
8. After two days, remove slides containing females, rinse briefly in
seawater and place into control medium (complete nutrients). Transfer
to 16-18'C if possible.
9. After an additional 3-7 days examine females under a compound
microscope and count the number of sporophytes.
10. Data are analyzed by analysis of variance (ANOVA) followed by
Dunnett's mean separation test to determine differences from the
control.
NOTE: We recommend making up four nutrient solutions.
1. Complete minus iron; for maintaining stock cultures.
2. complete plus iron; for recovery medium after exposure.
3. 1/8 strength minus trace elements and EDTA; for the exposure
medium.
4. an iron/EDTA solution for increasing the iron concentration
during gametogenesis.
IV-7
-------
Table 1. Recipe of nutrients to be added to artificial or natural
seawater for stock cultures and test medium. The test medium concentra-
tions are 1/8 strength of the stock culture concentration except for
EDTA. The concentrated stock solution is autoclaved at standard tem-
perature and pressure for 15 min (the pH is adjusted to 2.0 with HC1
prior to autoclaving to prevent precipitation).
COMPOUND
NaN03
NftH , PQ . *E*—O
2 u 2
Na2EDTA.2H20
NaC,H-0, (citrate)
O J 1
Iron0
Trace Elements
Na2Mo04.2H20
KI
ZnS04.7H20
NaV03
MnS04-H20
Vitamins
Thiamine*HCl
Bio tin
B12
AMOUNT/LITER
STOCK CULTURES CONCENTRATED STOCK*
127 mg
12.8 mg
2.66 mgb
1.03 mg
195 ug
24.2 ug
83 ug
21.8 ug
6,l_ug
0.61 ug
1.95 mg
1.0 ug
1.0 ug
12.7 g
1.28 g
266 mg
103 mg
19.5 mg
2.42 mg
8.3 mg
2.18 mg
0.61 mg
61.0 ug
10 mLS
*Use 1.25 mL/L for test medium (with the adjusted EDTA concentration)
and 10 mL/L for stock cultures. For test media an additional 2.5 mL/L
of the sodium bicarbonate stock solution is added (see Table 3).
The concentration is 20 ug/L for final solution of test media in
artificial seawater; EDTA is omitted entirely if this nutrient medium
is used in natural seawater.
CIron stock solution prepared by dissolving 1 g iron powder in 10 mL
concentrated HC1 and diluting to 1 liter with deionized water.
Acceptable stock solutions could also be made with ferric or ferrous
chloride. Iron is omitted for maintenance culture medium and is double
for initiation of gametes.
Trace elements are omitted for toxicity test medium.
Vitamin stock solution autoclaved separately in 10 mL sub-samples.
Each 10 mL contains 195 mg of thiamine.HCl, 100 ug biotin, and 100 ug
B12. Adjust pH to ca 4.0 before autoclaving for 2 min.
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Table 3^. Recipe for artificial seawater using GP2. The concentrations of
the salts have been adjusted to give a final salinity of 30°/00. The
original recipe calls for autoclaving anhydrous and hydrated salts
separately to avoid precipitation. However, if the sodium bicarbonate
is autoclaved separately (dry), then all of the salts can be autoclaved
together. Since no nutrients are added until needed, autoclaving is
not critical for effluent testing. To minimize microalgal contamination
the artificial seawater should be autoclaved when used for stock cul-
tures. Autoclaving should be for at least 10 min for 1 liter batches
and 20 min for 10 to 20 liter volumes (at standard temperature and
pressure).
COMPOUND GRAMS/LITER*
MaCl
Na2S04
KC1
KBr
N.2B407-10H20
MgCl2.6H20
CaCl2.2M20
SrCl2-6H20
HaHC03b
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
Generally made in 10 to 20L batches.
A stock solution of 68 mg/mL sodium bicarbonate is prepared by auto-
claving it as a dry powder and then dissolving it in sterile deionized
water. For each liter of GP2 use 2.5 mL of this stock solution.
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REFERENCES
Steele, R.L. and M.D. Hanisak. 1978. Sensitivity of some brown algal
reproductive stages to oil pollution. In: Proceedings of the
Ninth International Seaweed Symposium, pp. 181-190. A. Jensen and
J.R. Stein (eds). Science Press.
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PART V
COMPARISON OF THE SENSITIVITY OF LAMINARIA WITH
THAT OF CHAMPIA
V-l
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INTRODUCTION
At this point, Laminaria looks very promising as a toxicity test
species for testing single compounds and complex effluents. However, a
comparison of test results using Laminaria with that of the test using
Champia was necessary to determine the sensitivity of the new procedure
to toxicants. The comparison was made using two heavy metals (silver
and copper); two organics (pentachlorophenol and isophorone); and one
sewage effluent (East Greenwich STP).
MATERIALS AND METHODS
The procedures used for both Laminaria and Champia are describe
elsewhere in this report (Parts IV and I respectively).
RESULTS
The results of the comparison of Champia and Laminaria are shown in
Figures 1 and 2 and Table 1. The sensitivity of Laminaria to copper and
silver was not as great as that for Champia (Figure 1). In fact,
Champia was at least an order of magnitude more sensitive. However,
Laminaria and Champia were very similar in their sensitivities to both
of the organics tested (Figure 2) and the sewage effluent (Table 1).
The results of these early comparisons indicate that Laminaria will
be a useful toxicity test species. It was noteworthy that these two
algal species, which come from vastly different phyla, responded
similarly to the organics and sewage effluent. The gives support to the
use of either species to make preliminary generalizations about the
response of seaweeds to toxicants.
V-2
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Table 1. The effect of sewage effluent from the East Greenwich STP on
sexual reproduction in Laminaria saccharins and Champia parvula. The
values listed are the geometric mean (as percent effluent) of the
effect/no effect concentrations using no sexual reproduction as the
effect measured.
Date
Laminaria
Champia
September 9, 1986
September 15, 1986
1.8
3.7
3.7
1.8
V-3
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100
o
cr
i-
z
o
o
u_
o
0.
O
(T
O
0.
20 -
,1
40 60 80
O COPPER (^g/L)
100
10
20 30
' SILVER
40
50
Figure 1. The effect of copper and silver on sexual reproduction in
Laminaria aaccharina and Champia parvula. The effect for Champia is
only
represented
occurred.
by an arrow where the total absence of reproduction
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100
J^
t
o 80
/g/L) 1
i i i i i t
0 200 400 600 800 1000
ISOPHORONE (//M)
Figure 2. The effect of pentachloxophenol and isophorone on sexual
reproduction in Laminaria saccharina and Champia parvula. The effect
for Champia ia represented only by an arrow where the absence of
reproduction occurred.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Development of short-term Exposure
Tests for Marine Macroalgae for use in Effluent
Testing
5. REPORT DATE
November, 1986
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Glen B. Thursby
8. PERFORMING ORGANIZATION REPORT NO.
X117
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Botany Department
University of Rhode Island
Kingston, RI 02881
10. PROGRAM ELEMENT NO.
B101
11. CONTRACT/GRANT NO.
CR8.12070-01
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Narragansett
South Ferry Road
Narragansett, RI 02882
13. TYPE OF REPORT AND PERIOD COVERED
Final Nov. 1984 - Nov. 1986
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES „,,. •,-,,,.,.
iechniques developed during this cooperative agreement were
presented at three Effluent Testing Workshops sponsored by ERLN in 1985 and 1986.
16. ABSTRACT
A previous sexual reproduction toxicity test with the marine red macroalga, Champia
parvula, lasted two weeks. This report covers a modification to this procedure in
which males and females are exposed together to a toxicant or effluent for only two
days. The procedure was used successfully with single compounds and a variety of
complex effluents. A comparison of toxicity test results between the two-week and
the two-day exposure procedures was performed using single compounds. The two pro-
cedures compared favorably. Initial steps were also taken in the development of a
similar two-day exposure test with the brown alga, Laminaria saccharine. Laminaria
represent another phylum of algae than Champia, and has the additional advantage of"
being both economically and ecologically important. For the current study, the
feasibility of using Laminaria as a routine toxicity test species was verified. A
comparison was also made between the sensitivity of both species. The sensitivity
of Laminaria to copper and silver was not as great as that for Champia. However,
both species were very similar in their response to pentachlorophenol and isophorone,
as well as a sewage effluent.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Champia, Laminarja effluents
18. DISTRIBUTION STATEMENT
Release Unlimited
19. SECURITY CLASS (This Report')
21. NO. OF PAGES
86
20. SECURITY CLASS (This page)
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
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