8113

8113 MARINE MACROALGAE*
8113 A. Introduction
1. Occurrence and Importance
Marine macroalgae are important biological components of
the oceans and are found in coastal and intertidal zones down to
the depths of light penetration sufficient for photosynthesis. They
are classified in one of three phyla based, to a large extent, on
color derived from the major photosynthetic pigments present.
Members of Chlorophyta (green algae) contain chlorophyll aand
band
␤ -carotene. Red algae (Rhodophyta) contain phycoeryth-
rin, an accessory pigment that provides the reddish color ob-
served in most species of this phylum. Brown algae (Phaeo-
phyta) contain the brown accessory pigment fucoxanthin. Within
this phylum are the most structurally complex of all algae, the
laminarians (Order Laminariales). Laminarians include the large,
canopy-forming kelps of the world, such as the giant kelp Mac-
rocystis pyrifera.
Macrocystis pyrifera (see Section 10900, Plate 2A:E) forms
extensive coastal submarine forests that provide food, habitat,
and shelter for numerous species.
1
Giant kelp also is an
important economic resource that is harvested commercially
to obtain alginic acid, a thickening agent used in foods,
cosmetics, culture media, and other products. The economic
and ecological importance of Macrocystis pyrifera, as well as
its abundance, make it an attractive test species for monitor-
ing coastal water quality. Indeed, test methods using the early
life stages of giant kelp have been developed and found to be
sensitive to many toxicants found in the coastal environment.
2– 4
The Macrocystis pyrifera method has been used successfully for
monitoring and determination of compliance with National Pol-
lution Discharge Elimination System (NPDES) permits in Cali-
fornia and other regions along the Pacific Coast of North Amer-
ica since 1990.
5
2. References
1. NORTH, W.J., ed. 1971. The biology of giant kelp beds (Macrocystis)
in California. Nova Hedwigia 32:1.
2. JAMES, D.E., S.L. MANLEY, M.C. CARTER & W.J. NORTH. 1987.
Effects of PCBs and hydrazine in life processes in microscopic stages
of selected brown seaweeds. Hydrobiologia 151/152:411.
3. ANDERSON, B.S. & J.W. HUNT. 1988. Bioassay methods for evaluating
the toxicity of heavy metals, biocides, and sewage effluent using
microscopic stages of giant kelp Macrocystis pyrifera (Agardh): A
preliminary report. Mar. Environ. Res. 26:113.
4. SINGER, M.M., D.L. SMALHEER, R.S. TJEERDEMA &M.MARTIN. 1990.
Toxicity of an oil dispersant to the early life stages of four marine
species. Environ. Toxicol. Chem. 9:1389.
5. STATE WATER RESOURCES CONTROL BOARD. 1990. California Ocean
Plan; Water Quality Control Plan; Ocean Waters of California. State
Water Resources Control Board, Sacramento, Calif.
3. Bibliography
GRAHAM, M.H. 2004. Effects of local deforestation on the diversity and
structure of southern California giant kelp forest food webs. Eco-
systems. 7:341.
RAIMIMONDI, P.T., D.C. REED,B.GAYLORD &L.WASHBURN. 2004.
Effects of self-fertilization in the giant kelp Macrocystis Pyrifera.
Ecology 85:3267.
8113 B. Selecting and Preparing Macrocystis pyrifera Sporophylls
1. Life Cycle
The life cycle of Macrocystis pyrifera, like that of all lami-
narians, alternates between a diploid sporophyte and a haploid
gametophyte generation (Figure 8113:1). The conspicuous adult
sporophyte produces haploid, dioecious, biflagellate zoo-
spores in dark patches (sori) found on reproductive blades
(sporophylls) near the base of the plant throughout the year.
The zoospores are released and settle on an a appropriate
substrate, where they germinate and develop into microscopic
gametophytes. Flagellated male gametes are produced within
the antheridia of the male gametophytes and released to
fertilize eggs produced within oogonia on the female gameto-
phyte. The fertilized eggs develop into young sporophytes in
12 to 15 d.
2. Occurrence
Macrocystis pyrifera forests are found subtidally along the
Pacific Coast of North America from Central California south to
Baja California. Substantial populations also are found along
the west coast of South America from Peru south and off the
coasts of New Zealand, Tasmania, and the islands of the
subantarctic.
1
3. Collection and Maintenance of Test Organisms
Zoospores of Macrocystis pyrifera are obtained by subtidal col-
lection (usually by SCUBA divers) of sporophylls. The sporophylls
are found near the base of the plant, usually lack air bladders, are
thinner than vegetative blades, and have sori that extend to near the
* Approved by Standard Methods Committee, 1999. Editorial revisions, 2009.
Joint Task Group: Lan C. Wiborg (chair), Joseph R. Gully, Robert W. Holst,
Donald J. Reish.
1

margin of the sporophyll. The sori contain the mature and devel-
oping zoospores. Unless contrary to the purpose of the study, collect
sporophylls from several plants, in a region relatively free from
point and nonpoint sources of water discharge, and below the
thermocline, if present. Check local regulations and collection per-
mit requirements before obtaining sporophylls.
Once sporophylls have been collected, keep them damp but
avoid immersion in seawater because this will cause the zoo-
spores to be released. Keep blades out of direct sunlight to avoid
damage to the zoospores from ultraviolet radiation. Clean the
sporophylls by gently rubbing with fingers or soft bristle brush
and rinse with seawater filtered through 0.22-
␮ m filter. Layer the
sporophylls between paper towels to prevent any blades from
overlapping. Keep the sporophylls’ temperature between 9 and
12°C during transport and/or shipment to test facility using
newspaper-insulated chemical ice packs and a cooler. Stored in
this manner, the sporophylls can be used up to 24 h after
collection. However, an increase in the zoospores’ sensitivity to
copper after storage has been demonstrated with sporophylls
collected at one southern California kelp bed.
2
4. Predators, Parasites, and Diseases
Giant kelp is a valuable food source for many herbivores in the
coastal environment, including the opaleye (Girella nigicans),
the half-moon (Medialuna californiensis), and other fishes.
Many snails also feed on Macrocystis sp., including abalone
(Haliotis sp.), the black turban (Tegula funebralis), and the sea
hare (Aplysia californica). However, the most notorious grazers
are sea urchins (Strongylocentrotus purpuratus, S. franciscanus),
which can cause extensive damage to giant kelp forests when the
population explodes due to a lack of predation.
3
Figure 8113:1. The life cycle of the giant kelp, Macrocystis pyrifera.
MARINE MACROALGAE (8113)/Selecting and Preparing Sporophylls
2
MARINE MACROALGAE (8113)/Selecting and Preparing Sporophylls

Certain bryozoans, particularly Membranipora membranacea,
use blades of giant kelp as a substrate for attachment; do not use
encrusted sporophylls for toxicity tests. Morphological deformi-
ties associated with endophytic algae
4
and epidemic diebacks
from infection by virus-like particles
5
have been documented in
other species of kelp. Collect sporophylls for use in toxicity tests
only from plants that appear healthy and free from deformities.
5. References
1. DRING, M.J. 1982. The Biology of Marine Plants. Edward Arnold
Limited, London, U.K.
2. GULLY, J.R., J. BOTTOMLEY & R.B. BAIRD. 1999. Effects of sporophyll
storage on giant kelp, Macrocystis pyrifera, bioassay. Environ. Toxi-
col. Chem. 18:1474.
3. NORTH, W.J., ed. 1971. The biology of giant kelp beds (Macrocystis)
in California. Nova Hedwigia 32:1.
4. ELLERTSDATTIR, E. & A.F. PETERS. 1997. High prevalence of infection
by endophytic brown algae in populations of Laminaria spp. (Pha-
eophyceae). Mar. Ecol. — Prog. Ser. 146:135.
5. EASTON, L.M., G.D. LEWIS & M.N. PEARSON. 1997. Virus-like parti-
cles associated with dieback symptoms in the brown algae Ecklonia
radiata. Dis. Aquat. Organisms 30:217.
6. Bibliography
BOLD, H.C. & M.J. WYNNE. 1985. Introduction to the Algae, 2nd ed.
Prentice-Hall, Inc., Englewood Cliffs, N.J.
ANDERSON, B.S., J.W. HUNT, S.L. TURPEN, A.R. COULON &M.MARTIN.
1990. Copper toxicity to microscopic stages of Macrocystis py-
rifera: Interpopulation comparisons and temporal variability. Mar.
Ecol. — Prog. Ser. 68:147.
8113 C. Toxicity Test Procedures
1. General Procedures
Expose zoospores from mature sporophylls of the giant kelp,
Macrocystis pyrifera, to several concentrations of a standard
toxicant (CuCl
2
) and the test sample for 48 h at 15°C under a
specified light intensity and cycle without solution renewal
(static). At the conclusion of the test, determine the proportion of
germinated zoospores and the length of the resulting germ tube
microscopically.
2. Water Supply
a. Artificial seawater: The use of artificial seawater in this test
has not been evaluated, and its use can not be recommended at
this time.
b. Natural seawater: Collect natural seawater with a salinity of
33 ⫾3 g/kg from a location relatively free from point and
nonpoint sources of waste discharge unless the study warrants
the use of a seawater from a specific location. After collection,
filter using a 0.45-
␮ m membrane filter and store in the dark at 4
⫾1°C until used. Seawater held in this manner may be used up
to 14 d after collection. Filtered seawater may be held at 15 ⫾
1°C for up to 24 h prior to test initiation. During a test, the
salinity of all test solutions should be 33 ⫾3 g/kg and not vary
by more than ⫾2 g/kg among the test concentrations. Hypersa-
line brine produced from natural seawater may be used to adjust
the salinity of test solutions that are below the recommended
range, but extra controls and statistical procedures must be
employed in the test design and analysis. Guidance on using
brine to adjust salinity can be found elsewhere.
1
3. Exposure Chambers
Use petri dishes (100 ⫻20 mm) filled with 50 mL test solution
as test chambers. The composition of the exposure chamber
depends on sample type and study objectives. Preferably use
acid-cleaned glass petri dishes; dilution-seawater-leached dis-
posable plastic chambers, plastic food containers, and glass
beakers also have been used successfully. Place glass micro-
scope slides (approximately 7.6 ⫻2.5 cm) in test chamber before
adding test solution and spores. The slides serve as a substrate
for spore settlement and gametophyte development that can be
removed and analyzed microscopically at the end of the test.
This testing system is not suitable for volatile compounds.
4. Conducting the Toxicity Test
a. Preparation of test chambers for inoculation: Label test
chambers in a way that will facilitate blind analysis of the
biological endpoints at the conclusion of the exposure. Place test
chambers randomly in testing area to account for subtle differ-
ences in light intensity and temperature. Measure light intensity
at each test chamber using a planar light meter and adjust light
fluence rate and/or position of chambers as required to obtain a
fluence rate of 50 ⫾10
␮ Einsteins/m
2
/s (2000 to 3010 lux; 186
to 280 ft-c). Set the light cycle in testing area for 16 h light and
8 h dark for the duration of the test.
Prepare volumes of test solution needed to provide solution for
test chambers and water quality/chemistry measurements. Use a
minimum of five replicates at each test concentration. Selection
of sample test concentrations depends on the nature and intent of
the study; for guidance, see Sections 8010D and F. Seek a
concentration series that causes a partial response at several
concentrations because such a series provides valuable informa-
tion about the nature and precision of the concentration–response
curve. However, in some studies it may be more important to
select concentrations around specific exposure conditions of
interest. The concentration series used in the CuCl
2
standard
toxicity test is fixed at 0 (control), 5.6, 10, 18, 32, 56, 100, and
180
␮ g/L of copper ion (nominal).
Measure pH, dissolved oxygen (DO), salinity, and temperature
of each test sample and copper concentration. Acceptable salin-
ity values range from 30 to 36 g/kg. Ensure that solution tem-
perature is 15 ⫾1°C before inoculation with spores. The solu-
tions may be aerated according to established procedures if DO
is below 4 mg/L. When target water quality measurements have
MARINE MACROALGAE (8113)/Toxicity Test Procedures
3
MARINE MACROALGAE (8113)/Toxicity Test Procedures

been obtained, fill each test chamber with the appropriate test
solution and volume (100 ⫻20-mm petri dishes ⫽50 mL).
b. Spore release and inoculation of test chambers: Rinse
approximately 100 g wet weight of sporophylls (approximately
30 blades) with filtered seawater. Rub blades with the fingers or
gently scrub them with a soft bristle brush to remove debris and
epiphytic organisms. Rinse with filtered seawater and lay on
paper towels so no blades overlap. Blot dry using paper towels
and place in a room at 15 ⫾1°C for 1 h. Maintain 15 ⫾1°C
temperature for spore release and inoculation procedure. Desic-
cation of sporophylls in this manner will cause spores to be
released upon exposure to seawater.
Fill a clean glass 2-L beaker with 1 L filtered seawater. After
1 h of desiccation, place sporophylls into seawater for 1 h. The
water should become discolored as the spores are released from
the sori. After 1 h, remove sporophylls and allow spore suspen-
sion to settle for 30 min. This allows inactive spores and debris
to settle to the bottom of the beaker. Midway through settling
procedure, examine a small sample of the spore suspension
(collected from the upper 2 cm) microscopically to ensure that
the spores are motile. After settling, decant 250 mL of spore
solution into a clean 250-mL graduated cylinder or beaker.
Collect a sample of the inoculation suspension and determine
spore density using a hemacytometer. The target density of
spores in the test chambers is 7500 spores/mL. Calculate volume
of inoculation suspension required to obtain target spore density
using the equation:
V
i
⫽共7500 spores/mL⫻V
1
)/D
i
where:
V
i
⫽volume of inoculation added to test chamber,
V
1
⫽volume of test suspension in test chamber, and
D
i
⫽spore density of inoculation suspension.
Typically, the volume of the inoculant required to obtain 7500
spores/mL is ⬍1% of the test volume. If greater volumes are
required, the quality of the spores and consequent test results
should be suspect. Inoculate each test chamber with spore sus-
pension using a cut or wide-mouth pipet. Note the time, which
represents the start of the test.
c. Duration and type of test: The exposure of spores to test
solution is static, and the duration is 48 ⫾3 h. Continuous
temperature monitoring of a surrogate test chamber in the testing
area is recommended to ensure that proper temperature was
maintained during the test.
d. Test termination and endpoint examination procedures:
1) Slide preparation—Prepare slides for microscopic analysis
by carefully removing slide from petri dish with forceps. Save
remaining test solution and store it in testing area until final
water quality measurements (pH, DO, temperature, and salinity)
of each test solution concentration are made. Wipe excess water
from bottom and drain top by placing edge of slide on a paper
towel. Carefully place a cover slip (24 ⫻50 mm) on top of slide,
wipe excess water from around cover slip, and label each slide
with test chamber identification. Do not prepare more slides than
can be read in 30 min to avoid potential inaccuracies in length
determination due to drying of the gametophytes.
2) Percent germination endpoint determination—Place pre-
pared slides on a microscope set at a magnification of approxi-
mately 400⫻. Focus microscope on the plane where the majority
of gametophytes and spores are clearly visible. Using a labora-
tory counter with at least two counting levers, move across the
slide counting both germinated and nongerminated spores until
at least 100 spores have been scored. Record number of germi-
nated and nongerminated spores and calculate proportion of
germinated spores for the replicate.
A spore is considered germinated if the germ tube is longer
than or equal to the width of the spore case (about 3
␮ m).
Typically, the germ tube can be identified by an irregular shape
and the presence of cellular contents while the spore case usually
is circular and devoid of cellular material (Figure 8113:2). If an
object cannot be easily identified as a spore or gametophyte, do
not count it.
3) Germ tube length endpoint determination—Place prepared
slide on a microscope at approximately 400⫻magnification
equipped with a calibrated micrometer capable of measuring
ⱕ2.5
␮ m/micrometer unit. See Section 10200E for micrometer
calibration procedures. Blindly move to a random location on the
slide and locate the spore case of the germinated spore that is
closest to the micrometer. The selected germ tube should be
straight and in focus for the entire length of the tube. Move slide
and micrometer as necessary to overlay micrometer on germ
tube. The length of the germ tube is defined as the distance from
the edge of spore case at the base of the germ tube to the tip
(Figure 8113:2). Measure and record germ tube length rounded
to nearest whole micrometer unit. Once germ tube length has
Figure 8113:2. Examples of nongerminated (A and B) and germinated
(C and D) giant kelp zoospores and germ-tube-length
measurement of germinated zoospores (E). Zoospores are
considered germinated when the germ tube length is equal to
or greater than the width of the spore case. The length of the
germ tube is measured from the edge of the spore case to the
tip of the germ tube.
MARINE MACROALGAE (8113)/Toxicity Test Procedures
4
MARINE MACROALGAE (8113)/Toxicity Test Procedures

been recorded, blindly move to another field of view and repeat
selection and measuring procedures until 10 germ tubes have
been measured. If germination percentage is low (⬍10%), the
slide may be scanned for germinated spores, measuring at the
first 10 germ tubes identified. Calculate mean germ tube length
in micrometer units and convert mean value into microns using
the conversion factor derived from the calibration procedure.
Record this value as the mean germ tube length for the replicate.
5. Reference
1. CHAPMAN, G.A., D.L. DENTON & J.M. LAZORCHAK, eds. 1995. Short-
term Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to West Coast Marine and Estuarine Organisms,
EPA-600/R-95-136. U.S. Environmental Protection Agency, Off.
Research & Development, Washington, D.C.
6. Bibliography
DEYSHER, L.E. & T.A. DEAN. 1984. Critical irradiance levels and the
interactive effects of quantum irradiance and quantum dose on game-
togenesis in the giant kelp, Macrocystis pyrifera. J. Phycol. 20:520.
ANDERSON, B.S., J.W. HUNT, S.L. TURPEN, A.R. COULON,M.MARTIN,
D.L. DENTON & F.H. PALMER. 1990. Procedures Manual for Con-
ducting Toxicity Tests Developed by the Marine Bioassay Project,
Rep. No. 90-10WQ, October 1990. State Water Resources Control
Board, Sacramento, Calif.
THURSBY, G.B., B.S. ANDERSON, G.E. WALSH & R.L. STEELE. 1993. A
review of the current status of marine algal toxicity testing in the
United States. In W.G. Landis, J.S. Hughes & M.A. Lewis, eds.
Environmental Toxicology and Risk Assessment, ASTM STP
1179. American Soc. Testing & Materials, Philadelphia, Pa.
GARMAN, G.D., M.C. PILLAI & G.N. CHERR. 1994. Inhibition of cellular
events during early algal gametophyte development — Effects of
selected metals and an aqueous petroleum waste. Aquat. Toxicol.
28:127.
ANDERSON, B.S., J.W. HUNT &W.PIEKARSKI. 1998. Recent advances in
toxicity test methods using kelp gametophytes. In P.G. Wells, K.
Lee & C. Blaise, eds. Microscale Testing in Aquatic Toxicology.
CRC Press, Boca Raton, Fla.
CIE, D.K. & M.S. EDWARDS. 2008. The effects of high irradiance on the
settlement competency and variability of kelp zoospores. J. Phycol.
44:495.
8113 D. Data Evaluation
1. Statistical Analysis
The statistical analysis selected to evaluate the sample’s tox-
icity or the reference toxicant depends on the objectives of the
study (see Section 8010G).
The mean germination percentage and germ tube length in
seawater controls for each test and the ⌭C
25
/IC
25
and mean
square error (MSE) values derived from analysis of variance
(ANOVA) must be calculated for the reference toxicant test to
evaluate the quality of the test. If brine was added to any test
concentrations, recalculate toxicant concentrations and compare
response of brine controls with natural seawater controls using a
t-test. If control responses were statistically different (two-tailed;
⬀⫽0.05), interpretation of the data may become difficult and
may warrant repeating the test. If the data are used, perform
separate statistical analyses for unaltered and brine-enriched
treatments using the corresponding control data.
2. Toxicity Test Evaluation
a. General considerations: The following test acceptability
criteria are intended to help ensure that healthy kelp sporophylls
and spores and adequate dilution water were used, and the test
was performed in a manner consistent with the method de-
scribed above. Data derived from tests that do not meet these
criteria should be considered suspect and may warrant repeat-
ing the test.
b. Negative control minimums: The purpose of negative con-
trol minimum criteria is to ensure that the spores germinated and
grew to a minimal level consistent with those seen in tests using
healthy sporophylls, adequate dilution seawater, prescribed pro-
cedures, and qualified staff. The minimum mean germination
percentage and germ tube length of the control replicates in the
reference toxicant and sample tests should be 70% and 10
␮ m,
respectively.
c. Positive control response ranges: The purpose of positive
control response range criteria is to ensure that the spores’
response to a standard toxicant (copper) is consistent with that
observed in tests using healthy sporophylls, adequate dilution
seawater, prescribed procedures, and qualified staff. The ⌭C
25
for the germination endpoint of the reference toxicant test should
be between 18 and 158
␮ g Cu/L. The IC
25
in the germ-tube-
length endpoint of the reference toxicant test should be between
13 and 106
␮ g Cu/L.
d. Within-test variability maximums: The purpose of within-
test variability maximum criteria is to ensure that the variability
of the spores’ response, particularly among treatment replicates,
does not exceed the variability observed in tests using healthy
sporophylls, adequate dilution seawater, prescribed procedures,
and qualified staff. The measure of variability used for this
criterion is the MSE derived from ANOVA. The MSE for the
proportion germinated endpoint of the reference toxicant test
should be less than 63.3 deg, or 0.019 radians for arcsin square
root transformed data. The MSE for the germ-tube-length end-
point of the reference toxicant test should be less than 4.32
␮ m.
MARINE MACROALGAE (8113)/Data Evaluation
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MARINE MACROALGAE (8113)/Data Evaluation