8112

8112 PHYTOPLANKTON*
8112 A. Introduction
Phytoplankton are primary producers in the aquatic commu-
nity and, as such, are at the base of aquatic food chains. Because
of this, they must be tested in bioassays that predict and deter-
mine the potential effects of a substance on the aquatic environ-
ment. The same general principles and techniques used in deter-
mining biostimulation (Section 8111) are used to determine
toxicity to phytoplankton. The procedure applies to fresh water,
estuarine, and marine phytoplankton. See Sections 10200D–H
and 10300D for more information on phytoplankton.
8112 B. Inoculum
In addition to the marine or estuarine algae listed in Section
8111F.1b,Monochrysis lutheri Droop may be used. Maintain the
test species in full-strength media [Section 8010E.4c1) and Ta-
bles 8010:III and 8010:IV.A and B]. Test species must be in the
logarithmic growth phase; therefore, transfer them to fresh cul-
ture medium every 4 to 5 d.
8112 C. Test Conditions and Procedures
1. Maximum Specific Growth Rate
The maximum specific growth rate ( ␮
max
) is the greatest
specific growth rate (
␮ ) occurring at any time during an incu-
bation. The
␮
max
can be estimated from growth measurements. It
is related to the concentration of rate-limiting nutrient available.
The specific growth rate is defined by:
␮ ⫽
ln (X
2
/X
1
)
t
2
⫺t
1
where:
X
2
⫽biomass concentration at end of selected time interval,
X
1
⫽biomass concentration at beginning of selected time interval, and
t
2
⫺t
1
⫽time interval of selected time period, d.
Add test material to test vessels to give desired concentrations.
Prepare triplicate vessels for each concentration. Use dilutions of
culture medium to simulate chemical conditions of specific re-
ceiving waters. For optimum surface-to-volume ratios, see Sec-
tion 8111C.2a.
The
␮
max
occurs during the logarithmic phase of growth,
usually between Day 0 and Day 5. Therefore, measure biomass
at least daily during the first5dofincubation. Indirect measure-
ments of biomass, such as chlorophyll aor cell numbers, usually
will be required because accurate gravimetric measurements at
low cell densities are difficult. See Section 8111G.3cand
8111G.4 for methods.
Test a geometric series of concentrations initially (see Section
8010F.3b). After this preliminary test, progressively bisect in-
tervals on a logarithmic scale. Narrow the range of test concen-
trations to determine the concentration that reduces
␮
max
to 50%
of the control. This requires that two of the concentrations tested
fall on each side of the concentration that inhibited ␮
max
to 50%
(see Section 8010G).
Compare
␮
max
to that obtained in the synthetic freshwater or
artificial seawater culture medium. Regional and seasonal vari-
ations in quality make natural waters unsuitable as standard test
media for comparative toxicity tests. Therefore, use a synthetic
freshwater medium and/or artificial seawater. Add various con-
centrations of toxicants to the culture medium in triplicate and
inoculate with test species.
2. Other Tests
For other types of tests, such as those to determine effluent
requirements or compliance with water quality standards, take
dilution water from the receiving body near the outfall but
outside its influence. Remove undesirable organisms before
making growth rate tests with selected sensitive species (Section
8111D). Determine
␮
max
in test vessels and compare with controls
and EC
50
s based on percent of growth reduction. An alternative
approach that provides a number that may relate to natural condi-
tions should be reviewed.
1
3. Reference
1. MILLER, W.E., J.C. GREENE &T.SHIROYAMA. 1978. The Selenastrum
capricornutum Printz Algal Assay Bottle Test, U.S. Environmental
* Approved by Standard Methods Committee, 2000. Editorial revisions, 2010.
1

Protection Agency Rep. EPA-600/9-78-018. National Technical In-
formation Serv., U.S. Dep. Commerce, Springfield, Va.
4. Bibliography
ERICKSON, S.J., N. LACKIE & T.E. MALONEY. 1970. A screening technique
for estimating copper toxicity to estuarine phytoplankton. J. Water
Pollut. Control Fed. 42:R270.
WALSH, G.E. 1972. Effects of herbicides on photosynthesis and growth
of marine unicellular algae. Hyacinth Control J. 10:45.
GREENE, J.C., W.E. MILLER,T.SHIROYAMA &E.MERWIN. 1975. Toxicity
of Zinc to the Green Alga Selenastrum capricornutum as a Function
of Phosphorus or Ionic Strength, U.S. Environmental Protection
Agency Rep. EPA-660/3-75-034. National Technical Information
Serv., U.S. Dep. Commerce, Springfield, Va.
KOELMANS, A.A., C.S. JIM´
ENEZ &L.LUKLEMA. 1993. Sorption of chlo-
robenzenes to mineralization of phytoplankton. Environ. Toxicol.
Chem. 12:1425.
STRANGE, K. & D.L. SWACKHAMER. 1994. Factors affecting phytoplank-
ton species-specific differences in accumulation of 40 polychlori-
nated biphenyls (PCBs). Environ. Toxicol. Chem. 13:1849.
BROWN, L.S. & D.U.S. LEAN. 1995. Toxicity of selected pesticide to lake
phytoplankton measured using photosynthetic inhibition compared
to maximal uptake rates of phosphate and ammonium. Environ.
Toxicol. Chem. 14:93.
BENJAMIN, R.B., R. BROWN, III, M. FRAIZER, B.M. JOAB, R.C. CASEY &
S. KLAINE. 1998. Algal growth rate fluctuations observed under
uniform ambient test conditions using static and semicontinuous
assay techinques. Environ. Toxicol. Chem. 17:460.
KIRKWOOD, A.E., P. CHOW-FRASER &G.MIERLE. 1999. Seasonal mercury
levels in phytoplankton and their relationship with algal biomass in
two dystrophic shield lakes. Environ. Toxicol. Chem. 18:523.
PHYTOPLANKTON (8112)/Test Conditions and Procedures
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PHYTOPLANKTON (8112)/Test Conditions and Procedures