\
REPORT ON ALGAL TOXICITY TESTS ON
SELECTED OFFICE OF TOXIC SUBSTANCES (OTS) CHEMICALS
Michael A. Bollman1
Wanda K. Baune1
Shei 1 a Smi t h1
Kevin DeWhitt1
Larry Kapustka1
24 FEBRUARY 1989
Mention of trade names or commercial products does not constitute
endorsement for use.
1 NSI Technology Services Corporation
USEPA Environmental Research Laboratory
200 SW 35th Street
Corvallis, OR 97330
: USEPA Environmental Research Laborator,-
200 SW 35th Street
Ccrvallis, CR 97330
-------
REPORT ON ALGAL TOXICITY TESTS ON
SELECTED OFFICE OF TOXIC SUBSTANCES (OTS) CHEMICALS
Michael A. Bollman1
Wanda K. Baune1
Sheila Smith1
Kevin DeWhitt1
Larry Kapustka1
24 FEBRUARY 1989
Mention of trade names or commercial products does not constitute
endorsement for use.
1 NSI Technology Services Corporation
USEPA Environmental Research Laboratory
200 SW 35th, Street
Corvallis, OR 97330
2 USEPA Environmental Research Laboratory
200 SW 35th Street
Corvallis, OR 97330
-------
EXECUTIVE SUMMARY
Algal (Selenastrum capricornutum) assays were performed on 11
chemicals selected from a larger list of candidate chemicals
provided by the EPA's Office of Toxic Substances (OTS). Limited
chemical analysis of those chemicals that could be tested readily
at ERL-C was performed in support of the bioassays. Of the 11
chemicals tested, two (tetraethyl ammonium chloride and butyl
mercaptan) were judged non-toxic to the algae by virtue of the
calculated EC90 exceeding 1000 mg/L. Three chemicals were extremely
toxic (EC9„ < 10 mg/L; methyl vinyl ketone, octylamine, and phthalic
anhydride); three were highly toxic (EC80 > 10 mg/L but < 100 mg/L;
azosu1famide, methyl acrylate, and nitrobenzene); and three were
moderately toxic (EC90 > 100 mg/L but < 1000 mg/L; glyoxal, sodium
dodecylbenzenesulfonate, and glycidol). This work contributes to
the toxicity data base assembled on these chemicals. Previously,
algal toxicity information was lacking for these compounds.
i i
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CONTENTS
Executive Summary ii
I. Introduction 1
II. Chemicals Analyzed 1
III. Methods 1
A. Modifications to Procedures 2
1. Chelating Agents and Minimum Cell Counts at 96
hours 2
B. pH Adjustment 3
C. Derivation of Algal Assay results 4
1. Data Reduction 4
2. Statistical Analysis 4
a. Approach 5
IV. Chemical Analysis 6
V . Resu Its 7
VI. Discussion 7
References '
i i i
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TABLES AND APPENDICES
Table 1 Algal Assay Summary 10
Table 2 Chemical Results Summary 11
Appendix A ERL-C Standard Operating Procedure for the Algal
Assay
Appendix B ANOVA Tables and Regression Graphs
Appendix C Raw Data
Appendix D EDTA Test Results
iv
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I. Introduction
This report presents the results of the algal toxicity tests
performed on 11 chemicals selected from a list designated by EPA'5
Office of Toxic Substances (OTS). Important procedural
modifications were necessary to complete the tests of these
substances. This report describes the biological, chemical and
statistical methods used to complete the analyses. Interpretations
of toxicity are provided.
II. Chemicals Analyzed
The following list of chemicals was selected from a master list
provided by OTS. Feasibility of testing the chemical in an aqueous
matrix and limiting health and safety concerns were the primary
criteria for selection. To optimize personnel safety, the least
toxic chemicals were favored. Testing was performed between April
and September 1989.
g1yoxa1
sodium dodecy1benzenesulfonate
tetraethyl ammonium chloride
phthalic anhydride
azosu1famide
glycidol
oc ty1 amine
n i troben zene
methyl vinyl ketone
methyl acrylate
butyl mercaptan
Two other chemicals were candidates for testing. Methyl
methacrylate was not approved for testing by the Environmental
Research Laboratory - Corvallis (ERL-C) Health and Safety
Committee, "due to its possible explosive polymerization." Dibutyl
phosphate was not available for testing.
Methyl vinyl ketone, methyl acrylate, and butyl mercaptan, due to
their low vapor pressures and possible carcinogenic properties,
had to be tested in a ventilated exposure chamber. This special
requirement necessitated building an appropriate testing chamber
and delayed testing until late August 1989.
III. Methods
The test protocol requested by OTS was the standard procedure from
the Federal Register (Vol. 50; No. 188; Part 797; Sec. 797.1050,
1
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Algal Acute Toxicity Test). Since that publication, several
substantive methods modifications have been promulgated in EPA
publications (Greene, et al., 1988; Weber, et al., 1787). Attached
in Appendix A is a copy of the ERL-C standard operating procedure
for the algal assay test. This procedure with the specified
modifications requested by OTS (also in Appendix A) was followed
in testing these materials. Variances from procedures in these
documents included absence of subculturing, limitations in chemical
analyses (see Chemical Analysis section), and minimum control
yields and use of chelating agents, which are discussed below.
Although tests were counted every day, the final EC,„ value was
determined.using only the 96 hour counts. Counts for each day are
reported as "raw data," in Appendix C. The records of the daily
monitoring of the light intensity and shaker speed for each test
are also presented in Appendix C.
All test compounds were subjected to range-finding tests. If the
ECJ0 was greater than 1000 mg/L in the range-f inding test, no
definitive test was run.
A. Modifications to Federal Register Procedure
1. Chelating Agents and Minimum Cell Counts at 96 hours.
The procedure published in the Federal Register stipulates: "Algal
growth in controls should reach the logarithmic growth phase by 96
hours at which time the number of algal cells should be
approx i ma te 1 y ... 3.5 x 104/ ml for Se 1 enast rum. If growth in
controls does not reach this logarithmic phase within this 96 hour
period, the test is invalidated and should be repeated." The
procedure also states: "No chelating agents should be included in
the nutrient medium used for test solution preparation." The EPA
publications cited above define media with EDTA added as a
chelating agent. Recent ASTM proceedings concur that EDTA is
required to support sufficient algal growth. The ERL-C standard
Algal Assay Media contains 300 ug/L EDTA, and the growth in
controls does reach the log phase, although the number of algal
cells at 96 hours averages somewhat less than what is suggested in
the Federal Register. The proposed New Standard Guide for
Conducting Static Toxicity Tests With Microalgae (ASTM El218)
requires that for a test to be valid, 10® cells/ml must be present
at 96 hours.
As verification of the EDTA requirement, negative control tests in
media without EDTA failed to achieve the ASTM minimum growth
criterion (See results in Appendix D).
2
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B, pH Adjustment
In order to test Glyoxal , pH adjustment was necessary. The
analysis of this compound was more extensive than was originally
anticipated because of the tendency of the Glyoxal to cause a drop
in pH below the tolerable range of the test organism during the
course of the test.
During the first range finding test the pH of the 100 mg/L
concentration (the second highest) was above the lower limit of
6.0 for the green algae Selenastrum capricornutum. However-, the
pH of the 1000 mg/L concentration was below that limit (about 4.5).
Because the EC,, for that test fell between 100 and 1000 mg/L, it
was impossible to distinguish between pH caused toxicity and other
toxic effects. Accordingly, in compliance with ERL-C QA
requirements, the pH was adjusted in subsequent tests.
An initial adjustment to pH 7.5 failed to keep the pH within the
tolerance range for the duration of the test. Two more tests were
run in which the pH was adjusted oyer a longer period of time and
to a higher pH (9.5) to possibly "saturate" the buffering capacity
of the solution or to offset the drop below 6 until after the 96
hour exposure. We were hesitant to go ahead and run the definitive
test between 100 and 1000 mg/L not knowing at what point the pH
would fall below the lower limit and not knowing whether a
stabilized pH could be attained.
After the third attempt at stabilizing an adjusted pH above the
lower limit at the 1000 mg/L concentration with no success, we
decided to run the definitive test on an adjusted sample, fully
expecting that during testing some of the concentrations would fall
below the pH range of the algae. After testing, the pH of the
concentrations used in the determination of the EC#0 value were all
within the acceptable range for the algae (the data from
concentrations that were out of the acceptable range were not used
in the EC#9 determination), thus the pH had no influence on the EC90
value. Removing these data points was possible because the full
response range (no effect through complete inhibition) occurred at
concentrations that were within the pH range of the algae. Since
the sample was originally adjusted, however, the adjustment
solution may have influenced the ECS0 value.
No other compound required pH adjustment for a definitive test.
The highest two concentrations in the range-finding test of
phthalic anhydride were pH adjusted but because the toxicity for
that test fell below the adjusted concentrations no adjustment was
necessary for the definitive test
3
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C. Derivation of Algal Assay Results
1. Data Reduction
One ml samples were pipetted from the test flasks and counted on
an electronic particle counter (Coulter Counter, Model ZM). Counts
were multiplied by the appropriate dilution factor. These counts,
cells/ml, were then multiplied by the mean cell volume (MCV), and
a conversion factor (3.6 x 10~7) to get the mg/liter dry weight.
If a cell count for a negative control flask was 500 or greater
(minus the background) at a dilution factor of 200, the flask
contained >_ 10s cells/ml and was valid according to the proposed
New Standard Guide for Conducting Static Toxicity Tests With
Microalgse (ASTM E1218). The mg/L dry weight was calculated for
each flask and compared to the average mg/L dry weight of the three
control flasks after the inoculum had been subtracted in the
formula t/c, where t was the test cone entration yield in mg/L dry
weight and c was the control. This number was multiplied by 100 to
get the percent growth as compared to the control.
For example, in a test with an inoculum mg/L dry weight of 0.16,
a flask had a yield of 5.40 mg/L dry weight (minus inoculum = 5.24)
which was divided by the average contro1 yield of 32.23 mg/L dry
weight (minus inoculum = 32.07) to get 5.24/32.07 = 0.1634; this
number was multiplied by 100 to get 16.34 percent of control yield.
2. Statistical Analysis
After the cells per milliliter, milligrams per liter dry weight,
and percent effect were calculated using a Lotus spreadsheet
program, the median effect values were derived from a regression
analysis using the Statgraphics program (a commercial statistical
software package). The following is a brief explanation of the
methods used, and the reasoning those methods, for determining
toxicity test results.
Options for statistical analysis are many, and certainly other
approaches to data reduction could generate alternative
interpretations of the data.
The test concentration yields, expressed as a percent of the
control, were plotted against the toxicant concentrations in the
Statgraphics program . Regressions were performed using linear,
multiplicative, and exponential models on the percent of control
yield data. The model that had the highest r-squared value
(goodness of fit) was chosen from the options, and the EC„ was
calculated from that model.
4
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Fiducial limits1 (commonly mislabeled confidence limits) were
obtained mathematically using an in-house Lotus spreadsheet program
that calculates the limits from outputs generated from the
Statgraphics program.
a. Approach
As variables were entered into the Statgraphics program, asymptotic
portions were removed. Removal of these asymptotic portions
facilitated statistical analysis. ERL-C standard procedure
dictates removal of data sets that were either redundant because
they were equivalent to the control yield or showed complete
inhibition (all dead). All points associated with a particular
toxicant concentration were omitted when data was truncated. A11
concentrations above or below an omitted set were also omitted.
For example, if all concentrations up to and including 25 mg/L had
growth equivalent to the control, data for those concentrations
below 25 mg/L would be omitted. Similarly, if concentrations of
500 mg/L and over showed complete effect, all those greater than
500 mg/L would be omitted.
What constitutes "equivalent to the control" or "complete effect"
is often a subjective decision. Data truncation can profoundly
influence the estimated median effect. For this reason we present
all the raw data, in order to facilitate any recalculation desired.
To minimize interpretive decisions, it was originally decided not
to truncate the data at all for this project. However, due to the
1 Fiducial limits (intervals) are the 957. (two standard deviations) limit
on either side of a calculated number for the independent variable (the toxicant)
which lies along the horizontal axis. These are the toxicant amounts (limits)
between which (interval) there is a 957. probability that the 507. response will
occur.
Confidence limits (intervals) are the 957. (two standard deviations) limit on
either side of a calculated number for the dependent variable (the response)
which lies along the vertical axis. These are the response (as percent of
control yield) amounts (limits) between which (interval) there is a 957.
probability that (at that particular calculated toxicant amount) the response
will occur.
For example, an EC50 is 16.35 mg/1 with 957. fiducial limits of 10 and 23 mg/1.
There is a 957. probability that the calculated EC50 ( 507. response) of 16.35 mg/1
is between 10 and 23 mg/1. The confidence limits for that EC50 of 16.35 mg/1
may be 20 and 75. There is a 957. probability that at the toxicant level of 16.35
mg/1 the response is between 20 and 757..
5
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test design (the dilution scheme) of some tests, regression without
truncation was not optimum. The concentrations in the range-
finding tests were far apart relative to the "response window."
This caused the effective response area to be so small, median
estimates using all the points were at times anomalous.
Truncation was kept to an minimum in analyzing the data, and the
amount of truncation can be determined from Table 1 by looking at
the Degrees of Freedom (DOF) used in each ECst> calculation. The
degrees of freedom can be figured from the number of data points
minus one, so if an entry reads 14 DOF, that means that fifteen
flasks were used in data analysis, comprised of three replicates
of five different toxicant concentrations. The number of flasks
actually tested is the number of concentrations multiplied by the
number of replicates. If DOF + 1 is not equal to concentrations x
replicates, the data was truncated. A difference of 3 would
indicate one coneentration (three replicates) was omitted. The
actual data points used in the calculations can be seen in the
graphs in Appendix B. Again, raw data used in the regression
graphs is presented in Appendix C.
Three data points (one concentration, the highest one) were removed
for sodium dodecy1 benzenesu1fonate , methyl vinyl ketone and methyl
aery]ate. Removing the highest concentration when there is
"redundant death" is a common truncation practice. The highest
concentration for glyoxal was removed because the pH at that
toxicant level was below the tolerance range for the organism. All
other data points in the definitive tests were used.
IV. Chemical Analysis
ERL-C had no previous experience analyzing the specific organic
compounds tested in this project. The analyses we completed in-
house were on the original sample concentrations before testing
and on the final filtered solutions for each concentration. Some
sample levels were verifiable using gas or liquid chromatography,
others required verification by some other method.
The Chemical Analyses Summary (Table 2) presents the results for
the analyses completed. Analyses not performed include octylamine
and methyl vinyl ketone (the definitive test amounts were below the
analytical detection limit for our instruments), sodium
dodec y 1 ben zenesu 1 f orsate and tetra ethyl ammonium chloride (a
derivati2ation procedure to create an analyzable compound would be
required), glyoxal (a standard simply was not detectable with any
of our available instrumentation) , and butyl mercaptan (samples
were mistakenly disposed of before chemical analysis could
commence}.
6
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At present the unanalyzed test solutions (except for butyl
mercaptan) , both from before and after testing, are being held in
refrigerated storage at 4 degrees C. Additional resources are
needed to design methods to test these materials. A1ternatively,
samples could be forwarded to a designated laboratory for analysis.
V. Results
The ECS0's, fiducial limits, and selected test parameters associated
with the tests performed are presented in Table 1. Results from
both the range-finding and definitive tests are included in the
table for those materials on which both tests were run. Each entry
in the table has an accompanying analysis of variance (ANOVA) table
and regression graph, in Appendix B, that was used to generate the
calculated median effect value. The raw data used to generate the
graphs in Appendix B, including daily counts for the range-finding
tests, are in Appendix C.
Of the 11 chemicals tested, two (tetraethyl ammonium chloride and
butyl mercaptan) were judged to be relatively non-toxic to the
algae by virtue of the calculated EC90 exceeding 1000 mg/L. Three
chemicals were extremely toxic (ECse < 10 mg/L; methyl vinyl ketone,
octylamine, and phthalic anhydride); three were highly toxic (EC90
> 10 mg/L but < 100 mg/L; azosulfamide, methyl acrylate, and
nitrobenzene) ; and three were moderately toxic (EC#0 > 100 mg/L but
< 1000 mg/L; glyoxal, sodium dodecy1benzenesu1fonate, and
g1yc ido1).
VI. Discussion
Tetra ethyl ammonium chloride and butyl mercaptan did show toxicity
in range-finding tests at the highest concentration, but the
inhibition was in both cases less than 50"/. so no definitive tests
were run. An ECao of 1068 mg/L for butyl mercaptan and an EC90 of
1406 mg/L for tetra ethyl ammonium chloride are offered on the
Algal Assay Summary (Table 1); but these calculated median effect
estimates are extrapolations beyond the highest concentration, are
from single replicate tests, and have no fiducial intervals
associated with them. Because of this ambiguity, the toxicity for
these chemicals should be most properly described as "greater than
1000 mg/L" (>1000 mg/L). Since toxicity was observed, however; and
since the ECS0 value could fall quite close to 1000 mg/L if tested
with a more robust test design, the information was included.
The first entry for the range-finding test for phthalic anhydride
is quite dissimilar to the definitive test entry due to the fact
that the two highest concentrations (1000 and 100 mg/L) in the
range—finding test were pH adjusted while the lower four were not.
7
-------
These two high concentrations were the only ones out of the
organism's pH range. When all the data points were examined, two
distinct slopes could be seen. A toxic response was evident in the
lower unadjusted group (the second entry for the range-finding test
for that material), and the design of the definitive test was based
on that data. None of the range-finding test results should be
heavily relied upon due to the single replicate test design; in all
cases the results of a definitive test should be reported instead
of those from a range-finding test. The results of the range-
finding tests are included only to provide the data base on which
decisions about concentrations for the definitive test were made,
and to illustrate the correlation between the range-finding and
definitive tests.
All definitive test median effect estimates have fiducial limits
associated with them except for azosu1famide. The median effect
estimate for that compound had no upper fiducial limit due in part
to the fact that the EC50 fell relatively close to the highest
concentration (Statgraphics will not extrapolate a fiducial limit
beyond the highest coneentration); and in part because the second
highest concentration was not generally more toxic than the third.
The data look relatively sound (see ANOVA table and regression
graph in Appendix B), and an upper fiducial limit might be obtained
using another procedure.
S
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REFERENCES
Federal Register (Vol. 50; No. 188; Part 797; Sec. 797.1050, Algal
Acute Toxicity Test).
Greene, et al., Protocols for Short Term Toxicity Screening of
Hazardous Waste Sites. EPA/600/3-88/029. February, 1989.
Weber, et al., Short-Term Methods for Estimating the Chronic
Toxicity of Effluent and Receiving Waters to Freshwater Organisms,
Second Edition. EPA/600/4-89/001. March, 1989.
Standard Operating Procedures for ERL-C Bioassays. Site Assessment
Team, Environmental Research Laboratory - Corvallis, USEPA, 200 SW
35th St., Corvallis, OR 97330. 1989, unpublished.
New Standard Guide for Conducting Static Toxicity Tests With
Microalgae (proposed). ASTM E1218.
Statgraphics is a registered trademark of Statistical Graphics
Cor poration.
9
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ALSAL ASSkV SES'JJS SUHKhSY: CHEMICALS JESTED FOR THE OFFICE OF TOXIC 5U3STfiNCE3 (5TS)
DhTE: Os-Hsr-?C.
Table 1
Niiaber
fiiis-ber
HIGH
LOW
Decree:
CHEMISTRY
TEST
TEST
EC 50
LFL-
iiFL-
R-SSUASED
of
of
COMC.
COfsC.
of
:ool
NUriSEP
DATE
TYPE
(.55 ¦;L)
(sg/Li
Ug/L;
(R--2)
CCfiCS.
REPS,
iag/L)
(eg/Li
Freedom
OiVvXo i
GTP14bOO
04-03-35
D
143.96
0.00
343.59
32.29
5
j
1000
62.50
i < *
hi
GTN0960C
02-23-S9
F;
66.45
0.00
551.45
33.67
6
1
1000
0.01
Ssdiuf
07N18iC0
O5-0B-S9
D
70.27
45.11
95.42
9».72
5
3
250
15.63
til
dooec v'l ben zehe su! f o n ate
0TN1S600
05-01-35
F.
171,94
55.93
287.99
99.26
6
1
1000
0.01
5
Tet.-:=thy! a.*.a:niuc
OTN20SOO
0^-15-35
R
1405.9+
23.04
2733,76
71.05
6
1000
0.01
5
c-wo.-ide
rrtHsJli; anhydride
OTN23600
06-05-39
D
4,14
0,00
10.91
32.57
7
;
20
0.31
20
07N21600
05-23-St
F;
75.53
0.00
1353.33
53.03
6
1
1000
0.01
c
0,14m
0,06
0.35
75.73
i
1
10
0,01
*
rIC 2 u i Tc'.Cc
OTN25600
0 h-lk-Z-l
D
23.93
4.51
174.05
71.41
6
3
100
3.12
17
0TS25600
06-15-39
R
32,77
0.00
70.53
95. ii
6
1
1000
0.01
J4
B i ye: c I
QTM5601
06-26-3?
D
144.70
52,20
237.20
95.15
C
?
500
31.25
1-
OiWfcCl
C:-i9-3?
R
53.31
0,00
167.33
55.25
6
1
1000
0,01
Z
C'ct/is*ine
DTN25602
06-26-89
D
0.22
0.03
0.42
94.40
6
3
1
0.03
17
OTK25&C2
Oi-19-35
R
0.07
0.00
1.23
99,00
6
i
1000
0.01
S2
N; :r:fce' jere
OTN25iOO
07-24-3?
D
23.78
S. 58
7 - QC
e » L-
96.65
5
r
100
6.25
i*
OTiilicOC
07-17-8?
R
20.79
3,45
38.13
99.04
6
*.
looe
0,01
i-
fetny: v;ny! ketone
0TS3430S
05-25-35
D
0.13
0,05
0.21
85.93
r
.\
0.5
0.03
til
07N3iBCO
05-21-5?
R
0.12
0.00
0.61
59.33
6
1
1000
0,01
$2
McthyL acryisze
C"N37S0i
09-13-8?
d
18.57
3.74
33.41
94,35
6
3
100
3,13
i\i
0TN37301
09-12-39
R
15.53
0.00
46.73
96.03
0
i
i
1000
0.01
$4
r-utyi isrcapten
GTfs'37500
09-12-35
R
1065.3+
0.00
5473.24
92.44
6
i
1000
0,01
j
J Definitive
3 Range-Finding
LFL Lower Fiducial Licit
L'FL luper Fiducial L-sit
» Data was irinciirc U:e text!.
= ?id-jciai Lifits obtained sathesaticaliy.
tt Sutrci cf nng;-finding test (see iexii.
i EC'O ftbo^e high concentration isse test).
10
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CHEHIML RESULTS SUHffARY; CHEHICAL3 TESTES FOR THE OFFICE OF TOXIC SUBSTANCES (GTS) PATE: Fefc-90 TABLE 2
NQH1KAL ANALYSIS PRIOR ANALYSIS AFTER
CHEKI5TSY TEST TEST NUHBER CONCENTRATION TO TESTING TESTING ANALYTICAL
CHEMICAL MMBEJ? DATE TYPE* cf (RS/LJ
BD
0.0
BD
BD
0,0
31.25
BD
BD
0,0
BD
BD
0.0
HFLCI
Hitrooenrene DTN29c00 07-24-39
100
34,0
32.i
83.3
27.3
26.7
27,0
50
42,3
42.3
42.3
12.3
12.2
12.3
25
Tl ")
LI i L.
20.9
21.1
5.2
5.2
5.2
12.5
10,6
10.5
10,5
2.6
r e
L i fc"
2.5
6.25
5.0
4.9
4.7
1.0
1.1
i.i
HFLCi
Methyl
0TN37S01 09-18-89 D
6
100
67.7
63,3
68.0
BD
ED
0.0
acrylate
50'
42.6
40.6
41.6
FD
BD
0.!)
Lti
21.3
21.4
21.4
ED
BD
0.0
19 t,
Ui ^
10.3
10.2
10.2
BD
BD
0.0
6.25
6.0
6.1
6.1
BD
B9
0,0
3.125
3.1
3.2
0 i i
BD
BD
0.0
HPLCI
Butyl OTN37800 9-12-69
R
6
1000
541.0
331.0
436.0
wcaptan
100
BO
BD
0.0
10
BD
BD
0.0
1
BD
SD
0.0
0.1
BD
BD
C.O
0.01
BD
3D
0.0
Not
Avaliatle
HPLCt
I R ® Ranoe-finding; D = Definitive.
I High Perforeartce Liquid Chrosatograph
§ Ultraviolet Visible Spectroscopy
BD Below Detection Liirit. The final concentrations for aethyl acrylate and ail but the highest concentration
for glycidoi and butyl sercaptan could not be detected.
NA 'let available.
-------
APPENDIX A
ERL-C STANDARD OPERATING PROCEDURE FOR THE ALGAL ASSAY
-------
Hazardous Waste Assessment Team
N51-ES
ERL-C
Date i January 30, 19B9
T o:
Distribution
F rom:
Michael Bollman
Subject: Test Procedure -for OTS Chemicals
After careful examination of the correspondence between our lab
and the.Office of Toxic Substances (OTS) it has become evident that
OTS would.like us to follow procedures that differ markedly from
our standard operating procedures. This memo is to outline what
procedures we will follow for bioassays of all the OTS chemicals
which we test.
J. An algal range finding test is required on a logrithmic scale
from the saturation point of the compound in water down to the
detection level (e.g. 1000, 100, 10, 1.0, .1, .01 mg/Liter]. At
least 5 concentrations must be run. This test will be run using
only one replicate.
r. A]] testing must be done in glassware pre-conditioned with a
solution of the appropriate test concentration. To accomplish this
and tc provide enough solution for possible chemical analysis at
a later date (see #3 below), our procedure will be to mix up 300
mis of eac h test concentration in a 500 ml flask (exactly the same
procedure we do now but 6 times as much) the day before testing is
to go on (or the Friday before), and then on the day of testing 50
mis of each concentration is buretted into one of the three
replicate fiasks for each concentration, then rinsed through the
other two, and then discarded. Each replicate flask is then filled
with 50 mis of the proper concentration, and the remaining solution
from the 500 ml flask is poured into a 100 ml Nalgene bottle for
storage.
3. Samples of each concentration before and after testing are' to
be kept in frozen storage for possible future chemical analysis.
Beginning samples are obtained as above in #2, final samples are
obtained by combining all three replicates (after sampling for
testing on day four) and withdrawing approximate1y 100 mis.
In addi tion. after the final sample has been removed for storage,
an aliauot of the remaining pooled sample is centrifuged down (or
filtered) and the algal cells themselves saved in frozen storage
fc possible future chemical analysis.
-------
4. If the algal range-finding test shows toxicity (EC50) below the
lowest concentration tested above the highest concentration
tested, a definitive test will not be run. If an EC50 is obtained
within the bounds of the range-finding test a definitive test will
be run using 5 concentrations in which the ratio is 2.0 (e.g. 1,
2, 4, 8, 16 mg/liter). If a definitive test is run, the samples
held from the range-finding test for chemical analysis can be
discarded. If a definitive test is not run, they must be kept.
5. No chelating agents can be used in the media so we must mix up
same Algal Assay Media without EDTA. This media will be used for
all tests of OTS samples.
6. All stock solutions and serial dilutions must be made with
media, not R.O. water.
7. Both the range-finding and definitive tests must be counted
daily (i.e. 24, 4B, 72, and 96 hours). In addition, light intensity
and shaker oscillation must be recorded daily (this can be
incorporated into the sampling procedure). Tests should be sampled
at the same time each day, so a space will be made on the data
sheet to record this. To facilitate ease of these daily
observations, tests will be started on Monday and run through
Friday. Tentatively, testing for Glyoxal will begin on Monday,
February 27th.
Other parameters of testing will remain the same as usual; a 10,000
cell inoculum will be used, positive and negative controls set up
in triplicate, Lotus (algae program) and Statgraphics on the
results, subcultures of concentrations with counts under 1300
cells/ml at 1:20 dilution (actually the lowest concentration that
completely inhibited algal growth), etc.
In calculating the results, observations of cell size and number
in relation to biomass are to be included, and tests in which the
control yield is less than 1.5 x 10* must be repeated.
Distribution: Kapustka
Baune
DeWhitt
Nwosu
Smith
-------
Se I enastrum caprjcornutum
96-Hour Static
Bioassay
Chronic Toxicity
Introduction: Unicellular algae have been extensively used -for
toxicity bioassays. Their importance in oxygen evolution and as
primary producers stake them suitable environmental indicator
organisms. The procedure presented here is similar, with slight
variations, as those presented by Porcella (19B3) and, Horning and
Weber (1985). An inoculum (approximatel y 10,000 cells/ml) of
Selenastrum caprjcornutum is incubated for 96 hours with varying
concentrations of one of the following, diluted with algal assay
medium: surface water, ground water, or soil or sediment eluate.
A negative control of algal assay medium is used as a reference for
toxicity evaluations. Toxicity assessments are made by determining
an EC,6 value (the concentration causing inhibition of growth by 50
percent relative to measured growth in the control).
Room 274 - Environmenta1 Conditions for Culture and Test
Incubation
Environmental Chamber for Selenastrum caprjcornutum
Temperature: 24 +/- 2cC
Light: continuous, "cool white " fluorescent, 360-440 foot candles
(= 4 304 +/- 10*/. lux)
Shaker rate: 100 rpm +/- 107.
All references to room 274 indicate the above conditions.
Culture Methods: A sample of Selenastrum caprjcornutum culture
(ATCC Culture No. 22662) {which can be obtained from the American
Type Culture Collection) has been maintained at CERL by consecutive
aseptic transfer to 50 mis of liquid algal assay media (see AAM).
These transferred cultures are used to maintain a continuous stock
culture. Daily aseptic stock transfer consists of pipetting
approximate]y 1ml of a 3 to 7 day old stock culture, that has been
incubated in room 274, into each of two sterile, 125ml Erlenmeyer
flasks each containing 50mls of fresh AAfl. (Newly transferred
flasks are referred to as "isolates".) The grown stock culture is
taken from room 274, algal culture room, to lab 250 for isolation
transfer. A sterile 5ml pipet or an automatic pipet with sterile
tip and Eunsen burner are used to reduce contamination during
transfer. (Between 0.5 and 2.5mls can be used for daily transfer,
the lower the amount transferred, the longer the time period before
the culture can be used for testing purposes. The greater the
amount transferred the quicker the culture reaches nutrient
1imi tation.)
-------
Depending on the amount transferred, newly inoculated flasks
require 3 to, 7 days incubation, in room 274, before growth is
suf ficient to be used for • test er another stock trancfer. (When
lml is used consistently for daily stock transfer, growth for use
in toxicity tests or stock transfer is generally sufficient after
3 or 4 days.)
Monthly isolates from liquid culture or isolated plate colonies are
plated on approximately 1% AMM agar (see AAM agar below). A.flame
sterilized streaking loop is used to streak algae on the plate.
The plates are incubated on a shelf in room 274 until isolated
colonies can be seen en the plate. A single colony is then
transferred via a sterile streaking loop to 50rals of fresh liquid
AAM. Once this has grown, it is used for the daily isolate. All
isolate transfers, daily and monthly, are recorded in the Isolate
Book and Culture Log, located in room 274.
NUTRIENT STOCK SOLUTIONS FOR ALGAL ASSAY MEDIA
Spikes (1-5) for Liquid Aloal Assay Media (AAM)
Stock Spike 01 of 5
NaNQj 6.375g.
MgClt*6Ht0 3.041g.
CaCl,'2Hs0 1.103g.
Dissolve in 200 mis of R0 water and bring up to 250ml volume in
a volumetric flask.
Stock Spike 02 of 5
weigh out
dissolve in
R0 water
pipet out
H,B0s
MnClt*4Ht0
FeC 1.-6H.0
CoCl,'6H,0
NaMo0.*2H,0
NaEDTA'2H20
ZnCl,
0.464g.
1.038g.
0.399g.
0.357g.
1.Bl5g.
0.7 50g.
O.BlBg.
100 mis
100 mis
100 mis
1000 mis
1000 mis
100 mis
1000 mis
10 mis
10 mis
10 mis
1 ml
1 ml
10 mis
1 ml
Weigh out the amounts, dissolve separately in listed amounts of R0
water, pipet out the listed amounts into a 250 ml volumetric flask
and bring volume up to the 250 ml mark with RO water.
-------
Stock Spike *5 of 5
ng50/7H,D 3.675g.
Dissolve in 200 mis of RD water and bring up to 250ml volume in
a volumetric flask.
Stock Spike *4 of 5
K.HPO, 0.261g.
Dissolve in 200 mis of R0 Mater and bring up to 250ml volume in
a volumetric flask.
Stock Spike *5 of 5
NaHC03 3.750g .
Dissolve in 200 mis of RD water and bring up to 250ml volume in
a volumetric flask.
Procedure for Liquid AAM Preparation
The 250 ml stock solution spikes used for AAM should be prepared
quarterly. (The liquid AAM consists of 90 mis R0 water and 1 ml
of each of the 5 above mentioned spikes and the total volume
brought up to 1 liter volume with more R0 water.) Generally 16
liters are prepared at a time using a specified four liter
graduated cylinder. Each of the five stock solutions is filter
sterilized in to one of the RD water aliquots, using a 20cc syringe
filled to the 16cc mark, and a 0.2um Acrodisc filter disc. The
water and filter sterilized stock solutions are subsequently poured
into a prepared cubitainer. The pH of the total amount prepared
is adjusted to between 7 and 7.5 with a couple of drops of 1 N HC1.
An aliquot is submitted for chemistry analysis (approx. 75 mis) in
a labeled, 125 ml Nalgene bottle. A Sample Data Sheet is completed
and submitted with sample (See attached 'AAM Sample Data Sheet).
The AAM is stored in a cubitainer that has been rinsed with 107. HC1
then RD rinsed and labeled as AAM and with a chemistry number. The
chemistry number consists of the beginning ME. (the specific code
for media), the two digit week number of the year, and the 600
number for nutrients (see General S.O.P.). The cubitainer is
stored in the lab 250 refrigerator (approx. 4*C).
-------
Solid AAM Agar
Constituents
3.0g. of agar
250 mis of liquid AAM
The antibiotic spikes (below) are optional and are only used to
reduce contaminants associated with the algal cells. If these are
included they are not added until the above constituents have been
autoclaved.
0.5 ml of Penicillin G spike
0.5 ml of Chloramphenicol spike
0.5 ml of Streptomycin sulfate spike
0.25 ml of micronutrient spike
•Note: Only revive ATCC cultures en 1% AAM agar without
antibiotics.
Optional Antibiotic and Micronutrient Stock Spike Solutions
Penicillin G Spike
0.40g. penicillin G in lOOmls R0 water (»Bppm)
Chloramphenicol Spike
0.02g. chloramphenicol in lOOmls R0 water (»0.4ppm)
Streptomycin sulfate Spike
0.10g. streptomycin in lOOmls RO water (»2ppm)
Micronutrient Spike
Stock Spike 412 of 5 liquid AAM spike (lOOmls removed then filter
sterilized, see below)
Autoclave four reagent bottles at 121'C for 15 minutes and allow
to cool before filtering the antibiotic or micronutrient solutions
in to them. The above antibiotic and micronutrient spikes are
separately filter sterilized using lOOcc syringes and 0.2 um
Acrodisct.„. filter discs. These spikes should also be made up
quarterly and they are stored in the refrigerator in lab 250 with
the liquid AAM spikes.
Procedure for Solid AAM Agar
The AAM agar, for monthly isolates, is prepared by adding 3.0 grams
of agar to 250 mis of liquid AAM. In a 500 ml acid bottle, with the
cap loosely screwed, 250 oils of liquid AAM and 3.0g. agar (without
antibiotic and micronutrient spikes) are autoclaved for 15 min. at
121cC. This is allowed to cool slightly (not solidify) before the
plates are poured or the filter sterilised antibiotic and
micronutrient spikes are added (see Constituents). When antibiotic
spikes are added the media should be mixed well before the plates
are poured. Approximately 25 mis per petri plate is poured into
each of 10 sterile petri plates and allowed to cool and solidify.
These poured plates are then stored in the empty, plastic bag that
they came in. The bag of poured plates is kept in the refrigerator
in room 274 until a monthly isolate plate needs to be streaked.
-------
Ma teria 1 s
For a list of materials that are not standard laboratory equipment
and are specific to this test see the Selenastrum capricornutum 96-
Hour Static Chronic Toxicity Bioassay Protocol, located in the gray
notebook in office 2B9.
TEST PROCEDURE
Setting-up the Test
Preparation for this test takes place the day before the test is
to be Put-on. This consists of: labeling the glassware, filling
out the paper work, and acclimating the AAM diluent and alleged
toxicant to room temperature.
For a standard test, thirty-nine clean, sterilized and foam
plugged, 125ml Erlenmeyer flasks are necessary (see Dishwashing
5.O.P.). A standard test incorporates eleven concentrations at
the following intervals:
BO'/, toxicant- 60V.- 30*/.- 10V.- 6'/.- 3*/.- 17.- 0.6*/.- 0.3"/.- 0.1*/.- 0.017.
Each concentration is performed in three replicates. There are
also three positive and three negative controls.
(11 concentrations) (3 replicates) = 33 + 6 controls «= 39 flasks.
Each flask is labeled with the concentration and beginning at the
B07. concentrations and proceeding through the lower concentrations
the flasks are labeled one through thirty-three. The first flask
(B07. concentration, replicate number one) is marked with the chem.
number, and the test code (see General S.O.P.). The positive
controls are designated with the numbers 1,2, and 3. Each of these
three flasks has "pos." and "Zn*".
The negative controls are labeled "neg." 1,2, and 3. If more than
one test is put-on in a day, only one set of controls are needed.
The algal bioassay cover sheet and seven data sheets (7 data sheets
used in a standard test each of which contains results of two test
concentrations) can begin to be filled-out. The Test Code, Test
Mater, Chem. No., date sampled and date eluted can all be filled
in (see example cover sheet). The data sheets receive: Test Code,
Test Water, Chem. No., treatment, species, and concentration spikes
(see example data sheet). For a more complete explanation of sample
identification and designations, see General S.O.P..
-------
Both the AAM and test sample need to be removed from their cold
storage locations so that both equilibrate to room temperature
before the test is put-on. This prevents algal temperature shock
from occurring Mhen the inoculum is added. The test sample (i.e.
eluate, ground Mater, or surface water) and the AAM diluent'are
taken from the refrigerators (sample from vented refrigerator in
lab 250 or from 159C, and AAM from refrigerator in lab 250)* The
test sample is placed in • hood in lab 250/ The AAM is either left
in the cubitainer and put on a counter in lab 250 overnight, or is
dispensed into the flasks according to the dilution scheme on the
back of the cover sheet (see example cover sheet). The dispensing
of the AAM can be done the day before the test or the day the test
is put-on.
Putting-Dn the Test
Putting-on the test involves: dispensing the test sample and AAM
(AAM if not already dispensed); preparing, calculating, and
dispensing the inoculum; recording initial pH measurements of high
and low concentrations; checking test incubation conditions; and
completing the paper work.
The AAM i6 dispensed first using a 250ml buret labeled "AAM Only"
(or any clean buret), located in lab 250. The lower concentrations
require the additional use of 1ml and 5ml automatic pipets. These
are used to subtract small volumes that can not be measured as
accurately with the 250ml buret. It is not necessary to dispense
the AAM under a hood. The test sample is dispensed under a hood
in lab 250 from a clean 250ml buret and appropriate automatic
pipets for the lower concentrations (see example below).
V. concentration mis sample mis diluent (AAM)
eo 40 10
60 30 20
30 15 35
10 5 45
6 3 50-3.0«47
3 1.5 50-1.5*48.5
1 0.5 50
An extra IV. concentration is prepared in a medium size beaker and
used for the 0.1 and 0.01% concentrations.
0.6 0.3 50
0.3 0.15 50
0.1 5mls of V/. conc. 45
0.01 0.5ml of IV. conc. 50
-------
The negative and positive control flasks each receive 50 mis of
AAM. The positive control flasks also receive 1 ml each ZnCl,
spike solution. This solution is prepared as described below.
ZnCl, Stock Spike Solution
Weigh out 3.86 g. ZnCl, and place in a 500 ml volumetric -flask
before bringing the volume up to 500 mis Mith R0 Mater. Pipet one
ml of this solution into 1 L. volumetric flask and bring up to
1 L. volume Mith R0 Mater. This is the solution used for spiking
the positive control flasks. (One ml in 50 mis AAM produces
approximately a 74ug/L. 2n* solution.)
Measuring the Initial pH
Initial pH measurements are taken of the high (BOX) concentration
and low (0.01*/.) concent ration. The pH meter is calibrated before
readings are taken (see QA/ QC S.O.P.). The pH measurements are
read from a pH meter located under a hood in lab 250. These pH
values are recorded on the Cover Sheet.
INOCULUM
Inoculum Preparation
The 5e1enastrum capricornutum inoculum is prepared from a three to
seven day old stock culture obtained from the algal culture room,
room 274. In lab 250, the inoculum is prepared the day the test is
put-on, after the sample and diluent have been dispensed into the
125ml Erlenmeyer flasks. The stock culture is poured into a clean,
sterilized, foam plugged 500ml Erlenmeyer flask. This is diluted
Mith R0 Mater to around the 200 to 250 mark. Before this can be
counted, a IV. saline background count must be determined on the
Coulter Counter.
Coulter Counter Operation
The Coulter electronic particle counter needs to be turned on
fifteen minutes before counts can be made. Turn on the sampling
stand, the mean cell volume recorder, and the particle counter.
Change the mean cell volume recorder to corrected count and check
all settings with those listed on the index card taped to the
particle counter.
-------
Settings for ZM# 309B7 Coulter Particle Counter, specified for
«lg«e toxicity bioassay.
Full Scale
Current >700 x 1 Auto
Lower Threshold ¦ 5 Upper Threshold 99.9
KD ¦ 15.7
Dia." 4.76u Vol." 56.5u*
Manometer ¦ 500
This equipment requires training from qualified HWAT or Coulter
personnel and any questions a* to its operation should be checked
with the machine's operation manual located in the filing cabinet
in office 267 or with the proper staff members. Any operation
processes mentioned here are not intended to be inclusive of all
procedures that must be followed to correctly operate this
equipment. Monthly calibration is discussed in the QA/QC S.O.P.
1'/. Saline Preparation
First, a 207. NaCl solution is made by mixing 400g. NaCl with
1850mls of R0 water. Using a 200ml graduate cylinder, 200mls of
the 20*/. solution are mixed with 3800mls of R0 water to obtain the
IV. solution which is then filtered. The IV. saline is prepared and
vacuum filter sterilized four times through a 0.22um filter. The
filtered IV. saline is poured into designated saline bottles with
Oxford pipettor screw cap lids capable of dispensing lOmls
(generally set at the 9ml mark for Coulter Counter dilutions).
Background Counts
The Coulter Counter is "flushed" before determining a background
count. This is accomplished by first placing a tube with saline in
the Coulter sampling stand and raising the platform until the
electrode is Mell submersed into the saline solution. Close the
glass door and turn the lower knob to the fill position. Then,
turn the upper knob to the reset position and hold for
approximately 10 to 30 seconds before turning this knob to the
count position. Then immediately turn the lower knob back to the
closed position. Check the aperture for clogs and if necessary
remove debris with small brush. Check the "waste" and "fill" jars
in the back of the Coulter Counter (see Coulter Counter Operations
Manual). Using another Coulter tube with 9mls of saline, a
background count is determined. Place tube on stage and position
the electrode in the saline solution. Close the door. Turn the top
knob to the reset position until the counts read zero. Then turn
this knob to the count position and wait until the count is
completed. This is repeated five times. The five counts are
-------
averaged and this is recorded as the background count.
Counting the Stock Culture For Inoculum Use
Now, the stock culture to be used for the inoculum can be counted.
Nine mis of the IV. saline solution is dispensed into Coulter tubes.
One milliliter of RO water diluted stock culture is swirled and
then pipetted into a Coulter tube containing 9mls of 1'/. saline
(dispensed from saline bottle with Oxford 10ml capacity pipet lid
set at 9mls). This is mixed, by aspirating with the pipet, before
lml is pipetted out into another tube containing 9mls of saline,
so that a 1:100 dilution of stock culture has been achieved. The
Coulter Counter further dilutes this by Ii2, so that upon counting
a total dilution factor of 1t200 is reached. If the counts are
greater than 6,000, then more R0 water is added to the stock
solution, and another 1:200 dilution made and another count taken.
When the stock culture is dilute enough so that the counts are
between 4,000 and 6,000 (this ensures that the inoculum volume will
be close to 0.5ml, our standard inoculum volume] and the MCVs are
greater than 30, (our lower MCV limit) the stock culture is
acceptable •for inoculum use. Seven counts are taken and recorded
with their corresponding MCVs on the reverse side of the Cover
Sheet. The highest and lowest counts are not used to determine the
amount of inoculum to be added to each flask to create a 10,000
cells/ ml suspension (i.e. in 50ml test volume the inoculum must
distribute 500,000 cells per flask). The five intermediate counts
are entered into a program on the HP calculator to determine the
amount of stock culture to be inoculated into each flask, the
average MCV (Mean Cell Volume) and the milligrams/ liter dry weight
of algae (see Inoculum Calculator Instructions).
3. Enter background number, then press "STO" and then 6.
A. Enter test volume amount in mis (50) and press HST0", then 7.
Inoculum Calculator Instructions
(See Appendix for Programming Calculator),
1. Turn on Calculator
2. Press "f" (second function) and then "cl^ar
-------
5. MCVs and cell counts
Display
(1 of 5)
•nter
(2 of 5)
•nter
(3 of 5)
•nter
(4 of 5)
•nter
(5 of 5)
•nter
count and then .£+
MCV no. ENTER
count and then £+
MCV no. ENTER
count and then
count and then 2*+
MCV no. ENTER
count and then £+
MCV no. ENTER
MCV no. ENTER
3.000
2.000
4.000
1.000
5.000
6. Press "f" then A
The value displayed represents the volume (ml) of stock culture
corresponding to 500,000 cells (10,000 cells/ml) to be added to
each flask as an inoculum. Record this value and those generated
in steps 7 and 8 (below) in the inoculum portion of the Algae Cover
Sheet.
7. Press R/S
The value displayed is the average mean cell volume
6. Press R/S again
This value is the milligrams / liter dry weight algae in the
inocu1um.
•Note:
MCV x Dilution Factor x Counts x 3.6x10-7 ** mg./L. dry weight
Dispensing the Inoculum
The diluted stock culture that is to be used for inoculating the
test flasks is placed on a magnetic stir plate and a small 6tir
bar is placed in the culture flask. The stir plate is set on low,
number 2 setting. Using a 1ml adjustable pipet set to the volume
designated in step 6 (above), »ach test flask containing 50mls is
inoculated with 500,000 algae cells (i.e. 10,000 cells/ml).
All the flaBks are put onto a gray, Rubbermaid,.,,, cart and taken to
room 274 for incubation.
-------
Checking Environment*] Parameters in Rm 274
The lights are checked with a light neter located in room 274.
All general locations on the shaker table where flasks are to be
placed should be checked to make sure all areas are in the
acceptable light incubation range 360-440 foot candles. If the
light meter reads greater than 440, aluminum foil is placed on
areas of the bulbs until the range is acceptable. If the light
intensity is too low, then the aluminum foil is adjusted to allow
more light to reach the shaker table or the fluorescent bulbs are
changed and the lights are checked again. The shaker table is
turned on and the revolutions in a 15 second time span are counted.
This number is multiplied by 4 to get the rpms (revolutions per
minute). If this is not between 90 and 110, the rate is turned up
or down accordingly. The rpms are then checked again. Both the
light range and rpms of the shaker table are recorded in room 274
in the test incubation conditions book. The flasks are arranged
on the shaker table in numerical order from front to back, left to
right, and left to incubate for 96 hours.
The Test Preparation portion of the cover sheet is filled in with
the initials of the technician that executed each specific
procedure. The cover sheet and attached data sheets are hung on
the wall in lab 250 on a hook that corresponds to the day the test
is to be counted. Example: if the test is put-on Thursday, then
the paper work would be placed on the Monday hook (to be counted
96 hours 1ater}.
Counting the Test
Sampling
The test is counted after 96 hours of incubation in room 274. The
sampling procedure for counting the test is initiated by taking one
rack of Coulter tubes per test to be counted and a one milliliter
automatic pipet with disposable tips to room 274, The flasks are
examined for precipitates that may have formed during the test
incubation. If precipitates are present, the test must be counted
by chlorophyll & fluorescence (see procedure below) and electronic
particle counting. If a test contains precipitates and is also a
highly colored sample, so that fluorometry would be ineffective,
it must be counted directly using a hemocytometer and a microscope.
Tests containing precipitates are noted on their cover sheets.
A rack of Coulter tubes is used for each individual test that
is to be counted in a day. There are forty tubes in a rack and
usually thirty-three flasks per test. That leaves seven extra per
rack, six of which are reserved for the positive and negative
controls.
-------
The left, front tube is labeled with the chemistry number of the
test and is by convention designated as the number one position,
and is used for the number one flask (BOX concentration, replicated
number one). Each flask is swirled before sampling. One
milliliter is pipetted from this flask and dispensed into this
Coulter tube. Proceeding to flask number two. a one milliliter
aliquot is removed from the flask and pipetted into the second tube
in the first row. This process is repeated for each flask.
Aliquots from flask one through ten are located in the first row,
eleven through twenty in the second row and so on. Using this
convention, each concentration occupies ji designated spot in the
rack. For example, on a standard test the IV. concentration
replicates, flask numbers 19, 20 and 21, are located in the last
two positions of row two and the first position of row three in the
tube rack. - Pipet tips are changed after each test, as long as
sampling is from low algae.(less green) to high algae and the test
is to be terminated that day, and also between the negative and
positive controls. Obviously aberrant flasks within a test should
receive new tips. Flask numbers one and thirty-three (highest and
lowest concentrations) from each test, along with the Coulter
tubes, containing test sample, are taken via gray cart to lab 250
for counting and final pH measurements.
Counting
The Coulter particle counter should be turned on fifteen minutes
before its intended use. The electrode should be "flushed", the
settings checked, counts set on corrected, and a 17. saline
background count determined as explained in the preceding inoculum
section. The background count is recorded on each data sheet. Nine
milliliters of saline are dispensed into each tube containing one
milliliter of sample (i.e. 1:20 dilution upon counting).
The dilution factor (20, 200 or 2000) is recorded on the data sheet
as the concentrations are being counted. Three counts are Jtaken of
each replicate tube. The counts and corresponding MCVs are recorded
on the data sheet under the appropriate concentrations (see example
data sheet). If the counts are too high (above 35,000), a dilution
must be made. This is accomplished by pipetting one milliliter of
the 1:20 dilution into another tube and dispensing 9 mis of 17.
saline into it, thereby creating a 1»200 dilution. Further 10k
dilutions (to ls2000) may. be necessary at times. Once the counts
are sufficiently high enough at a particular concentration to
require additional diluting of 1(200, the rest of the succeeding
tubes (i.e. lower concentrations) Mill probably require a dilution
factor of 200 also. This is carried out by transferring one
milliliter of each 1»20 diluted tubes remaining to be counted into
corresponding positions on a parallel rack containing Coulter
tubes. The 9mls of 17. saline are not added until just prior to
being counted, so that settling does not occur causing erratic
results.
-------
The rest of the tubes are then counted and recorded on the data
sheet with the new 200 dilution factor noted in the appropriate
kite. The positive and negative controls arc counted and recorded
on a separately labeled data sheet. The positive control usually
only requires a 1:20 dilution where as the negative control
generally needs to be diluted 1:200. Flask number 1 (high
concentration) and flask number thirty-three (low concentration)
from each test are used to obtain final pH measurements. The pH
meter located in a hood in lab 250 is calibrated before these are
measured and recorded on the Cover Sheet. The technician
responsible for sampling and counting, places their initials in the
appropriate lines of the Cover Sheet (see example).
Subculture
If the counts at any given concentration are <25,000 cells/ml or
<0.45 mg/L dry weight (approximately <1500 counts at a dilution
factor of 20), then a subculture to determine lethality (algicidal
vs. algistatic results) must be performed. This is accomplished
by removing 0.5ml from each of the three replicates and placing
them into a 500ml Erlenmeyer flask containing 100ml of fresh AAM.
This is incubated for seven days in room 274. The flask and a data
sheet are labeled with the sample identification and "subculture"
concentration specification. The subculture data sheet is hung in
lab 250 on the hook labeled with the day it is to be counted. If
the yield after seven days is <25,000 cells/ml or <0.45 mg/L dry
weight, the solution is algicidal. This is calculated at present
on the APPLE Computer (training by HWAT personnel required).
Calculations
The data obtained from the algae bioassay toxicity test using the
Electronic Coulter Particle Counter is entered in to a Lotus 123
Program developed in house. This program is extensive and requires
training from HWAT personnel. The program calculates the mg/liter
dry weight in each flask using the equation:
dilution factor x cell counts X HCV X 3.6xl0"T «
caprjeornutum (mg/L dry weight)
Each flask is treated as a single data point. The test yield for
each flask is normalized and compared directly to the negative
control yield, example: B0*/. toxicant contains 20'/. AAM, so the
expected "no effect" response would be 20/i of the negative control
value. This is used to determine the percent effect. Each data
point is then transformed to give log percent effect and log
toxicant concentration, and these are input into a Stat Graphics
Program (training required) and an ECM value graphically generated.
-------
Chlorophyll ft. Fluorescence
Any test in Mhich a precipitate has formed in the test solution
must have a chlorophyll ft. fluorescence determination performed.
The fluorometer is located in lab 250. Six milliliters per
replicate are required to execute this procedure. A five ml
automatic pipet set at 3ml is used to remove two 3m1 aliquots from
each flask, Mhich are dispensed into a separate Coulter tube rack
in the same order and corresponding locations as described for
Coulter Counter sampling. Some technicians Mill combine the
sampling by removing two 3.5ml aliquots from each flask (totaling
seven mis, one of which is used for Coulter counting and six for
fluorometry determination). The fluorometer needs to be turned on
fifteen minutes before its intended use. This equipment requires
training from qualified HWAT personnel. Measurements of
chlorophyll & fluorescence are made after first adjusting the
fluorometer with a RO water blank. This is accomplished by placing
6 mis of RO water into an appropriate fluorometer test tube and
putting this tube into the fluorometer chamber. The "blank" knob
is then adjusted until the scale reads zero. The RO water is
discarded and 6 mis of test solution are placed into the
fluorometer test tube and this tube placed in the chamber. With the
range set on "auto", approximately thirty seconds are required
before the scale can be read. The machine will automatically cycle
through the series settings. If the reading is off scale and the
filter setting is X100, change the filter setting to XI by flipping
the pointer switch. If after another thirty seconds the reading
is still off scale, the sample must be diluted Ii2 with RO water
before the results can be read. Once the reading remains on scale;
the filter setting, series, upper scale reading, and dilution
factor (if any) are recorded on a test labeled fluorometry data
sheet (see fluorometry data sheet example). The series being used
is indicated by a red light next to the series numbers on the light
board located on the front panel of the instrument. The filter
setting is recorded from the pointer switch setting and the upper
scale reading is from the scale needle using the top scale. Filter
settings of IX in conjunction with series settings of min. sens,
or X3.16 are not to be used. One reading is taken and recorded for
each replicate (three per concentration). The RFU's (relative
fluorescence units) are calculated using the HWAT's group HP
calculator (see instructions below). This program is completed on
each replicate reading.
-------
RFU Calculator Instructions
1. Turn on calculator, press "g" then blue "clx" button.
2. Enter series # then press "STO", then press 1.
3. Enter filter setting # then press "STO" then press 2.
4. Enter upper scale reading #, then press "ENTER".
5. If dilution factor does not equal 1, key in dilution factor and
pres "x" (multipiication key).
6- Press "f" (second function) then "B".
7. Calculator stops and displays RFU, this is recorded on the
fluorometer data sheet.
B. If you want to change the series or the filter setting, key in
the new number and press STO 1 or STO 2 respectively and proceed
Kith step 4 for next calculation.
The RFU's are recorded on the fluorometry data sheet. The negative
control RFU's are averaged, then the percent effect is calculated
using the Algal D/E Calc program on the APPLE computer (training
is required). The transformed percent effect (TPE), log,0 toxicant,
and log,e effect are calculated on a LOTUS 123 spread sheet'from the
disk labeled: Algae, SOS test programs (training also required).
-------
Hemocytometer Counting
Samples that contain precipitates and are too dark or colored for
fluorometry, nust be counted with the use of m hemocytometer
counting chamber and a nicroseope. This allows for direct
examination of the cells. Appropriately place the cover slip over
the grids en the hemocytometer slide. Use a capillary pipet to
remove algal cells from a well nixed flask. Pipet an aliquot into
the hemocytometer slide channel chamber. Be sure that no air
bubbles remain in the chamber. Count the cells in the nine
diagonal grids under the microscope using high power (2X).
Calculate the cells/ml using the following equation:
cells/ml=
(# o-f cells in 9 grids) / (9 grids) X (25 grids total) X 10,000
Once the cells/ml are calculated the counts versus concentration
can be plotted to determine the EC«, value.
-------
APPENDIX—CALCULATOR PROGRAMMING INSTRUCTIONS
The calculations for the inoculum may be programmed onto a
hand held calculator for convenience. Our lab uses a Hewlett-
Packard 11C programmable calculator, to which the following
instructions apply.
Turn on calculator. Press g P/R to get into programming mode.
The PRGM annunciator Mill appear in the display. Press the keys
GTO, ., and 000 in sequence. This sets the calculator at line 000
without affecting other programs already in the calculator. If you
want to get rid of all previous programs in the*calculator, press
f CLEAR PRGM.
Key in the keystrokes listed in the KEYSTROKES column. The
calculator will display what is in the DISPLAY column. The
function of each set of keystrokes is explained in the
DOCUMENTATION column.
More instructions for programming may be found in the HP-11C
Owner's Handbook and Problem Solving Guide.
KEYSTROKES LINE NO DISPLAY DOCUMENTAT'N
f LBL A 001 42,21,11 Names and
defines start
of program
Finds mean of
cell cts
RecalIs bkgrd
Subtracts bkgrd
from eel 1 ct
Finds r»c i proc a 1
of cell ct
Multiplies
10,000
by reciprocal
of corrected
counts =
(cts-bkord)
g x 002 43,0
RCL 6 003 45,6
004 30
1/x 005 15
1 006 1
0 007 0
0 00B 0
0 009 0
0 010 0
x Oil 20
2
012
2
divides
-------
0
0
»
RCL 7
x
R/S
xXy
R/S
1
EEX
4
6
EEX
CHS
7
x
g RTN
013
014
015
016
017
016
019
020
021
022
023
024
025
026
027
02B
029
030
031
032
0
0
10
45,7
20
31
34
31
1
26
4
20
3
48
6
26
16
7
20
43,32
by
200
Recti1b test
vol
x test vol
Stops, displays
mis inoc
mean of MCVs
Stops, displays
MCV
Multiply
by
10,000
Multiply
by
3.6
x
10
-7
Displays mg dw
Ends program
-------
TEST CODE: ,
TEST WATER: $ar/~ /> j " /
CHEM NO. f>H/J m oo9
DATE SMPLED: Of I 5" ^9
DATE ILUTED: O 3/p"7 1ft
Sigh erne. cane.
INITIAL pH: *7. "7. /
FINAL pH:
£2.
?,5L
STOCK CULTURE DATA:
DAYS GROWTH: Mi
INOCULUM:
TEST PREPARATION:
TEST WATERS:
DILUTION MEDIA: fA)A
INOCULUM: "Xi/fJ
INOCULATED: ^UA/
SAMPLED: M ft
COUNTED: MP\
<,r\fY)a^CLirf\ r • ALGA
-jo*
/). i&iX
V? 3.
n. S~73
AAM BATCH M £//3 3 fOO
CELLS/ML
KG/L DRY HT.
HCV
MLS 3HCCULLK
TEST/CONTAINER VOLUME:
# REPLICATE FLASKS: J/ tOrtS & 3rcfS €#•
DILUTION RAKGE: % /o Q.O/ %>
NOTES: fa iten Ujt s//c Sfitr^k fj'fi /£-
-------
ST WtSR
-lEffla.Ti /•sn-h'.'-h/Off/Jr , BtaB:,
EH. «: pplhl30OO9
m.
DAyJ^oVIctounoI^
12?
COUNTS I MCV 1
COUNTS
MCV I COUNTS
hhhhmhh
rmmLibkci
wimmn
H
I I I I M
H
u
I
H
¦ I
ai^iiftuir'i^iLirsinrii^iBiinRgir'ig
anizEnHB m utmnmufin wi^smnrnvm
nwE&mmvmwwLn&mmnwxiKsaimvak
II 1 1 II 1 M
i
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1
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f 1 1 1 1 II 1 1
4H4.
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,
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1
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1
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»
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2
1
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1 II 1 II 1 1 1
1
2
1
1
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-------
Loh as'o
SAMPLE DATA
Location Notes: —.
's initJa/5 ——assau rncd\uyn
Rnrorrlnr ^ '
Recorder
mo day* Jajc AAA\
I 11 II I I yrcpArt<
Dmn
RocnlvBd
Date
Oulpul
13 T
a X
1ci
l£ q'
Origin
Code
Adjustment |
Lab. No.
WV No.
Sample
Description
in
b
_j
X
E
n
2
3
-------
Ifest Code: Test Ifater:
her.. Mo.: ^technician: Date: Species
sxicant
Replicate
Series
Setting
Filter
Setting
Upper Scale
Reading
Dilution
of Saqple
Calc. EFU'
^ •
1
V
3
'ofi
1
V
fi U
1
30
I
10
l
V
1
t
fi
n.
1
1
3
V
.
1
1
V
3
1
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P
V
3
1
3
V
3
»
1
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V
3
1
V
V
Q
1
7
L /
*v
-------
APPENDIX B
ANOVA TABLES AND REGRESSION GRAPHS
-------
jcegressi or s
- L'i: 'if.' i a: r Zlf ; '• I - VF
®eptrae"t vsri &cie : ; IPC? ft:CTfti4£C»0. OTI ririefender.-t variable: 3 DJDF ft:0?h.14£00.
Standard T Frot.
?5:irT-r: I s 1 i - a -t e Error Value Level
5.31135 0.357054 13.375c 1.04E2E-7
?.354!iE-3 1.37527E-3 -6.8155 4.649431-5
:! cit
Analysis of Variance
Scarce
Sjt. of Squares
»f
Heart Square F-Eatio Profc.
Level
f-.c-de 3
29.731805
1
29.731805 46.455137
100005
Errcr
6.4001114
10
.6400111
Total (Co:r.)
36.131917
11
Ccrrel atici Coefficient = -0,907121
F-squarfd = 82.29 percent
S1nd. Error of Eft. B 0.8000!'7
tirz:r.:' c! 3 IV,} A:C7;i4fX.CTkM 14:•!«* c.r, 3 K3f P:C7-i4c-X'.CTnlTHf-A
! ! ~ i i : i i i i - r~~i • i i i 1 1 : I i i i i 1 !
\ ! ' J
j \ \ i
— \ • -!
\
MOF fl: 0TA14 6CO. OTfirTl4t^CO,
- j&fyA/miif Terr
^ 0^' & ) *f/t-
-------
Si"r. H'iiyiis "cdf. •
X a 9*f-> a*fcX;
• h ¦
:.,Jer.t va:i at'.e'• C7h14c'00^BHI
prpe'ndent variable: A.'0TA146OC.OTSTC'9t
Standard
te: l£t:ir.ate Irrcr
T
Value
Prot.
Level
t ej-. 4.3::-E5 0.451032
-c.l45cl-r 1.099261-3
9.57991
-5.55445
6.63435E-4
5.009581-3
Analysis of Variance
•-e Suit of Squares Df
30.420143 1
3.8877722 4
Hean Square
30.420143
.9719430
F-Iatio Prob. Level
31.298278 .00501
iCcrr.)
34.307916
:f.a*.ion Coefficient = -0.941637
?- Irrer of 1st. = 0.9S5872
i-
i
i-
K
r
RfSr«Jicr. of fis0TO14WC.01TJ.096X or, *01514600. OlMtSWO
i I 1
i . i
T
"i ! r
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4 '6 8
*OTO146OO.OTFTO96C0 ( 66.4774,50)
72S?—
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(o -ft*)
10
(X 100)
-------
:t£5:cr. Analysis -¦¦¦(&¦: jti: V = a*b>
eide^t variatie: P:I7h1?s-;0.|
i^Fendent variatie: h:OTh1SdOC.CThTISs
ar?:?:
5t aniard
Error
T
Va i up
Prob.
Levei
e:: if
Fe
4 m Z i A
.< M
h, 0 i 5 :¦ 7
0.C564S3
25,3474
-13,3S72
2.0S4i3E-iv
1.03771E-7
Analysis of Variance
r re
Sum of Sijares
Df
Kta" Square
T-Fati o
Frot. Level
tl
12043.250
1
12043.250
179.218
.00000
-•
671.967f9
10
67.15875
al C Corr. i
12715.236
rela'icr, Coefii ci er.: = -0.973217
.a. Error of Est. = 1.19749
'nre::-::-' of P:";!?:•>?.c. tCT-Tir^-v
i- \ •
t-.
I 4—
r
.t
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1—
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V i
N.v
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9: 9C 120
A: 0TG1 StX\ OTAT: ttOO (70 2£5
SoDjurt Thenar^ 'BexT&+?_suc?VAJ47^r
V&?wr/t&' TS=5T
70.3 /^/
150
(yt-KO
-------
TwHOtA/*- 7fe7"; SZ>B£
fgression Analysis Y ¦ ti»(a»bX)
^=!ggg^"^ 1 —^—¦ I ¦ ¦ !¦¦ II .i i n. -
"tpendent variable erendtnt variabjtt H0TA18600.0TlTi86 ^VA/f
Standard T Prob.
£araneter Kstiiate Irror Value Level
itercejt 4.651? 0.0760193 61.1911 4.271961-7
Slope -4.300441-3 1.832751-4 -23.2111 2.041791-9
iance
mrc# Sub of Stuarts Pt Mean Square f-Iatio Prob. level
¦del 14.67531 1 14.67531 538.75652 .00002
Irror .110441B 4 .0276104
tal (Corr.) 14.98574B 1gP'rr~"*
'"irrelation Coefficient ¦ -0.996308 ^;»|uaiJ8 ¦*.'86 wriw't y
¦nd. Error of 1st. > 0.166164
£:0TA18600.OTttl8600 on H0TA18600.OTSTl8600
150
120
D
t
0
T
A
o
(X 100)
/7Z ^
HOTftl6600.07*116600
72=57"
-------
i 5 s;:' H'.i, us; s
e^der.t variable: HiCTHSOtOOl
dependent variable! ft:OTft20e>CO. 07FT20s
V
Stsrdard
T
Prcb.
£ * It *
Est s - o-. e
Error
Vai ue
level
i:: e i:
4.7??"i
0.07710&2
£¦1. 4e5?
4.195041-7
r f
-5.cr?3cZ-4
1.873241-4
-3.13284
0.0350885
Analysis of Variance
i r r f
Sum of
Squares I>f
Mean Square
F-Rstio Prob. Level
i?:
2757928 1
.27B7925
9.8147039 ,03509
c r
1136225 4
.0284056
¦' Cc-rr.)
.3924153
¦: flow or. Coefficient = -0. E423S4
¦•a. Irror ci 1st. = 0.16854
14!
Hgrwior of 0T;S.::-V..C7I.Jon A!CT^L'OvX.C17 72?cX
i ¦ I
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1
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c
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n
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A: 0TA20W0. 0TET20SOJ (j. 405913,50)
e77rYu dtHcTCiP^
R/bu*€'~'F>*W- TZZT"
ecSo =
(n*0
10
o. i00>
-------
Secession Analysis
i c exp(a+bX]
Dependent variable:
tIndewndent variablt: t:0TA236OO.DTAT236
Parameter
Estimate
Standard
Irror
T
Value
Prob.
Level
Intercept
SI ope
4.46115
-0.132638
0.120311
0.0137838
37.08
-9.62279
9.741961-9
Analysis of Variance
Source
Hodel
Irror
Sua of Stuarts
16.269709
3.338348
tf Mean Square T-Iatio Prob. Level
1 16.269709 92.998050 .00000
19 .175703
Total (Corr.)
19.608057
Correlation Coefficient « -0.910904
Stnd. Error of Est. » 0.419169
Agression of *:07A23600.0TAK23600 on D:0Tft236OO.OTfiT23SOO
0
T
A
2
3
6
0
0
0
T
A
F
t*
3
6
0
0
8 12 16
&:0TA23600.0TAT23600 <4.14,50)
1^7?///77I 'e~ 7^57^
(p-n)
-------
ifgression
Analysis -3^m
H^^^odel
Y « txp(a+bX)
Depended
var i abl e:
BfPendent variable: P:0TA236O0.0TAJT23
Standard
T
Prob.
Parameter
Isti mate
Irror
Val ue
Level
intercept
4.10427
0.472631
8.68383
9.679321-4
SI ore
-2.44809E-3
1.15191-3
-2.12526
0.100744
Analysis of Variance
Source
Model
Irror
Sum of Squares Df
4.6205259 1
4.2690409 4
Mean Square
4.8205259
1.0672602
F-Iatio
4.5167297
Prob.
Level
.10074
Total (Corr.)
9.0895666
Correlation Coefficient c -0.728242
Stnd. Irror of 1st. » 1.03308
Regression of D:0TA236OO.0TARR236OC on H:OTA23600.0TfiKT23600
2f/j
D 160
6
J
A
£ 120
o
o
6
T
jj 80
S
2
3
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0
0 40
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1 '1
1 '
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1 1 1
1 1
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-
i...
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T I"""!'
4 1.
J
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4 6 8
t: OTA23600.CTAIT23600 (79.5394,50)
10
(X 100)
Jfrrrfct-iC
ec6: - ^ 5 ^
-------
"Ten fir.oJys;s - r>; rm i cat i vt ir.:-3rii s
r.t variables 1 ftiPTft236C»O.OTIndeBPr.rfpni variable: 1 WO? ft!OTft236CO.
Standard T Prob.
t-iixiie Irror Value level
2. fi73S 0.0842333 34.6415 0.0133723
-0. J13425 0.02S3365 -18.2957 0.0347616
7;.e Intercept is equal to Los a.
Analysis of Variar.re
Sum ef Squares Df Hear. Square F-Eatio Prob. Level
2.850C4 1 2.85004 334.73124 .03475
.0035144 1 .0085144
err.)
2.8535572
a*.i:n Cc-efficient « -0.93S51
rcr cf £si. B C.0522736
K-ggjared s 55.70 percent
JksrMsicn cf 1 Ir.Zr AtOTMSeOC.OTVmiMC or, 1 JFCP ft:CTM:£-X>.07JP.T21£>:
S j i I I j I I
I I I
t I i
J
_ \
X2 0.4 0.6 o.e
1 LROF A:0TC23f0C-.0rdXI2:«iy u7
Tfo/tyujc. fyHy&VOc
-------
Dependent variable:
dependent variable: D:OT&25600.0TfiI'T25
* Standard T Prob.
Parametpr Istimate Irror Value Level
Intercept* 4.83465 0.13475£ 35.87? 0
SiOFP -0.274202 0.0433777 -6.32127 1.016341-5
* NCTI; The Intercept is **ual to Log a.
Analysis of Variance
Source
Model
Irror
Sun of Squares ®f Mtan Square F-Iatio Prob. Level
1.897746 1 1.697746 39.958411 .00001
.759888 16 .047493
Total (Corr.)
2.657634
Correlation Coefficient * -0.845029
Str.d. Irror1 of 1st. = 0.217929
Regression of D:OTfi25600.0TftDK25600 on D:(JTfl25600.0TftI>T25600
10?
0
T
A
2
c
6
0
0
b
T
P
*
2
5
6
0
0
4?
27
I 1 1
J-
-Is \
1 1 1
1 1 1
111
1 _
-. \
•- \ ¦
— \ • .
u \ ¦
\
1
•
-
•
-
- — -
1 1 1
1
f"T--
...I 1 1
1 1 1
III
20
/bogau=tfzS;£c-
40 60 80
P: 0TA25600. 0TA6T256OO (28.9266,30)
100
-------
-ion Analysis
— del s V = expC a+bX)
frit varia'rit: -5 ThKI A I Independent, variable!. -5 TfiKI A'OTA256CO
standard T Fret.
:er l5tj*.ate Irror Value Level
Hi 4.41165 0.084455 52.2179 '1.946821-5'
-C.CI*2437 1.679671-3 -8.10974 3.91895E-3
Analysis of Variance
Suit, ef Squares
1.773422
.0808946
It Hear Square
1 1.773422
3 .0269649
F-latio
65.767887
Prob. Level
.00392
'.SIT. )
1.6543165
itior. Coefficient » -0.977944
Irrcr o: 1st. = 0.16421
•egress: c of -5 TAH A:07»2560C..07<»F.25&K> or, -5 TAXI fls07A25600.0TWT25fr»
i—i—i—r
i
*-
V.
• I t • i
I
I
1
J
•- ¦
i
• ¦¦ ¦
X
i
.. i
i-
i
i
:
F-
b
u
! I ' I ! 1 I
i ! i i i I i i i
• H
1 , , , "
20 40 60 80
-5 TAKE A:OTA25600.0TAPT256
-------
iegressicn Analysis
ode]• Y * txr
Dependent
V
abi e:^H9B
ndependent variable: D:0TA2S6O1.0TADT25
Standard T
Prob.
Pa:arret e:
Isti mate
Irror Value
Level
Intercert
5.37624
0.163577 32.8666
6.727951-14
SI c p e
-0.0101191
6.33841-4 -15.9647
6.383541-10
Analysis of Variance
Source
Model
Irror
Sum of Squares tt
~4.63819 1
2.276612 13
Mean Square
44.63819
.175139
F-tatio
254.67235
Prob. level
.00000
Total (Corr.)
46.915000
Correlation Coefficient * -0.975433
Stna. Error of 1st. = 0.416497
degression of D:0Tfi25601.0TWR256Ol or, D: 0TA256C1. 0TMT256C1
IK
120
' \ ' ¦¦ 1
;,v\
1.1 11
1 1 1 1
| 1 1 1 1 | 1 1 1 1
!
-
•
. \ •
\
W- "
-
-
V
\
a —
1 1
,,
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1 1
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-
1 1 J 1 U ¦¦¦•I1', i|'.„J.ii!i'iiiiid
100
200 300 400
#:0TA256Oi;OTf»T256Ol (144.6957,50)
500
ec^ /V
-------
regression Analysis
noaei: Y » exp(a+bX)
>ependent variable!
I Independent variable: DsOTA25601.0TAJT25
'arameter
Standard T
Estjmate Irror Value
Prob.
Level
intercept
>] ope
4.64588 0.238112 19.5113
-0.0137661 5.60329E-4 -23.7212
4.068511-5
1.872741-5
Analysis of Variance
Source
Model
rror
Sun of Squares Pf Mean Square
152.42646 1 152.42646
1.0835473 4 .2708868
F-Iatio Prob. Level
562.69425 .00002
Total (Corr.)
153.51001
orrelation Coeffioient ¦ -0.996465
Stnd. Irror of 1st. > 0.520468
100
ion of D:0TA256O1.0TAM25601 on D:0Tft256O1.0TAFT256Oi
-j—I—I—I—j—I—I—I—|—I—I—I—|—I—I—T
iiu*J L
J I L
1
J I L
10
(X 100)
4 6 8
U0TA256O1.OTAFT25601 ($3.3092,50)
^3.3 ^
(<£>- '7")
-------
degression Analysis
odel! Y = *xp( a+bX)
dependent variable:
Independent variable: r:0TA256O2.0TADT25
V
Fa:aneter
Isti mate
Standard
Irrcr
T
Ual ue
Prob.
Level
J nterceyt
SI ope
4.76897
-3.82159
0.109725
0.232712
43.463
-16.422
0
1.950241-11
Analysis of Variance
Source
Model
Irror
Sum of
Squares Pf
30.10368 1
1.766024 16
Mean Square
30.10368
.111627
F-latio Prob. Level
269.68220 .00000
Total (Corr.)
31.889706
Correlation Coefficient * -0.971593
Stnd. Error of 1st. = 0.334105
Recession of t:0TA256O2.CTft5F25602 on p:0TA256O2.0TAPT256-C2
/A
?FF/A//r-/vc'
0.4 0.6 o.e
J:0TA256O2.0TACT256O2 ( 0,2242,50)
£*S6~ *?/-
/, oz - .*&-)
-------
lent varsatle: -3 TAKE ft1
'.dependent variable: .-3 TAKE A:QTh25602
Standard T Prcfc.
ter Estimate Error Value Level
rzeit 4.5i:t5 0.619451 5.9926 0.105264
-14.0476 1.41222 -9.9472 0.0637856
Analysis of Variance
Sun, of Squares Df Mean Square f-Katio Prob. Level
116.28277 1 118.26277 98.94671 .06379
1.1954189 1 1.1954189
(Corr.)
119.47819
atjon Coefficient » -0.994935
Error of Est. = 1.09335
iejr«sitr of -3 Tfitt A:CTA25602.CTAWi25602 on -3 IfiXI flsCTR25602.0TWfT25602
p-
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0.2 0.4 0.6 0.8
-3 TAXI A:OTft25602.0TAFT256<}^0711i50)
*$/*¦
(***')
-------
¦ei er,ae"'"t vana'tie: fi:D~H25600. 07IiF.29600In(ieperident variable: A: 0 TPi 2 5 c- C'0. 071-7256
Standard T fret,
1st irate Error Value Level
r.tercfi: 5.155;'i 0.143295 36.2 Ele 1.8S736E-14
;:M -0.054127 2.77625E-3 -19.4564 5.23S72E-11
Analyst5 0/ Variance
ojree
Sum of Squares
H
Mean Square
F-iatio
Frob. Level
led?;
51.08725
1
51.06725
380.11066
.00000
r:c:
1.747212
13
.134401
"ciaj (Ccrr.) 52.83445" ^4
or re!at ion Coefficient = -0.933326 ' I-iiuirtd * 96.69 Mrcent
¦ir.d. Error of Est. = 0.366607
of Q:?Tt,25oX'..CTI'>2V:o:.' or. P:0Tfc256X.OTDT256CO
A:0TA2560C.0TtT25b0O
Mr7ic&?jjje2j£~
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E-C& - 23. $ tj/i.
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rien Analysis -
models K e exjCa*t.X>
fnt variat:*: -5 TAXI Afl
[.Slndepender.t variable: -5 TAKE A:GTfi29600
Estirade
Standard
Error
. T_ .
Value
Frob.
Level
}•
4.8554 5
-0.0453765
0.116159
2.58527E-3
41.7855
-17.552
3.01635E-5
4.03126E-4
Analysis of Variance
Sum of Squares Df Hear Square F-Satio Prob. Level
15.71436 1 15.71436 306.07180 .00040
.1530262 3 .0510087
Corr.)
15.867383
Aion Coefficient » -0.995166
Irror of 1st. » C.225851
Itsrjsr.sr. of -5 TAKE fi:0?fc2S600.0TM52963J or -5 Tfltt d:CTA2960C-.OTRT29€OC^
! 3 i 1 I I i i ~i 1 i r
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-5 TAKE A:OTA29600.0TF.T2960p2Ci>?Sl8f50;
fyn&e'-rrW"' 7&zt~
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100
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:ejress;or, Analysis -"Linear model! Y = a+b*
't^erde'i' vtriatie! 3 PrOr A:CTh34800. OTindependert variable; 3 I»F 0 F A:0ThJ4?C'C,
Standard T Prob.
I5tiirate .Irror Value Level
rte::e;: 114.752 8.078E6 14.204 5.59762I-E
:!cf? -503.321 55.?52e -6.95705 4,191541-c
Analysis oi" Var
i anee
5o*r:e Sum of Squares Df
-cde I 21620.074 1
Cr:c: 2676.63?£ 10
Mean Square
21620.074
267.6640
F-Iatio Pr?t.
80.767
Level
.00000
ret*; < Corr.) 24296.914 , 11
^crrel ati o~ Coeff i ri e-rt = -0.943307
E4.r,a. Irrcr ;.f 1st. = 16.3611
n-Jiuirid «
88.98 ptrcrnt
tartiuc* oi Z SKI ftsCTi?4?X:.0TI.?34SCC- or. 3 MOF ft:6Ta3480C.OTMKC
t':
0
0
0.05
0.15
0.2
0.25
3 BROF A:OTft34BOO.0TTT34&>^o. 1292,50')
tfzTTrti- tern**
fie-fy hi T/tr'S / «>r
P/?cP/~eP tcrua..
= -
(.t$- , z / )
-------
-r = ;or, Analysis
>:ae;: I ¦ exu a»CA,
•n't variable: -2 TAKE ftl
independent variable: -3 TAKI A:0TA3480C
:e:
Esi: nsa-t e
Standard
Irror
T
Value
Prok.
Level
4 .Til "A
-£.54555
C.156842
0.27C297
30.1383
¦24.2163
0.0211151
0.026274
Analysis of Variance
:e
Sur, of Squares
25.68105
.0437924
It Mean Square f-Fatio Frofc. Level
1 25.68105 586.42783 .02627
1 .0437924
' Ccrr.)
25.724846
itior. Coefficient « -0.999148
Irror of 1st. & 0.209266
Serene- of -3 TAKI A:CT4^X.07j>FJ4?00 0r -3 TAXI A:0Th34&>D.0TPT348X
m—r
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ZZse" -/Z
(6-.it)
-------
refr;:-" fl*.a."ss:s
/iiode): Y * txF'a+bX)
E'3f; vs.-: a tie; £:VH|
J F 375 T^O I ride Fende r. t variable-' A:C7A37&00. 07ST37S
Standard
T
Fret.
B^.eier Estimate
Error
Val ue
Level
erceft 4.55552
0.036957
123.977
2.53S65I-8
Tt -6.304551-4
9.01451E-5
-6.99362
2.19941-3
Analysis of Variance
rce
Sur of Squares
Pt
Mean Square
J-JEatio Prob. level
i
.319707
1
.319707
46.913489 .00220
CT
.0261447
4
.0065362
?S5T6SSior. cf a: C-T^?-7&-X;. OTT F37K-I- or. fi>0TA3?G%. OTI.T^&X
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d.ObJjEi', 50)
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E5J I" 'W,'|6J as J S - il-;a: a;.' 1
.'it variable: 3 IrC? ft:?7A37S01.0T1 ndependent variable! 3 MO? A!0TA37S01.
Standard T Frob.
£ stinsate Error Value level
53.7765 2. 75626 =29.3055 2.930531-13
-1.65706 0.106301' -15.5154 9.0J352S-10
Analysis
of Variance
S'jm cf Squares
Pf
Mean Square
f-Batio
Frcb. Level
11563.S3?
1
11963.635
243.726
.OOOJO
646.35634
13
43.71572
iCzrr.) 12615.136 14
::or. Cceffirier.t = -0.974045 F-gauared g 94.PS gerce^t
Irr?r ef Est. " 7.05)22
}ii7nsiz-n :f 3 KC? A:CTSj7SCl,CTK3?8Cl en 3 WD? A807ftS?K>i.G71>T3?K'l
TTj i i i i j 1 i i T j i i i I i i i i i j
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model: Y = exf< a+bX)
¦ fjf
• cT i ot . f • 1 t
!"! ndepender.t variable: i ISO? A:07A37EC1.
¦a-c.t:
Est i mate
Standard
Irrcr
T
Val ue
Prob.
Level
:e::ejt
4.85209
-0.063103?
0.332781
7.40391-3
14.7006
-8.5230b
6.827691-4
3.392891-3
Analysis of Variance
ie 1
Sum of Squares
30.391136
1.2550959
Df Mean Square F-Katio Prob. Level
1 30.391136 72.6425S2 .00339
3 .4183653
;a; s'Ccr:.)
31.646222
¦re lot: or- Coefficient = -C. 979965
¦.a. Irrcr of Ei". = 0.646512
hsressic. of 1 1*0? A:9T&S?»l.CTB.?7«i or, 1 I>P.3F A:0TA37&?!.CTET37&C-3.
40 60 60
1 MOP A:OW3?801.0TST37801ii5i53ii5c.;;
/frA/s-e-r)'U&P&' 7&>T~
(o-&)
-------
'V i ^ ". V . *
APPENDIX C
RAW DATA
-------
TEST CODE:
TEST HATER: (rlyn V a /
CHEM NO. . n-TP H boo
DATE SAMPLED: '
unit uvikw.
high core.
INITIAL OH: 7,3
low oonc.
7. *
FINAL dH: # 9 T'
7/
STOCK CULTURE DATA:
INOCULUM:
MEDIA: AAM
Selenastrun
ALGA
DAYS GROWTH: 03
^ /O^
CELLS/KL
J
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est gg n-r-nve?r?s caw. wmer a-r/» ^oi?
SHARER MtMER ?// TEST raiVKLAL
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TEST VPJTR:
C^ola Jl/n XQ /
TESTCDIE: ^ CTn Vn ? Z"?4
Selenastrm
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TEST CODE: UT0~L
TEST WATER:
DATE SAMPLED:
DATE ELUTED:
bigh oanc.
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notes: *T&zr j - ?fa>£cZuie-
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TEST WATERS: ^
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NOTES: rZlJjA^c
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TEST CODE: £TOlfL#lB>
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APPENDIX D
EDTA TEST RESULTS
-------
FOLLOW UP SI DE-BY-SIDE ALGAL COMPARISON TEST OP NEGATIVE CONTROLS
k]TH AND W1THDUT EDTA AND WITH AND WITHOUT TRACE CUPPER
Tr.is test was performeo in six replicates each and three counts
were takers o* each flask.
(6 flasks each x 3 counts each = IB counts per test)
ALL VALUES EXPRESSED
IN CELLS / ML
TEST 1
TEST2
TEST3
TEST £
AAfi
AAM
AAT1
AAf.
W/ EDTA
W/0 EDTA
W/ EDTA
W/D EETA
W/ CU
W/ CU
W/0 CU
W/0 CU
I. 23B0200
18680
2260400
21540
2. 2409400
19360
2299300
25120
^^ v wD V s'%'
1B400
2348600
19220
4. 37254<:0
20460
2101 BOO
26320
5. 2916400
2054C
2012500
26040
6. 40572CO
21520
2142400
26720
7. 345520:
16S40
2176400
19660
E. 363B200
18440
2266200
20000
9. 3776000
16440
2220200
1932C
10. 3011BOO
21160
2914400
376B0
l:. 326000;.-
2064 0
2677000
36340
x 2 • * S1B n.*1 0
20440
2913400
3F-360
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23120
2993000
236 20
14. 2^13400
23100
2977400
25940
15. 2543A00
232S0
300B000
26420
16. 2565200
135S0
3636400
7; 900
17. 2963600
14160
3B62000
7 61B '•
IE. 2310000
1476C
3B65400
7BS60
AVE.
AVE.
AVE .
AVE .
3069167
19264
2715422
34406
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