EPA 600/3-81-005
February 1981
later Laboratory Comparison
Acute Testina ?et
hv
Arfiond v.. LerVe
Research Aauatic Biologist
Preoared for Office of Pesticides and Toxic Substances
-------
TECHNICAL REPORT DATA
(Please read Insmicrions on the reverse before completing)
I. REPORT NO.
EPA-600/3-81-005
3. RECIPIENT'S ACCESSION NO.
160??2
4. TITLE AND SUBTITLE
Interlaboratory Comparison Acute Testing Set
5. REPORT DATE
FEBRUARY 1981 ISSUING DATE.
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Armond E. Lemke
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND AOORESS
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, Minnesota 55804
10. PROGRAM ELEMENT NO.
1 1. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND AOORESS
Same as above
13. TVPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A multiple laboratory (6) set of tests was conducted using a single Test Standard
Method. The tests consisted of static and dynamic aquatic bioassays with two
species of fish and static tests with Daphnia magna in duplicate. The extreme
values for any one test were one order of magnitude while the extremes of the means
were only a factor of 12.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENO5O TERMS c. COSATI Field/Grouo
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report;
UNCLASSIFIED
21. NO. OF PAGES
20. SECURITY CLASS (This page/
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (R.v. 4-77)
PREVIOUS EDITION IS OBSOLETE
-------
Comprehensive Report
Inter laboratory Comparison
Acute Testing Set
Mans additions to the environnent, bv clan or mistake, of materials not
normallv found "here have increased tremendous lv. '-'arious offices of the
n.S. Environmental ?mtection Agency are charged with regulation of various
sources, i.e., Office of °esticides ^rosrams. A relativelv new unit, the
Office of Toxic Substances COTS') , is chareed with -nakins judgments on the
effect of those materials not regulated elsewhere.
The or? staff and personnel in the Office of Research and Development
CHRP) have been worVine on a set of toxic itv testina protocols to he
reouested for r>remarket testing of materials which appear to he potential
hazards. Office of Research and Development research labs have performed
round-robin tests on such protocols. This report is a compilation of the
acute tests which were part of the first -iroup.
wethods and -'aterials
°rotocols for acute static and dvnanic ( f low-th roueh) tests with fathead
minnows and rainbow trout were conducted with a pesticide endosulfan and a
metal, silver nitrate (AgW!^) • (?ach chen.ical was run in duplicate for
each species and test type for a total of 16 tests.) Four contract
laboratories and two E"A research laboratories participated. The final data
set contains % 96-hr LC50 values. Also two acute ^8-hr tests with Paphnia
were conducted us ins the same protocol with slight modifications by
each laboratory (12 each £8 hr LC50 valuesK
Th.e protocol, used was AS™ Draft 7, April II, l°73, of 3ronosed
Standard Practice for Conducting ?asic Acute ^oxicitv Tests with rishes,
Macroinvertebrates and Amphibians.
-------
-7-
rontractors were asked Co use natural waters and to provide their own
fish usinz the safeguards necessarv as per the orotocol. rach contract
lahoratory was visited once hv the proiect officer durir? actual testing.
~>ata handling in the reports was as reauested in the supolied protocol.
To insure as much as possihle a normal situation they were asked in the
hiddins» process to supolv oeoole with experience in toxicity bioassavg,
natural waters, and an adenuate analytical cornnetence, as oer the attached
ratine sheet (Figure 1). Mo effort was ^ade to correct small Differences in
techninue or interpretation of the Drotocol during the duration of the actual
testin?. ?ach successful contractor was told to report as oer the orotocol
with a seoarate section in the reoort for problems with interoretat ion.
therefore, each contractor was asked to regard the work and test results as
if clarity of the protocol affected the data variance. ue would he asked to
testifv in court as to their validity and to approach all internal decisions
on. this basis. As an example, if it was found that the analytical work was
in ouestion, the contractor was asked to do what was necessarv if he vas to
testicv in a court room rather than having the oroiect officer say what he
?houli do.
In this reoort, °6-hr Lf5ns used for statistical comparisons, are those
reported bv the participating lahoratories with only random checks as to
accuracy.
The evaluation criteria used to select the contract lahoratories
follows. It was deened necessarv to have experienced people so problems with
.he Protocol would he identified. Therefore a hish priority was Driven to
famiiiaritv with anuatic toxicity testing in general and the protocol in
particular.
^ . Technical Fvali!ation Criteria:
criteria to he used in the evaluation of proposals is set
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-3-
forth below:
Figure 1
Maximum Points
a. Oualification of personnel 15
h. Suitability of water (preference will he siven 15
to natural waters)
c. Familiarity with aauatic bioassavg 15
d. Familiarity with ASTM procedures l^
e. Adequacv of bioassay facilities 10
f. Availability of analytical enuipment ln
z. Competence of analvtical personnel 1^
h. Ahilitv to meet timetable 5
Toxicants
All toxicants were provided bv the aovernment. They were reaaent ?rade
silver nitrate and technical srade endosulfan. -Mso provided were purified
samples of endosulfan I and II to be used by the experimenters as a
analvtical standard. The purified reference «annles were obtained from the
EPA Standards Hranch.
Solvents
All of the laboratories dissolved the silver nitrate in distilled water
and then added or -netered the reauired amounts into the test containers.
The endosulfan was dissolved in acetone bv all but Lab ^. They
saturated ultra-pure water by stirring and then filtered and used the
filtrate as their hiehest concentration.
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-u-
Anparatus and Test Setup
The protocol is not specific concerning the tvpes of apparatus to he
used in maintaining temperature in flow through or static tests. The
eouinment to he used in making UP the toxicant water mixtures necessarv, is
also not specified. Table 1 shows how they were accomplished by each
lahoratory.
Co-istruct ion
All lahoratories followed the protocol in the use of materials for
holding and piping of test waters, test chamhers and heating equipment. All
test tanks were either glass jars or aquaria constructed with silicone and
all piping was rigid pvr or aged hlack iron. Diluter construction was nearlv
all glass with, a minimum of tygon used for ioints.
Chambers
Glass chamhers of sufficient size (10 1 volume) were used for all static
tests .
Class aouaria made with a minimum of silicone glue and containing 20 to
30 liters volume with covers to prevent fish (trout esoeciallv) from escaping
were used hv all lahoratories.
Volume Control
All lahoratories maintained suitable volume control in all of the
constant flow tests by the use of dilution delivery eouipment as noted in
Table I. This is probably the least troublesome of all requirements in the
protocol. The usual diluting enuirvnent bv its intrinsic nature vor'^s best at
a rate which delivers '->• to 6 turnovers into a chamber of the reauired size.
The maintenance of concentration control is considered to be verv
-------
Table I
I.ahoratorv Test ^acti1 ities
Ter.oerati)re
Control Method
Lab in
1
i
A.
7
il
5
c,
£tat ic
Tests
Tenoerature
control roon
'-'at?r baths
Trout water
batb , ^at'iear
tenoerature
control room
Vater hath
'•'ater xath
'ernneraturs
Control room
"low Th rough
Tests
Flow rate
Flow r i r r
Flow rate
1 and. vater hgch
"low rate
57 low rate
"^e^oerature
and clow
Ueatin5
"tain! ess
coil ^
Class water
beater
Class water
heater
Class aouaria
beater
Class water
beater
Stainless
coil '-^
Flow "n-trou?^1
TOX icant
Prenarat ion
ne foe
d i I u t e r
voun t / n. run
-------
•"6 *~
important during bioassay test ins?. Table 2 has a compilation of Che overall
concentrations reported by each laboratory comparing their nominal vith
analyzed concentrations. Most of the variation in initial static and flow
through numbers is analytical and appeared in the lower concentrations.
The final static values reflect losses by precinitation, oreanic binding
or in the case of endosulfan, degradation or volatilization.
Table 3 presents a compilation of the source of all test animals of all
of the participants. The-sources of the trout are most varied with all of
them being purchased either as eggs or fingerlings. All except two raised
their own fathead minnows and all had lab reared Oaphnia.
••.'ater ouality parameters for the dilution waters are summarized in Table
7. All particinatin* laboratories except number 2 have soft water. All
water samples were from natural sources with no ch lorination.
Results
Tables ^, 5, and f> present the analyzed part ic inat ing laboratory
sunolied LfSOg FOr all of the tests (total of 120). These results were used
as given by the reporting laboratory.
Random checks were made of the data in those cases where the preferred
orobit analvsis was not used. Results were different only at the third
significant digit, (Example 22.5 by probit and 22.3 bv Lichfield Wilcoxson)
so all results were used as submitted. All data are in micrograms (us) per
liter with 95% confidence interval immediately below the LC5n.
Statistical <">ucput
Regressions were run on the test sets vs. various water ouality data
from the laboratories. ^o significant correlations were found with the
-------
Table 2:
Toxicant deliverv and concentration control
and acute Daohnia
Lab in
I
2
3
L.
e:
6
1
2
3
/,.
5
6
Silver
Static Studies Initial
ino% + 10%
inn'/ + ins:
inn*/: + 102
Qn% + 10%
i r\M j. i S7
ino% T m%
Endosul Fan
Static Studies Initial
=>5% + 12%
100% + 15%
03^ + i^y.
i nn" 4- 90"
100% ~ 12'-
100% + 12%
Recoverv % rinal
«0% + 25%
a 5% + 30%
85% + 20%
70% •»• 10%
—
30% + 20%
Recoverv % Final
30% •*• 7n%
40% + 100%
20% •(• 30%
^~
50% + 50%
10% + 50%
Constant Renewal Studies vean Recoverv %
Silver Pndosulfan
1
2
3
ti.
5
6
100% + 12%
ino% + 10"
100% + 11%
100? + 20%
100% + 20%
100% + 9"
100% + 10%
100% + 10%
100% + 15%
100% •(- 12%
100% -i- 15%
100% -i- 12%
-percentages reoorted were developed bv comnarina nominal with
analvtical values as reported bv each laboratorv.
-------
•4
Table 3
Test Organisms
Source and Sizes Used
Lab ID
1
2
3
4
=5
6
Fathead
Size
0.5-1.5
* low
through
test
0.5-0.7
static
tests
Not
given
0.5-0.7
bath
0.05-0.
flow
th rou2h
test
0.2-0. 5
stat ic
test
0.5-0.9
bath
.5-1 z
bath
Minnows
Source
Z Lah
reared
f. Lab
reared
Ferder Fish
hathery
Baltic OH
2 '
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Table 4
Daohnia Acute Tests 48 hr LC50S ue/1
°5% Confidence Interval
LAP AeW-j AeW-j "ndosulfan "ndosulfan
Test I Test 2 Test 1 Test 2
1 0.66 0.3P* • 218 282
0.46 - 1.3 0.32 - 0.47 PI - 572 245 - 328
2 45 iQ 250 630
43 - 47 45 - 54 210 - 300 550 - 720
2.2 ,/ 2.0 740 378
\s
.Q - 2.S 2.3 - 3.6 521 - 1051 220 -
0.00 1.03 266 15?
0.78 - 1.08 0.86 - 1.24 241 - 2
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fABLF 5
Lndosul tan 96 hr LC'JOs tor All Fish Tests
Values are |i<|/l Active Ingredient
Lrtb 10 KICK
FlIOF
FH'J
FHS
RTCF
KTCF
RTS
RTS
I 1.2 1.91 2.10 3.451 .69 .75 l.2IOa .89
.99 - 1.701' 1.19 - 2.96 1.70 - 5.86 2.00 - 7.14 .25 - 1.00 .48 - 1.06 .85 - 1.51 .69 - 1.13
2 .29 .45 2.10 3.20 .17 .29
.19 - .44 .30 - .53 1.7 - 2.6 2.7 - 3.8 .12 - 24 .24 - 35
6 .75
.54 - .04
.49 .80
.40 - .60 .64 - 1.0
3 .76 .73 1.70 1.48 .30 .27 1.34 2.43
.64 - .90 .60 - .89 1.42 - 2.04 1.33 - 1.64 .25 - .36 .17 - .44 1.11 - 1.62 2.15 - 2.75
4 .81 .80 1.9 .97 .26 .41 1.30 .63
.67 - 1.00 .67 - 1.00 1.82 - 2.03 .78 - 1.19 .23 - 1.00 .33 - 1.00 1.14 - 1.58 .47 - 1.00
5 1.67 1.57 1.35 1.20 .32 .42 1.69 1.63
1.54 - 2.34 0 - 2.57 .75 - 2.44 .82 - 2.19 .27 - .38 .36 - .49 .77 - 3.69 1.37 - 1.93
1.00 3.20 2.50 .26 .24 .69 .79
.8 - 1.1 2.9 - 3.7 2.2 - 3.0 .23 - .29 .19 - .34 .65 - .75 .72 - .89
O
a Nominal no arid lysis
b All secondary values are 95# confidence Intervals.
FlCF = Fathead minnow constant flow test
FlIS = Fathead minnow static tost
KTCK a Rainbow trout constant flow test
RTS = Rainbow trout static tout
-------
TAIILF 6
SI Ivor '.)6 hr LC50 for all Fish Tests
Values aro IHJ/I Ay
l.al> II) RIO" FllCK FUS FHS RTCF KTCF KTS RTS
I 10.'JO ' 11.75- 30.43 .' 22.66 ' 17.87 I6.30a 19.92 ' 31.00 ^
9.31 - 12.77d )0.01 - 11.99 24.07 - 35.08 18.81 - 27.09 12.80 - 28.75 12.80 - 19.20 16.10 - 31.53 26.1 - 36.2
2 150 110 230 270 ^ 240 170 - 240 200 L.-
130 - 180 100 - 130 IflO - WO 220 - 320 200 - 290 ' 150 - 200 180 - 320 " 220 - 360
3 11.1 b 8b 13.0 ' 19.6 < 14 12C 48 ,.. 54
8.6 - 14.3 11.4 - 16.7 17.7 - 23.8 12.2 - 16.1 42.9 - 53.8 36.5 - 80.0
4 5.3 3.9 *s 6.7 <• 12.3 6.9 V 8.4 11.8 ^ 108.9« ^'
4.92 - 5.73 3.52 - 4.22 5.20 - 8.62 11.40 - 13.50 6.48 - 7.40 5.94 - 11.91 9.9 - 14.0 8.39 - 180.2
5 5.70 5.6 12.4 9.74 8.5 9.72 72.09* 84.4"
0 - 7.19 4.52 - 6.30 7.45 - 12.4 0.49 - 11.19 7.10 - 10.2 8.36 - 11.34 60.3 - 08.3 70.9 - 100.2
6 6.3 5.0 10.3 / 8.7 , 11.5 9.7 > 24.6 22.5
5.8 - 6.8 4.2 - 5.7 9.6 - 11.1 7.8 - 9.6 10.8 - 11.8 9.0 - 10.3 23.6 - 25.8 20.9 - 24.7
a N<«n I na I
k fstlnicitu loss lluiii 502 killed at highest concontratIon.
c Fstlniato more tlun 502 dead at lowest concentration.
^ All secondary values are 95£ confidence Intervals
* Heavy aeration.
FlICF = Fathead inlnnnow constant flow test
FHS * Fathead minnow static test
KICF = Ha Inbow trout Constant flow test
UTS =• lidlnbow trout static test
-------
Table 7
Laboratory Water Oualitv Parameters^*
Parameter
Aluminum
Arsenic
Barium
!*oron
Cadmium
Calc ium
Chromium
Cooper
Iron
Lead
Maenes ium
Manganese
Mercury
Potass ium
S e 1 e n i un
Si Iver
Sod ium
?inc
Bicarbonate
Carbonate
Chloride
"luoride
Hvdroxide
Mitrate
Nickel
^hosnhate
Sul fate
VH3
Dissolved C>2
"^•Hardness
Al'j
Lab 3
0.0116
0.0025
<0.0l
<0.005
0.009
3.0
0.026
O.OS
1.!
72
- 7.I
Sorine
Lab 4
O.OS
<0.0[
<0.25
0.07
<0.002
11.6
<0.005
0.01
O.OP
<0.02
4.1
<0.005
<0.0005
0.8
<0.0025
<0.005
6.3
o.oi
45.5
0.0
11.1
0.0°
0.0
o.?
<0.05
<0.05
8.7
6.0
46.1
37.3
136
Lab S
<0.005
14.7
<0.005
<0.005
0.01
<0.04
3."
<0.005
-------
I 7
Table 8
Part A
Interlaboratorv
Analysis of Variance for Silver Results
Fish
Due to HF SS MS=SS/DF
Laboratories "5 251407. 50281.
Test Tvnes 3 14294. 4765.
Combined 15 17328. 1155.
=!rror 24 5086. 212.
Total 47 288115.
Rows are Levels of Laboratories
Lab
I
2
3
4
5
6
Col.
Means
11.4
130.0
9.6
4.6
3.2
5.6
27.4
FMS
26.5
250.0
14 .Q
9.5
11.1
9.5
53.6
Part
Cell
Columns are Levels of Tests
Row
RTCF
17.1
205.0
13.0
7.7
9.1
10.6
43.7
RTS
7.5.9
260.0
51.0
11.3
78.6
23.6
75.1
Means
20,
211,
22.
a,
25,
12,
49.9
Dooled St. Pev .
14.6
Lab ID
I
2
Part C
Individual 95 Percent C. I. for Level Means of Laboratories
(Rased on Pooled Standard Deviation)
1*1*1
I*t*t
!*!**!
1*1*1
1*1*1
•[*T**T
50.
100. 150. 200. 250. 300.
Sets
Part n
Individual 95 Percent C. I. for Level Means of Tests
(Based on Pooled Standard Deviation)
• --------- -»• --------- + --------- + --------- •"• --------- •*•
T ***** T ***** T
I ***** I ***** I
J *****J***** 1
4.
15.
30.
45.
60.
!*****•[*****•[
+ --------- +
75. Qo.
-I-
105.
-------
Table °
Part A
Tnterlaboratorv
Analysis of Variance for Fndosulfan
Results of Fish Onlv
nue to DF
Laboratories 5
"fest Tynes 3
Combined 15
F.rror 24
Total 47
SS
19.418
8.688
3.481
32.586
.400
5.139
.579
.145
Part R
Cell Means
Sows are Levels of Laboratories Columns are Levels of Tests
Row
Lab ID FHCF F^S
2.775 .720 1.050
2.650 .230 .645
1.126
.885
1 .231
1
2
3
4
5
f,
Col.
Means
Pooled St.
1.555
.370
.745
.805
1.620
.875
' _QQ5
nev. =
2.775
2.650
1.5°0
1.435
1.275
2.850
2.006
.381
RTCF
.720
.230
.285
.335
.370
.250
.365
1.050
.645
1.885
.965
1.660
. 740
Means
I.S25
1.158
1.179
1.153
Part 0
Individual 95 Percent C. I. for Level Means of Laboratories
(Rased on Pooled Standard Deviation)
jK If) +• -I- >-- -f -I 4- --_- 4.
\ !*************•[*********-.•:***•[
2 i*************i************* T
3 T*************I************* i
4. I ************* T ************* I
5 T************i************i
f) I ************* I ************* J
4. 4,- 4, 4, 4. 4 4,
.60 .80 I.00 1.20 1.40 1.60 1.80
Part D
Individual 95 Percent C. I. for Level Means of Tests
(Rased on Pooled Standard Peviation)
rest Sets * •*• * * + •*• +
1 I****i***i
2 i****!***i
3 T*** T **** X
i !***•[****•[
4. ... +. 4. +. 4- +
0.00 .50 I.00 1.50 9.00 2.50 3.00
-------
Table 10
Part A
Analysis of Variance For S
Results at all Acute Tests
Due to DF SS
Laboratories 5 217661.
Test Type 4 36184.
Combined 20 4S444.
Frror 30 9832.
Total 59 309120.
43532.
9046.
27.72.
328.
are Levels of Laboratories
Lab 10
1
2
3
6
5
6
Col.
Means
FHCF
11.4
130.0
".5
4.6
5.7
5.6
27.8
FHS
26.5
250.0
Cell Means
Columns are Levels of Tests
16,
o
11.
53.9
STCF
17
205
13,
7
0
10
43.7
25.9
260.0
51 .0
60.3
78.6
2.3.6
83.2
Danhnia
.5
47.0
2.5
I .0
_o
11.7
10.6
Sow
weans
16.3
178.4
13.6
16.6
21,
12,
43.9
Pooled St. Dev. = 18.1
Lab ID
1
2
•>
u
S
6
Part C
Individual 95 Percent C. I. for Level Means of Laboratories
(Rased on Pooled Standard Deviation)
^ ----------- 1 ---------- + --------- + --------- 4. --------- j ----------
!**!**!
I**I**T
!**!**!
n.
40.
80.
H
120.
^
160.
-I
200.
240.
Part D
Individual 95 Percent C. I. for Level Means of "fescs
(Rased on Pooled Standard Deviation)
rest Sets +• --------- + --------- •*• --------- +• --------- > --------- + --------- •*•
[ -[***->!****];
2
3
20.
1
40.
60.
80.
100.
120.
-------
it-
Table 11
Due to HF
Laboratories 5
Test Tyne 4
Combined 20
^rror 30
Total 59
Part A
Analvsis of Variance for Endosulfan
Results at all Acute Tests
SS
32987.
11878P8.
112356.
140094.
1502335.
MS=?S/DF
ft5P7.
29ftQ74.
661*.
4P70.
'ows are Levels of Laboratories
Lab ID
1
2
FHCF FHS
1.6 2.8
.4 7. ft
.7 L.6
.3 1.4
1.6 1.3
.9 2.8
1.0 2.1
°ooled St. Hev. = 70.5
Col
12
.3
.3
.4
.2
Part 3
Cell Means
Columns are Levels of Test Tvoes
Sow
RTCF 9TS Oaohnia Means
.7 1.0 250.0
.6 440.0
1.9 559.0
1.0 212.0
1.7 30ft. 5
.7
1.2
350.0
352.9
51.2
88.8
112.7
43.1
62.3
70.q
71.5
Lab tr>
[
2
Part C
Individual 95 Percent C. I. for Level Means of Laboratories
(*ased'on Pooled Standard Peviation)
. + * > +, + -i
-[****•>***** vr* I ************* J
^****•***->*•*** J************ 1
I************I************T
************J************J
J************ •[ ************ £
I************I************I
30.
65.
LOO.
135.
170.
205.
Sets
-100.
Part D
Individual 95 Percent C. I. for Level Means of Tests
(Rased on Pooled Standard Deviation)
+ --------- +• --------- + --------- +• --------- +
•[***!***!
-[***!*** I
x***I ***!
0.
100.
700.
300.
400.
4.
500.
-------
-17-
various common water oualitv narameters and endosulfan toxicitv. The silvar
toxicitv had a Q5* or ereater correlation with ^ar-^ness an^ aT'alinitv with
all tests exceot the rainbow trout statics which were ahnut *P°- indicating-
other factors involved. Onlv slis^t correlation was noted with n'* .
A two wav Analvsis of Variance C.-iO") was computed with test tvoe "eans
vs. lahoratories Seine considered. -i.nv out outs for silver and endosulfan
data are found in Tahle 8 and °resr>ec t ivel v for fish acutes onlv and in TaM
I1"1 and Tahle 11 for all fish and nanhn ja acute tests in conh inat ion .
Discuss ion
Literature search efcorts OF the stafp of tue rnvironr-ental 'esearc'i
T,ahoratory-nuluth as nart of twe criteria document process uncovered videlv
scattered Lr"-0 values for sone materials, ('runos, 'ersonal communication).
rxnec tat ions for this hioassay inter la^oratorv comnarison were in the ranee
o ? one order of ^a^nitude considering the "reedor" of invest isacors for source
of tp = t anirals, water rualitv, invest isa tors exr;er ierce and ot^er less
t a PI 2 i Vs la fac tors .
Statistical
~^he. Analvsis presented in Tables Q-'l show values with ~uch less
variation than exnected. The data used are the reoorted. I,r5ns ?ron "^ahle ^
and f\ resoec t i"el v . ""he nunher presented in eac5^ colunn in ^art ^ of eac'"
tahle is the ^ean of the two tests o? a li': f,\\ tests + the nooled. standard Deviation and enables visual
n of Jata output he c we en lahoratories.
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Visual comparisons of tests types are presented in Part 0 of Cables S-ll.
Scales of Part C. and Part 0 of Tables 8-11 are different. The sraphs f°art
C) show that all of the laboratories overlap ranges except lab 1 and lab 3
for endosul^an. The neans of these two laboratories at 1.^8 upr/l and 1.89
us/I are less than a factor of two for all tests. This compares with the
neans of all constant flow rainbow trout tests of 0.39 tie/1 versus 2.117 ucr/1
for the fathead statics for a difference of iust over 5 times.
Interlahoratory vs. Species
'•'ithin species conparison of Static and Constant rlow tests are similar,
being 2.3 times (O.P2 vs. 2.12) for fathead minnows and 2."7 times (0.385 vs.
1.162) for the rainbow trout. These values are based on the initial analyzed
concentration of the static tests and on the analvzed mean of the initial ?nd
Final concentrations of flow-through tests. Analvsis of the end of the
static tests reveal a 3i">2-R<"i£ loss of toxicant ma!
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che analyzed data of all concentrations vs. the nominal numbers of each
concentration for laboratorv 1. Complete nrobit anrl binominial analyses were
accorolished separately rather tban na'rohit analvsis as a
similar machine method. Laboratorv U. used the Lich field. U'ilcoxson hand, »ran'n
estimate method. As a check on this and to verifv the various methods all of
r data was recalculated from the raw survival numbers usins the sucnlied.
vzed concentrations. In all but 1 cases, Fathead Constant clow 2 and
Rainbow '''rout Constant ?low 2, data was obtained by at least one other
method. The largest difference found was 16 v.s. 19.6 for the Fathead Static
2. The largest error is 22". All others were 'ess than 10".
Several results need further c lar i f •'cat i on . The acnarsnt hish results
l^a.° 'jff/l for laboratorv L in the second rainbow trout static is a case in
ooint. "^he nar t ic inat ine laboratorv noted this result. "''hev checked all of
their analvsis and nreoaration of toxic solutions and found no gnoarent
errors, "o lahoratorv was either low or *~ ish in results vith hoth chemicals
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difference is so ereat ( aooroxitnately 200 tines) chat the effect is similar
to t^at of ^ardness with silver, '''his soecies difference co^nresses the
Difference between the fish tests, as the hardness rioes between the
laboratories tests, where waters are aooroxipately the sane.
Considerations
Potential, sources of Lahoratorv test variability include; fish source,
fish size, water aualitv, test chanhsr aeration rate, method of toxicant
nreoarat ion and ->ethod of data analvsis. r-'ater nualitv, discussed
nreviouslv, causes vide variations if the toxicant under test is labile to
water hardness differences i.e. silver or other heavv metals. ?ish size is a
cactor esoeciallv in static testing. Several of the laboratories were found
to use larger than recommended :ish when those obtained earlv in the test
oroeram grew and renl acements were not available. These laboratories Vo. "*
and Mo . 5 renort sone of the highest values for silver. The larser fish
reouired heavv aeration to hold the oxvsen at the recornmended level and. the
two factors interacting annear to account for the decreased toxic itv. Lahor-
atorv 5 acco"iol ished another test vith smaller fish and no aeration after the
deadline cor the regular test set and reported an LC?n nf 12 'ip/As ./I in.
conoarison to the ^ean of 7S :ig/Afr /I renorted earlier. "'"he varyin?
sources of test fish aonear to contribute little bias to the results.
As noted, la'^oratorv ? oreoared its endosul ?an toxicant solutions bv
na''in.2 a saturated vater solution and then diluting txis to ^ake L;ie
concentration series. "eter-iin in=r whether this '-ad anv real effect is
difficult. Although t'-'ev report the lowest ne^n ;or all of t'^e ^ndosulfan
tests, in bots of the fathead static tests thev are near or ecua! to the
Least toxic values.
N'uch of the cost of conducting '-ioassavs is in the sunr'ort renuired for
of Lr5-s ,:si^
an
.lvsis o^ test conditicrs. ^->l- 12 presents the ccr.ar.on
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4. Trout have a propensity for lumping out of test containers
necessitating covering of the containers, making observations and
sampling more difficult.
5. Every effort should be made to test as many species as possible if
protection of the environmental life forms is to be accomolished
The large difference between species of over 200 times is much
greater than either the variation in test type results or between
laboratorv results of two to five times.
References
Andrew, R. W. , P. V. Hodson, and D. E. Konasewich (Editors). Toxicity to
Biota of Metal Forms in Natural Waters. Proceedings of a Workshop.
Duluth, MN October 7-8, 1975. Published bv Standing Committee on the
Scientific Basis for Water Quality Criteria of the International Joint
Cotnmiss ion.
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indicating that randomization of analytical and bioassay procedures was
taking place as expected. Upon receipt of this report this author asked them
to have another person on their staff run another check with the same
results. Further perusal of the report of laboratory & indicates that
perhaps the first result is slightly low and the second slightly high as the
mean is very close to that of other participating laboratories. Another data
set is that of Lab 2 in the silver data. As mentioned briefly previously
this laboratory used hard well water of about 300 mg/1 as CaCOj while all
of the rest of the participating organizations had water of 100 mg/1 CaC03
or less.
Metal toxicity is affected by water quality as shown by various
reports. A report edited by Andrew et. al. (1975) presents an excellent
discussion as to the reason for this, and the data from laboratory 2 is in
agreement.
Special Considerations
1. If metals are to be tested, use of the results must be eauated
with the type of water in question whether as test or receiving
water.
2. Animal size is quite critical as indicated in the protocol, but
following this part of the method is much easier with a species
that is small naturally than with one which rapidly becomes larger
i.e. fathead minnows vs. rainbow trout.
3. Constant flow tests produce results with less variability because
they are not subject to fluctuating conditions of various fectors,
such as aeration, precipitation and volatilization.
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Tahle 13
Comnarison of Calculations by Various Methods
the Same nata Set
Laboratory 4
Test
PHCF i
FHCF 2
F^S 1
F^S 2
9TCF 1
?TCF 2
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Table 12
Nominal vs. Measured Concentrations
ue/1 Silver
Laboratorv I
Mor".inal __ Measured _ Nominal _ Measured
Silver
FHCF I FHCF 2
11.28 10. 98 12.22 11.75
10.09 - 12.06 Q.31 - 12.77 11.02 - 13.55 10.01 - n.
S I
33.50 30. 43 24.15 22.66
. 12 - 40.13 24.07 - 35.88 ?0.15 - 28.00 18.81 - 27.00
23.50 17.87 16.38 no
21.3 - 26.1 12.80 - 28.75 12.80 - 1Q.20 analysis
RTS I RTS 2
28.74 10.92 32.95 31 .«0
20.80 - 38.80 16.10 - 31.53 27.7 - 36.9 26.1 - 36.2
Danhnia 1 Daphnia 2
.39 no .63 .66
.32 - .47 analysis .45 - 1.5 .46 - 1.30
Endosul fan
FHCF 1 FHCF 2
1.24 1.20 1.01 1.91
1.0 - 1.6 0.99 - 1.70 0.8S - 1.19 1.19 - 2.Q6
FHS 1 FUS 2
2.
1 .93 -
0 .
0.62 -
I.
0.85 -
220
188 -
35
5.08
RTCF I
86
I .0
RTS I
21
1.51
^anhnia
257
2.10
1.70 - 5.86
0.69
0.25 - 1.08
no
analvs is
1
218
91 - 527
3.34
2.35 - 4.17
0.81
0.62 - 1.0
0.94
0.73 - 1.17
251
215 - 297
no
analys is
RTCF 2
n.75
0.48 - 1 .06
RT^ 2
0.80
0.69 - 1.13
OaDhnia 2
282
245 - 32S
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warrented unless the soecies in ousstion is of oarticular interest in a
certain situation. Lone term tests with Daphnia Tiagna vith pndosulfan
(renorted elsewhere by Meheker) would not have nredicted the values for
the fish.
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Conc lus ions
1. T.aboratorv studies accomplished bv knowledaeable personnel ^ollowins a
standard protocol with reasonable orecision, should produce results
which are within the standard deviation of similar tests of like nature
accomplished by a single laboratory.
2. Oualitv of the test waters should be of concern when the test material-
is known to have a chelatine effect as that found with meca Is and hard
water or wich the effect of oH as with some orqanics such as
or?anophosohate insecticides. If nothing is known of these tvne of
reactions of the test material -lulciole tests with different waters
would be exoedient.
3. Careful attention to detail as por example fish, size has more effect on
reproducibi1ity than any protocol by itself provid iris such protocol is
biolo2icallv complete.
4-. ^iolocial effectiveness and completeness should have top orioritv as
poor biological information cannot be improved by statistical analysis
but <*ood biological work cannot be harmed bv ooor statistical work
providina the raw unworked data is also presented.
T. Tr limited funds are available the most information "or protection of
the environment can be sained, by doing short term tests on more and
•diverse species rather than bv expenditure of funds to set a more
precise answer for a sinjle species. A lonser tern test is not
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY
200 S.W. 35TH STREET
CORVALLIS. OREGON 97333
SUBJECT: Silver and Endosulfan December 15, 1988
FROM: Alan V. Nebeker
ERL-Corvallis &*
TO: Charles Stephan
ERL-Duluth
I would like to confirm that you have interpreted the data
presented in Lemke(1981), Nebeker(1982), and Nebeker et al.
(1983) correctly.
Table 2 in Nebeker (1982) and Table 4 in Lemke (1981) give
results of the same tests, except that Nebeker's Table 2 contains
results of two additional acute tests on endosulfan (one' from ABC
and one from WAPORA) and one less test on silver (from WAPORA).
Other differences between the tables are due to recalculation of
values (such as for ABC for silver) and roundoff (such as for
ERL-Corvallis for silver). In addition, the value of 0.63 for
silver from ERL-Duluth should not have been included in Table 2
in Nebeker (1982) because it came from the same test as the value
of 0.66 (as per Table 12 in Lemke 1981).
Table 1 in Nebeker et al. (1983) contains four 48-hr ECSOs from
tests on silver with D_. ma ana. The third and fourth tests were
side-by-side tests of the effect of food on the toxicity of
silver. The first and second tests were conducted as part of the
round-robin study and are the same as values reported for ERL-
Corvallis in Lemke (1981) and Nebeker (1982). The hardnesses for
the four individual tests are given in Nebeker et al. (1983);
the value of 60 mg/L given in Table 1 in Nebeker (1982) is a mean
of hardnesses that were obtained in a variety of acute and
chronic tests, which tests were reported in both Nebeker (1982)
and Nebeker et al. (1983). Thus the results of two acute tests
conducted on silver with D. maana at ERL-Corvallis were published
in Lemke (1981), Nebeker (1982), and Nebeker et al. (1983).
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