Insect Inte rlaboratory Toxicity Test
Comparison Study for the Chironomid
('Paratanytarsus sp. ') Procedure
(U.S.) Environmental Research Lab.-Duluth, MN
Mar 84
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EPA-600/3-84-054
March 1984
INSECT INTERLABORATORY TOXICITY TEST COMPARISON STUDY
for the
CHIRONOMID (Paratanytarsus sp.) PROCEDURE
by
Armond E. Lemke and Richard L. Anderson
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, Minnesota 55804
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TECHNICAL REPORT DATA
,flease read Instructiont on the reverse before completing)
1. REPORT NO. 2.
EPA-600/3-84-054
3. RECIPIENT'S ACCESSION NO.
PB8® Ifinn? 5
4. TITLE AND SUBTITLE
Insect Interlaboratory Toxicity Test Comparison Study
for the Chironomid (Paratanytarsus sp.) Procedure
5. REPORT DATE
March 1984
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Armond E. Lerake and Richard L. Anderson
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
same as above
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A test method guideline for the chironomid Paratanytarsus sp. was evaluated.
Six laboratories participated in the interlaboratory comparison study. Three items
were compared, including start-up and maintenance of a rearing colony, a 48 hr acute
test, and a 28 day life history chronic. All participating laboratories were able to
start and maintain the rearing colonies. Chemicals used for testing were trichloro-
phenol and acenaphthene. Forty out of an expected total of 48 test results were
reported.
The 17% failure rate appeared to be related to the volatility of the chemical
in the acute tests and to an unexplained test water problem at specific laboratories in
the chronic tests. All participants recommended the preparation of a set of forms for
recording data and training rather than more detail in the guidelines would improve
testing efficiency.
17- KEY WORDS AND DOCUMENT ANALYSIS
J DESCRIPTORS
b. IDENTIF IERS/OPEN ENDED TERMS
c. cosati Field/Group
IB. DISTRIBUTION STATEMENT
Release to public
IB. SECURITY Ct^SS (This Report)
Unclassified
21. NO. OF PAGES
18
20. SECURITY CLASS (Tttit patt)
Unclassified
22. PRICE
EPA Perm 2220-1 (R*v. 4-77) previous edition is oooliti
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
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Researchers in aquatic toxicology recognize that there are wide
differences in aquatic species sensitivity to chemicals. Intuitively, the
species dependent difference in sensitivity cannot be adequately measured
with only a few test species. At this time, most of the test method
development has been with fish. Only a few fish food organisms like Daphnia
have had wide use as test subjects and, because of their use, testing methods
and rearing procedures are available.
The need for invertebrate test species has lead to the development of
the procedure evaluated in this report. The test species is a
parthenogenetic insect Paratanytarsus sp. of the family Chironomidae in the
insect Order Diptera. The insects are commonly called "midges". Members of
this family are found in all freshwater and some saltwater habitats. Their
usual mode of living is either on or below the surface of sediments or
periphyton. The genus Paratanytarsus has been found in Australia, Japan and
North America.
Objectives: The objective of this work was to evaluate the adequacy of the
Paratanytarsus rearing and testing guidelines (Appendix A and B) by a group
of laboratories that have a strong background in aquatic toxicity testing.
Chironomids have not been tested extensively but all the selected
laboratories had chironomid rearing and testing experience although not
necessarily with Paratanytarsus. All the laboratories were requested to make
comments on the rearing and testing guidelines, both as to operating
problems and clarity. These comments would be incorporated into the
guidelines .
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Procedures
before start: A Request for Project was developed, advertised, and the
responses were evaluated by a panel at the Environmental Research Laboratory-
Duluth (ERL-D). Five laboratories were chosen. Amador part of the
selection criteria was the suitability of the respondents test water. All
the laboratories were asked to present information on heavy metals and
pesticides in their water supplies and evidence that the water would support
Daphnia cultures. All participants presented information of a satisfactory
water supply. Common physical measurements for the test water from each of
the selected laboratories are listed in Table 1.
A one-day informational conference was held at ERL-D and the principal
investigator for each laboratory was present. One of the authors (RLA)
described the toxicity test procedures and demonstrated the manipulative
techniques that appeared in the guidelines. At the end of the day a question
and answer session was held. After this meeting, the participants were told
to use the rearing and testing guidelines and onlv minimum guidance was
given. This was done to allow the method to stand by itself and to show
guideline deficiencies. Also, at the close of the meeting each investigator
was given stock supplies of th*» two chemicals that were to be tested and
chironomid eggs from the ERL-D colony to start their own rearing colony.
Chemicals were reagent grade trichloropheno 1, and reagent grade acenaphthene.
The chemicals were purchased from the manufacturer, Eastman Kodak Chemicals,
and reported as 99? .pure.
Test ing
Each laboratory was to establish a culture and conduct two static 48 hr
acute tests and two flow-through life tests with each of the chemicals for a
total of 8 tests per participant. Analytical chemical methods were left to
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the participants but those who requested help were assisted with methodology
as the necessary analytical work was only an incidental part of the tested
protocol. Methods used by each group are shown in Table 2.
Each laboratory was to analyze the chemical at the beginning of the
acute tests and at the end of 48 hrB of exposure. In the life cycle tests,
the chemical was analyzed at least twice each week with the samples being
alternated to assure that all containers were analyzed.
Riological effects of che exposures were reported as LC50s for the acute
tests and as effect and no effect concentrations for the life cycle tests.
It was also required that a discussion of all problems, guideline ambiguities
and a general commentary on the guidelines be produced.
No deadline was set for the completion of the tests and extensions were
granted on an individual basis by specific request concentrations and a control to determine the
LC50 rather Chan conducting a preliminary, range finder test.
The reported values for the static acute tests and flow-through life
cycle tests are in Tables 3 and 4, respectively. Forty-eight tests were to
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be run. Acceptable results were obtained in AO tests and in two of the 40,
the range numbers were not available because the required partial kills did
not occur.
Acenaohthene was more difficult to test than trichlorophenoI. All acute
test failures and one of the life cycle failures were with thitf chemical.
The onlv life cvcle test failures occurred at OKS. The principal
investigator reported non-toxicant related death at all concentrations and
the control. OSU did not report an effect concentration in one set of life
cycle tests with both chemicals. In each case, a very low replacement Lime
in the test chambers was reported. Several of the laboratories repeated the
experiments and the numbers reported to the Project Officer are those the
principal investigator considered satisfactory.
Discussion
These tests, with the oarthenogenetic chironomid Paratanytarsus sp.,
were done to determine the utility of the rearing and testing guidelines. A
three part guideline was provided and each will be discussed separately.
R<»arinn: The first part of the guideline describee procedures for
establishing a colony. The goal of the first section was to rear chironomids
in sufficient mmhers to provide larvae for the acute and eggs for the life
cycle tests. This section had a two-fold purpose. First, it provide'! a
check on the quality of the rearing water and, second, it provided an
opportunitv for the principal investigators to become faniliar with thn life
cycle and biological requirements of the chironomid. This task was
accomplished by all of the laboratories and no difficulties were reported
with the rearing guideline as it was written.
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Acute Testa: The second task was for each of the participant *, by
following the guideline, to conduct the acute 48 hr testa with the two
chemicals. Ml 1 nhorator i en were able to get an LCSO with t r ich 1 oropheno 1
hut. two laboratories, OSH and CAl.. did not ^et a SOT kill with *cenaphthene
although thev tried several times. Of those completing the ao'naphthene test
one, the order, in increasing sensitivity, was 2.00 »»$/l (MAS), 1.62 (st with acenaphth<*ne rep<>rted that thev were
unable to yet n kill with a saturated solution. Alt participants reported a
loss of chemical during the 48 hr tc?st period. Th i s loss rsav <*\jlain the
wirfe difference in LOSOs between the laboratories. Hovever, the interlaoora-
cory results sh< v that the LCS0 values were similar. Only ERt.-O showed a
wide variance between the two tests. The naximm variance with the other
three sets was 77, or less. The mator problem in the acenaphthene tests was
probably volatilisation or breakdown of the chemical rather than the ani«als
or guidelines.
The sensitivity order for the acute trichlorophenoI test one was il.O
(CAL), M.9 (SRI), 21.6 (0KS), 21.R »e variation between the
high and low reported value is 11 for teat one and 26 for teat two. Of the 6
data seta, 3 of the intralaboratory data aeta overlap in 951 confidence
interval (C.t.) between test 1 and teat 2, 2 do not overlap and 1 set doea
not have a confidence interval.
In nummary, for al! the acute teats, 10 data aeta were reported and 6 of
the 10 aeta show an overlap of the *)«>* C.I between teat 1 and teat 2 for a
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cheniral. Of th.» renainin 4, 1 »hov no ovt-rlao and one *et rlo^s not have a
C.I. and cannot he juried although inspection of the single val'je and a
comparison to the first test results indicate* that an overlap of the C.I. is
probable. In *>wr»ary, the acute data »hov that in bf) or 70Z of the tests, a
laboratory will produce data that is internally consistent. This slso shows
that the guideline can produce data that i» precise hut, that other factors
can iwoact the accuracy of toxicity test data. The factors which affect
i nt er I ahc at or * comparison* are discussed later in this report.
Chronic Tests'. The I if* cvc\e values for the six laboratories (Table U)
are reported a« effect and no effect concentrations. -rhe reported effect and
no effect concentration* are affected bv the exposure concentrations selected
hv each laboratory. To facilitate cotripir i Kin , geometric wins of the effect
and no effect concentration* were determined. For acenaphthene, the
teo"*<*trie we a-. ran*e* frrxt 0.01* 1 to 0.91 m*/ I and 0.024 m*/I to 0 ,4Q
**/1 t or te»t one arvl t**>. Par trichlorophenol, the ran^e of geometric means
is between 0.)l *t/l to V.O m^,f\ and 0. "1ft to 2.'i m$/i. One laboratory,
f>KS, wa* unable to conduct the flov-throu^h tests and OSU did not obtain a no
effect value apparently because of extreiaely low flow in the test tanka and
subsequent volatilisation of toxicant fro* the tanks.
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Summary
Sources of variation in toxicity teats with aquatic organisms can be
grouped into five main areas. They are the dilution water, test organisms,
chemical characteristics, test conditions, and chemical analysis. Variations
in each of these 5 areas can result in little change in reported values
between the laboratories if the factors balance each other or Lar^c variation
if all of the factor effects are in a single direction. Therefore, a
determination is needed of the problems found in this test series to
distinguish errors in the method guideline from the 5 non-method type
errors.
Forty-eight tests were to be completed in this project. Four acute
values were not reported and one 95% confidence limit was not calculated.
Four life cycle values were not reported and two did not develop effect
concentrations and one did not develop an effect concentration. A failure
rate of 12% is not unusual over a set. of tests with biological organisms,
dilution water problems, chemical solubility, and exposures to an
inappropriate concentration series ail contribute to failures and data
variability.
Examination of the individual laboratory reports reveal that the major
difficulties were with the test chemical characteristics, dilution water or
test animal selection. The low ERL-D trichiorophenoI acute LC50 value was
apparently caused by using fourth instar larva rather than third inatar as
suggested in the guide lines. All larva which nulted into pupa died and this
apparent life cycle related death skewed the data. If the low ERL-D
trichlorophenol results are not considered the range of the extreme values is
about six fold rather than 26.
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Those laboratories having problems with acenaphthene reported that the
chemical apparently volatilized from the test chambers. Losses up to 90% of
initial concantrat ion were reported. This chemical related problem would be
particularly important in static tests. Losses up to 90% of initial
concentration were reported. Volume to surface ratios probably added to the
volatilization effects because test chamber shape was not specified.
The OKS laboratory could not complete a chronic test apparently because
of an undetermined problem in their water supoly. Static rearing and static
acute tests were completed because they are "batch" events and are less
dependent on variable water quality. The test water was suspected because in
the longer flow-through tests even the controls did not survive.
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Conelusions
The guidelines are in three sections: culture methods, acute test
mathods, and life cycle test methods.
The culture method was completed by ail participants. The only
difficulty reported was in selection of the correct instar for the acute
tests in a crowded culture tank. More tanks with fewer animals would correct
this problem. With this exception, the culture method guidelines appear
satisfactory.
The acute test methods also appear to be satisfactory as those tests
which were not completed were caused by the chemical being only sparingly
soluble or volatile and and not by misstatements in the guidelines. All
participants were able to show satisfactory survival of animals in the
controls. It was reported that the protocol suggestion about using sand as a
substrate during acute tests is not needed. Although the participants in
this laboratory comparison series did not find the use of sand as a substrate
to be important in these tests, others have found it useful and it should be
included in the protocol as an alternative to the bare beaker method.
The life cycle exposure guideline should be modified to reflect the
problems encountered by the participants. The following changes appear
appropriate: (1) Start test by having all systems in operation; (2) take
samples for chemical analysis; (3) shut down or divert flow and add eggs to
test chamber; (4) run as static for 48 hrs; (5) take samples for chemical
analysis; (6) start flow.
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Recommendations of Participating Laboretories
The participating laboratories were asked to recommend changes or
describe problem areas in the test guidelines. They had no difficulties in
the rearing of the animals. It is recommended, however, to not overpopulate
the culture tanks when planning for acute tests because the larger instars
are more difficuJt to distinguish under crowded conditions.
The chronic tests gave the most problems to the participating labora-
tories. Their recommendations included a more detailed description of the
chambers, a narrower range on the flow rate, treating the first 48 hrs of the
test as a static to prevent loss of the pelagic larvae, and allowing more
eggs to be used in starting tests. The use of more eggs would reduce egg
manipulation time. Another suggestion was to use pupal cases from which the
adults had emerged as a measure of reproduction which would eliminate the
problems with retaining and capturing the adults in a flow-through system.
If pupal cases are used, the effect of the toxicant on adult emergence could
not be determined. This may be a sensitive endpoint and the researcher must
make the decision about the loss of that data. It was also suggested that,
rather than removing thlT adults, egg production in the original test chambers
be monitored and compared with the control as this appeared to be a sensitive
measure for the chemicals tested in this series.
Several of the laboratories are private testing organizations.
Determining the cost of performing a test is an important oroblem in their
bidding process. These laboratories recommended the following:
(1) The client organization is asked to produce a set of data forms
which reflects the actual data needed during each day. These forms shou.'.d
include spaces for such items aa aniwal numbers, number of routine chemistry
analyses, nAmber of analytical chemistry analyses, times for sample
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collecting and any other data such as physical conditions of the test systems
needed bv the client. For an acute exposure, they recommend a three sheet
set with one sheet for the initial day, 24 hr and 48 hrs of exposure.
(2) In, the life cycle exposures, a set of dafa forms for each day .plus
an initial day and a final compilation sheet complete with the statistical
formulas enumerated in such a way as to allow filling in the spaces with the
required information and performing the associated arithmetic. Enumeration
of the number and type of chemical analyses, the days the analyses should be
done, and quality assurance needs such as slope and correlation (R^) of the
standard curves are also requested.
(3) All participants requested that some training should be given prior
to the conducting of any test. All participants were of the opinion that if
training and a set of data fortius were prepared the guidelines were
sufficient.
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Table 1. Quality of Exposure Water for Paratanytarsus
sp. Toxicity Tests
Test Hardness
Facility Temperature pH as CaC03 Alkalinity
osu
19.9-22.1
7.6
650
127
OKS
19.4-20.5
8.r-8.2
148-170
150-161
NAS
20
6.5-7.7
40-60
20-30
RRI
19-22
7.6
46
37
CAL
19 .3-22 .A
7.2-7.75
640-735
120-205
ERL-D
20-21.5
7.6-7.8
47-
43-44
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Table 2. Methods of Toxicant AnaLysis
Acenaphthene
Trichlorophenol
osu
Fluorescence spectrophotometer
Gas chromatograph solvent extract
OKS
Fluorescence spectrofluororaeter
Gas chromatograph solvent extract
NAS
Capillary gas chromatograph
Capillary gas chromatograph
SRI
High pressure liquid chromatography
High pressure liquid chromatography
CAL
Gas chromatograph
Gas chromatograph
ERL-D
Fluorescence spfictrofluorometer
Auto analyzer direct colortnetric
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Table 3. LC50 Acute Values with a 95% Confidence Interval for the
Chironomid Paratanytarsus sp.a
Test
Facility Acenaphthene Tr ichlorophenol
osu
No Results
No Results
21.8
45.4
16.7-28.4
35.0-59.0
OKS
1.62*
1.65
23.6
27.2
1.45-1.81
1 49-1.86
15.2-41.5
6.9
NAS
2.00
2.09
10.3
16.3
1.69-2.36
1.83-2.39
8.4-12.7
13.1-20.7
SRI
.06
.07
41.9
65.1
sC
O
1
o
.06-.07
37.9-59.8
52.0-81.4
CAL
No Results
No Results
43
41
30-61
29-58
ERL-D
.14
.47
3.7
2.5
.10-.19
.41-.53
2.6-5.4
.4-15.8
a Values oresented by participants
* All values are mi?/1
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Table
4. Life Cycle Effect Values
for the
Chironomid
Paratanytarsus sp.
a
AcenaphLhene
Trichlorophenol
OSU
>.8*
.49**
5.0
>10
.3-.8
2.5-10
OKS
No
Results
No
Results
NAS
.23
.035
.72
.69
.18-.30
.027-.044
.55-.93
.50-.94
SRI
.014
.024
<.93
.76
.01-.02
.02-.03
.49-1.18
CAL
.095
.057
1.49
2.6
.06-. 15
.03-.U
1.3-1.7
1.6-4.2
ERL-T)
.91
.40
.11
.38
1.30-.64
.27-.58
.18-.53
.22-.65
® Values presented hv participants
* All values are m«/l
** Geometric mean of no effect/effect concentration
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