Peer Review Draft Risk Evaluation for Trichloroethylene Systematic Review Supplemental File: Data Quality Evaluation of Environmental Hazard Studies CASRN: 79-01-6 February 2020


PEER REVIEW DRAFT. DO NOT CITE OR QUOTE
SEPA
United States	Office of Chemical Safety and
Environmental Protection Agency	Pollution Prevention
Risk Evaluation for
T richloroethylene
Systematic Review Supplemental File:
Data Quality Evaluation of Environmental Hazard Studies
CASRN: 79-01-6
CI. H
CI CI
February 2020

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33
Table of Contents
Data Type
Acute (0-96 hour); Aquatic; Inver-
tebrates
Other; Aquatic; Fish
Acute (0-96 hour); Aquatic; Fish
Acute (0-96 hour); Aquatic; Fish
Acute (0-96 hour); Aquatic; Fish
static
Acute (0-96 hour); Aquatic; Fish
flow-through
Other; Aquatic; other South
African clawed frog
Acute (0-96 hour); Aquatic; Inver-
tebrates
Acute (0-96 hour); Aquatic; Plants
Acute (0-96 hour); Aquatic; Plants
Reference
Leblanc, G. A.. 1980. Acute toxicity of priority pollutants to water flea (Daph-
nia magna). Bulletin of Environmental Contamination and Toxicology 24:684-
691
Barrows, M. E.,Petrocelli, S. R.,Macek, K. J.,Carroll, J. J.. 1980. Bioconcen-
tration and elimination of selected water pollutants by bluegill sunfish (Lepomis
macrochirus).
Buccafusco, R. J.,Ells, S. J.,Leblanc, G. A.. 1981. Acute toxicity of priority
pollutants to bluegill (Lepomis macrochirus). Bulletin of Environmental Con-
tamination and Toxicology 26:446-452
Geiger, D. L.,Northcott, C. E.,Call, D. J.,Brooke, L. T. eds. 1985. Acute toxi-
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chloroethylene, trichloroethylene, 1,1,1-trichloroethane, and methylene chloride
to fathead minnows. Bulletin of Environmental Contamination and Toxicology
20:344-352
Alexander, H. C.,McCarty, W. M.,Bartlett, E. A.. 1978. Toxicity of per-
chloroethylene, trichloroethylene, 1,1,1-trichloroethane, and methylene chloride
to fathead minnows. Bulletin of Environmental Contamination and Toxicology
20:344-352
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green algae: A new assay. 1:223-228

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tebrates
Other; Aquatic; other amphibian -
wood frog and green frog
Other; Aquatic; other amphibian -
american toad
Other; Aquatic; other amphibian -
spotted salamder
Other; Aquatic; other amphibian -
frog
Other; Aquatic; other amphibian -
frog
Acute (0-96 hour); Aquatic; Inver-
tebrates
Other; Aquatic; Invertebrates
Acute (0-96 hour); Aquatic; Plants
Acute (0-96 hour); Aquatic; Inver-
tebrates
Other; Aquatic; Fish
Acute (0-96 hour); Aquatic; Plants
Yoshioka, Y.,Ose, Y.,Sato, T.. 1985. Testing for the toxicity of chemicals with
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of Environmental Contamination and Toxicology 47:101-109
McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects
of chlorinated solvents on four species of North American amphibians. Archives
of Environmental Contamination and Toxicology 47:101-109
McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects
of chlorinated solvents on four species of North American amphibians. Archives
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LETHAL TOXICITY OF HYDROCARBONS AND CHLORINATED HY-
DROCARBONS TO TWO PLANKTONIC CRUSTACEANS THE KEY ROLE
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TEST METHODS OF LEVELS I AND II OF THE CHEMICALS ACT ON
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TEST METHODS OF LEVELS I AND II OF THE CHEMICALS ACT ON
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2127844
Acute (0-96 hour); Aquatic; Plants
2127941
Acute (0-96 hour); Aquatic; Inver-
tebrates
2128165
Acute (0-96 hour); Aquatic; Plants
2298399
Acute (0-96 hour); Aquatic; Fish
static
2298399
Acute (0-96 hour); Aquatic; Fish
flow-through
2298399
Chronic (>21 days); Aquatic; Fish
2307041
Other; Aquatic; other
2800252
Other; Aquatic; Fish
2801609
Acute (0-96 hour); Aquatic; Plants
2801609
Acute (0-96 hour); Aquatic; Fish
Bacsi, I.,Toeroek, T.,B-Beres, V.,Toeroek, P.,Tothmeresz, B.,Nagy, A. S.,Vasas,	95
G.. 2013. Laboratory and microcosm experiments testing the toxicity of chlo-
rinated hydrocarbons on a cyanobacterium strain (Synechococcus PCC 6301)
and on natural phytoplankton assemblages. Hydrobiologia 710:189-203
Dobaradaran, S.,Mahvi, A. H.,Nabizadeh, R.,Ramavandi, B.,Nazmara, S.,Zarei,	98
S.. 2012. BIOASSAY COMPARISON OF TRICHLOROETHYLENE (TCE)
TOXICITY ON DAPHNIA MAGNA (D. MAGNA) BEFORE AND AFTER
ULTRASOUND AND PHOTOLYSIS PROCESSES. Fresenius Environmental
Bulletin 21:1533-1538
Lukavsky, J.,Furnadzhieva, S.,Dittrt, F.. 2011. Toxicity of Trichloroethylene	101
(TCE) on Some Algae and Cyanobacteria. Bulletin of Environmental Contam-
ination and Toxicology 86:226-231
Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J.	105
A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute and
chronic toxicity of 10 chlorinated organic-compounds to the american flagfish
(jordanella-floridae). Archives of Environmental Contamination and Toxicology
20:94-102
Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J.	108
A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute and
chronic toxicity of 10 chlorinated organic-compounds to the american flagfish
(jordanella-floridae). Archives of Environmental Contamination and Toxicology
20:94-102
Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J.	Ill
A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute and
chronic toxicity of 10 chlorinated organic-compounds to the american flagfish
(jordanella-floridae). Archives of Environmental Contamination and Toxicology
20:94-102
Rayburn, D. J. Deyoung, J. A. Bantle, D. J. Fort, R. Mcnew. 1991. Altered	114
developmental toxicity caused by three carrier solvents. Journal of Applied
Toxicology 11:253-260
Hayashi, M.,Ueda, T.,Uyeno, K.,Wada, K.,Kinae, N.,Saotome, K.,Tanaka,	118
N.,Takai, A.,Sasaki, Y. F.,Asano, N.,Sofuni, T.,Ojima, Y.. 1998. Development
of genotoxicity assay systems that use aquatic organisms. Mutation Research
399:125-133
Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF	121
TRICHLOROETHYLENE TO SALTWATER ORGANISMS. Bulletin of Envi-
ronmental Contamination and Toxicology 37:830-836
Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF	124
TRICHLOROETHYLENE TO SALTWATER ORGANISMS. Bulletin of Envi-
ronmental Contamination and Toxicology 37:830-836

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138
142
146
149
153
156
159
162
165
Acute (0-96 hour); Aquatic; Inver-
tebrates
Acute (0-96 hour); Aquatic; Fish
Acute (0-96 hour); Aquatic; Plants
Other; Aquatic; Invertebrates
Other; Aquatic; Invertebrates
Other; Aquatic; Plants
Other; Aquatic; Invertebrates
Acute (0-96 hour); Aquatic; Fish
Acute (0-96 hour); Aquatic; Inver-
tebrates
Acute (0-96 hour); Aquatic; Plants
Other; Aquatic; Fish
Acute (0-96 hour); Aquatic; Fish
Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF
TRICHLOROETHYLENE TO SALTWATER ORGANISMS. Bulletin of Envi-
ronmental Contamination and Toxicology 37:830-836
Dierickx, P. J.. 1993. Comparison between fish lethality data and the in vitro
cytotoxicity of lipophilic solvents to cultured fish cells in a two-compartment
model. Chemosphere 27:1511-1518
Bacsi, I.,Gonda, S.,B-Beres, V.,Novak, Z.,Nagy, S. A.,Vasas, G.. 2015. Alter-
ations of phytoplankton assemblages treated with chlorinated hydrocarbons: ef-
fects of dominant species sensitivity and initial diversity. Ecotoxicology 24:823-
834
Houde, M.,Douville, M.,Gagnon, P.,Sproull, J.,Cloutier, F.. 2015. Exposure of
Daphnia magna to trichloroethylene (TCE) and vinyl chloride (VC): evaluation
of gene transcription, cellular activity, and life-history parameters. Ecotoxicol-
ogy and Environmental Safety 116
Vidal, M.,Basseres, A.,Narbonne, J.. 2001. Potential biomarkers of
trichloroethylene and toluene exposure in Corbicula fluminea. Environmental
Toxicology and Pharmacology 9:87-97
Ando, T.,Otsuka, S.,Nishiyama, M.,Senoo, K.,Watanabe, M. M.,Matsumoto, S..
2003. Toxic Effects of Dichloromethane and Trichloroethylene on the Growth
of Planktonic Green Algae, Chlorella vulgaris NIES227, Selenastrum capricor-
nutum NIES35, and Volvulina steinii NIES545. 18:43-46
Yoshioka, Y.,Ose, Y.,Sato, T.. 1986. Correlation of the Five Test Methods to
Assess Chemical Toxicity and Relation to Physical Properties. 12:15-21
Yoshioka, Y.,Ose, Y.,Sato, T.. 1986. Correlation of the Five Test Methods to
Assess Chemical Toxicity and Relation to Physical Properties. 12:15-21
Yoshioka, Y.,Ose, Y.,Sato, T.. 1986. Correlation of the Five Test Methods to
Assess Chemical Toxicity and Relation to Physical Properties. 12:15-21
Tsai, K. P.,Chen, C. Y.. 2007. An Algal Toxicity Database of Organic Toxi-
cants Derived by a Closed-System Technique. Environmental Toxicology and
Chemistry 26:1931-1939
Schell, J. D. J.. 1987. Interactions of Halogenated Hydrocarbon Mixtures in
the Embryo of the Japanese Medaka (Oryzias latipes).
Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund,
M. D.. 2005. A Comparison of the Lethal and Sublethal Toxicity of Organic
Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127

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3665276
Chronic (>21 days); Aquatic; Fish
Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund,
M. D.. 2005. A Comparison of the Lethal and Sublethal Toxicity of Organic
Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
iro
3683110
Chronic (>21 days); Aquatic; In-
vertebrates
Leblanc, G. A.,Surprenant, D. C.. 1980. The Chronic Toxicity of 8 of the 65
Priority Pollutants to the Water Flea (Daphnia magna).
176
<

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Study Citation: Leblanc, G. A.. 1980. Acute toxicity of priority pollutants to water flea (Daphnia magna). Bulletin of Environmental Contamination
and Toxicology 24:684-691
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 7508
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1: Test Substance Identity High x 2 2 Chemical is identified by name.
Metric 2: Test Substance Source Low X 1 3 All chemicals tested were purchased from commer-
cial chemical suppliers, but the study does not spec-
ify what supplier provided TCE.
Metric 3: Test Substance Purity Low X 1 3 Study reports a minimum purity of 80 percent for
all chemicals tested, but does not specify what the
purity is for TCE.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
High X 2 2 Negative control consisted of the same dilution wa-
ter, test conditions, and test organisms, but contain-
ing no test substance of co-solvent. When appropri-
ate a solvent control was also used.
High X 1 1 Mortality among water flea control populations
never exceeded 10 percent in any test.
High X 1 1 Five daphnids were randomly placed in each 150 mL
test solution within 30 minutes of the solution prepa-
ration.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara-
tion
Low
Metric 8: Consistency of Exposure Administration
Metric 9: Measurement of Test Substance Concentra-
tion
x 2
Medium
Low
x 1
x 2
It appears the volatility of TCE might have been
taken into account in the test methods, but it's un-
clear. The study reports that, generally, "The tests
were also conducted in unreplicated 500 mL solu-
tions containing 15 daphnids if dividing the solution
into triplicate test vessels presented a risk of the loss
of the test substance through volatilization or if va-
pors of the substance posed a high health risk to the
investigators. In addition, these vessels were covered
with plastic wrap secured with an elastic band."
Only minor uncertainties
Measurements were not reported and the test sub-
stance is volatile.
Continued on next page

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. continued from previous page
Study Citation: Leblanc, G. A.. 1980. Acute toxicity of priority pollutants to water flea (Daphnia magna). Bulletin of Environmental Contamination
and Toxicology 24:684-691
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 7508
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 10: Exposure Duration and Frequency
High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High x 1
Exposure duration is 48 hours which is OECD TG
202 recommended. Only one dose for the 48 hour
period as this is an acute study.
5-8 exposure groups were used for each chemical. No
range finding was conducted to determine an appro-
priate exposure, but it appears they were appropri-
ate enough to establish an LD50.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Low
Group
Metric 16: Adequacy of Test Conditions
High X 2 2 Daphnia magna less than 24 hours old were used in
these toxicity tests from laboratory stocks cultured
at EG&G, Bionomics.
X 1 3 Study didn't report whether test organisms were ac-
climatized.
X 1 3 It appears there were 15 daphnia in each test con-
centration for TCE and no replicates to avoid losing
TCE to vitalization. OECD TG 202 recommends at
least 20 and separated into 4 different vessels.
High X 1 1 Ph, dissolved 02, temp, and 2ml per organism
were all appropriate for daphnia magna according
to OECD TG 202.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
Mortality data from these toxicity tests were used
to calculate a median lethal concentration and its
95 percent confidence limits based on nominal con-
centrations.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2
Procedures
Metric 20: Outcomes Unrelated to Exposure High x 1 1
Domain 7: Data Presentation and Analysis
Continued on next page . . .

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. continued from previous page
Study Citation: Leblanc, G. A.. 1980. Acute toxicity of priority pollutants to water flea (Daphnia magna). Bulletin of Environmental Contamination
and Toxicology 24:684-691
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 7508
Domain Metric Rating^ MWF* Score Comments^
Metric 21: Statistical Methods High x 1 1 The LC50's and 95 percent confidence limits were
calculated utilizing a moving average angle method
(Stephan, personal communication) when possible.
With the the moving average angle method, the
nominal test concentrations were transformed to log-
arithms and the corresponding percentage mortali-
ties to angles. Each group of three successive angles
was then averaged and the LC50 was estimated by
linear interpolation between the successive concen-
trations whose average angles bracketed 45 degrees.
When test data did not meet the moving average an-
gle method requirements, the LC50's were estimated
by probit analysis by converting the concentrations
to logarithms and percentage mortalities to probits
and then calculating a least squares linear regres-
sion analysis. Finally, if the data did not permit a
probit analysis, then a binomial probability analysis
was performed on these data. Calculations were per-
formed with a Hewlett-Packard Hodel 9815A calcu-
lator programed to scan the data base and to select
the most appropriate of the three methods described
above for the calculation of an LC50. If no mortal-
ity was observed in water flea populations exposed to
a nominal concentration of approximately 500 mg/
L, no additional concentrations were tested and the
LC50 was estimated to be greater than the highest
nominal concentration tested (>530 mg/L). LC50's
and 95 percent confidence limits were reported after
rounding to 2 significant figures. No correction was
made for control mortality.
Metric 22: Reporting of Data Medium X 2 4 Data for most but not all outcomes by study group
were reported but these minor uncertainties or limi-
tations are unlikely to have a substantial impact on
results.
Metric 23: Explanation of Unexpected Outcomes High x 1 1 No unexplained outcomes reported
Overall Quality Determination"'"
High
1.6
Extracted
Yes

Continued on next page . . .

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. continued from previous page
Study Citation: Leblanc, G. A.. 1980. Acute toxicity of priority pollutants to water flea (Daphnia magna). Bulletin of Environmental Contamination
and Toxicology 24:684-691
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	7508
Domain	Metric	Rating^ MWF* Score	Comments^
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Barrows, M. E.,Petrocelli, S. R.,Macek, K. J.,Carroll, J. J.. 1980. Bioconcentration and elimination of selected water pollutants by
bluegill sunfish (Lepomis macrochirus).
Data Type: Other; Aquatic; Fish
Hero ID: 18050
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High
High
Low
X 2 2 Chemical identified as 1,1,2-trichloroethylene
X 1 1 Each compound was received in individual, sealed
vials from, New England Nuclear, Boston, Mas-
sachusetts, and held under refrigerated conditions
until used.
X 1 3 No purity of test chemical was reported. Liquid gas
chromatography was preformed during the experi-
ment and purity of the chemical could be determined
then, but it wasn't reported in the paper.
Domain 2: Test Design
Metric 4:
Metric 5:
Negative Controls
Negative Control Response
Metric 6: Randomized Allocation
High
High
Low
x 2
x 1
x 1
Concentrations of TCE in fish tissue were measured
in controls and compared to test species.
Method for allocation was not reported.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High
tion
Metric 8: Consistency of Exposure Administration
High
x 2
x 1
"Steps were taken to minimize vitalization. Each
test aquaria was a closed system for the volatile
organics. Each fish was wrapped in Para film" at
each sampling to minimize the volatilization of each
chemical from thefish tissue during the combustion
process."
Continued on next page

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. continued from previous page
Study Citation: Barrows, M. E.,Petrocelli, S. R.,Macek, K. J.,Carroll, J. J.. 1980. Bioconcentration and elimination of selected water pollutants by
bluegill sunfish (Lepomis macrochirus).
Data Type: Other; Aquatic; Fish
Hero ID: 18050
Domain
Metric
Rating1
MWF* Score
Comments' t
Metric 9: Measurement of Test Substance Concentra- High
tion
x 2
Metric 10: Exposure Duration and Frequency
High
Metric 11: Number of Exposure Groups/Spacing of Ex- High
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
x 1
x 1
Half-life was defined as the period of time required
for one-half of the mean chemical residues measured
in fish tissues at equilibrium or at the end of the
exposure period to be eliminated following termina-
tion of exposure. The specific activity of e lch car-
bon 14-hibelcd stock solution was measured radio-
metrically prior to test initiation. During each test
exposure, representative water and fish samples, in-
cluding samples from control aquaria, were collected
Oil days 0, I, 2, 4, 7, 10, 14, 21 and 28 (if exposure
continued for the entire 28 days) and analyzed for
14 (-residues or metal concentrations as appropri-
ate. Fish were sampled on days 1, 2, 4 and 7 to
estimate the half-life of chemical residues in tissues.
Exposure was for 14 days. OECD recommends 28
but shorter is ok if it takes less time for the fish
residues to reach a steady state in relation to the
chemical in the water. Authors mentioned that this
was the case. Study used a continuous flow through
system for the 14 days.
"The levels of exposure were selected on the basis of
acute toxicity datagenerated at Bionomics and were
intended to be sublethal during thecontinuous expo-
sure period."
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Medium
Metric 14: Acclimitization and Pretreatment Conditions Medium
Metric 15: Number of Organisms and Replicates per Medium
Group
x 2
x 1
x 1
Minor reservations about the source of fish.
Three populations of bluegill sunfish (Lepomis
macrochirus) were obtained from a commercial fish
farmer in Connecticut. May not all be the same age,
but length and weight was documented, and age may
not be a big factor in determining BCF.
Fish were maintained in the holding facilities for a
minimum of 30 days prior to the initiation of the
study. Minor uncertainties in the details provided.
Study started with 100 organisms per exposure
group, and took fish out 5 fish on each sampling
day. OECD recommends having enough to remove
at least 4. Unsure the number of replicates.
Continued on next page

Metric 17: Outcome Assessment Methodology
Unacceptable
X
2
8
BCFs, which measure bioconcentration, were out of
scope for the environmental hazard assessment por-
tion of the risk evaluation. These were assessed else-
where in the risk evaluation. Also, in this study BCFs
and half-lives were reported for each of the chemi-
cals. Assessment was not as sensitive as it should
be for calculating a BCF - OECD recommends not-
ing if both sexes are used, differences in growth and
lipid content between sexes should be documented to
be non-significant before the start of the exposure,
in particular if it is anticipated that pooling of male
and female fish will be necessary to ensure detectable
substance concentrations and/or lipid content. This
was not noted.
Metric 18: Consistency of Outcome Assessment
Medium
X
1
2
Incomplete reporting of minor details of outcome as-
sessment protocol execution.
Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test Design and
Low
X
2
6
OECD recommends noting If both sexes are used,
Procedures

differences in growth and lipid content between sexes

should be documented to be non-significant before
the start of the exposure, in particular if it is antic-
ipated that pooling of male and female fish will be
necessary to ensure detectable substance concentra-
tions and/or lipid content. This was not noted.
Metric 20: Outcomes Unrelated to Exposure
Medium
X
1
2
Data on attrition and health outcomes unrelated to
exposure were not reported for each study group.
Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
High
X
1
1
A steady-state bioconcentration factor (BCF) was
calculated as the quotient of the mean chemical con-
centration measured in fish tissues during equilib-
rium divided by the mean measured chemical con-
centration in water during the entire exposure period
(Table 111).
Continued on next page . . .

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. continued from previous page
Study Citation: Barrows, M. E.,Petrocelli, S. R.,Macek, K. J.,Carroll, J. J.. 1980. Bioconcentration and elimination of selected water pollutants by
bluegill sunfish (Lepomis macrochirus).
Data Type:	Other; Aquatic; Fish
Hero ID:	18050
Domain	Metric	Rating^	MWF* Score	Comments^
Metric 22: Reporting of Data	Medium
Metric 23: Explanation of Unexpected Outcomes	High
x 2
x 1
Not all regressions, lipid content, and weights were
reported, but BCFs and half lives were reported for
all chemicals.
No unexplained outcomes.
Overall Quality Determination"'"
Unacceptable
4.0
Extracted
Yes


** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Buccafusco, R. J.,Ells, S. J.,Leblanc, G. A.. 1981. Acute toxicity of priority pollutants to bluegill (Lepomis macrochirus). Bulletin of
Environmental Contamination and Toxicology 26:446-452
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 18064
Domain
Metric
Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High
Low
Low
x 2
x 1
x 1
The study says all chemicals tested were purchased
from commercial chemical suppliers, but does not
specify where TCE came from.
Study reports a minimum purity of 80 percent for
all chemicals tested, but does not specify what the
purity is for TCE.
Domain 2: Test Design
Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
High
Low
High
X 2 2 Both negative and solvent controls were used.
X 1 3 There were many chemicals tested and do not give
details about negative control response, although it
says control mortality was recorded.
X 1 1 " Ten fish were randomly selected from a test popu-
lation and added to each test jar within 30 min after
the addition of the test chemical or stock solution."
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Medium x 2 4
tion
Metric 8: Consistency of Exposure Administration High x 1 1
Metric 9: Measurement of Test Substance Concentra- Low x 2 6
tion
Metric 10: Exposure Duration and Frequency High x 1 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Low x 1 3
posure Levels
Volatile chemicals were capped, but it is unclear
whether headspace was minimized in the jars and
with the jars capped could have had low DO con-
tent.
Nominal concentrations were used and were not
measured. TCE is volatile.
Exposure is 96 hours which is OECD TG 203 rec-
ommended.
Study says that the test was conducted according to
EPA's "Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians" which says for
static tests you must have 10 organisms in each
treatment divided into at least two test chambers;
not sure how they got the exposure concentrations
used of what the exposure concentrations were.
Continued on next page

-------
. continued from previous page
Study Citation: Buccafusco, R. J.,Ells, S. J.,Leblanc, G. A.. 1981. Acute toxicity of priority pollutants to bluegill (Lepomis macrochirus). Bulletin of
Environmental Contamination and Toxicology 26:446-452
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 18064
Domain
Metric
Rating^
MWF*
Score
Comments^
Metric 12:
Testing at or Below Solubility Limit
Low
X
1
3
Test substance concentration was not reported, and

it was reported that some concentration there was

undessolved chemical.
Domain 4: Test Organism

Metric 13:
Test Organism Characteristics
Medium
X
2
4
Test animals utilized were young of the year bluegill

(L. macrochirus) obtained from commercial fish sup-

pliers within the continental United States.
Metric 14:
Acclimitization and Pretreatment Conditions
Medium
X
1
2
While it was reported that there was a 48 hour time

where fish were observed and not used if had >3

percent mortality, it was not reported whether they

were held for 12 days in the lab before they are used

for testing.
Metric 15:
Number of Organisms and Replicates per
Medium
X
1
2
Minor uncertainties around number of organisms

Group

used.
Metric 16:
Adequacy of Test Conditions
Medium
X
1
2
Minor uncertainties around housing conditions

(headspace in jar, DO cones).
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology High x 2 2 24 and 96 hour LC50s
Metric 18: Consistency of Outcome Assessment High x 1 1
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Low x 2 6 Study did not provide enough information to allow
Procedures a comParison °f environmental conditions.
Metric 20: Outcomes Unrelated to Exposure Low x 1 3 Study did not provide enough information about
health outcomes of each study group.
Domain 7: Data Presentation and Analysis
Continued on next page . . .

-------
. continued from previous page
Study Citation: Buccafusco, R. J.,Ells, S. J.,Leblanc, G. A.. 1981. Acute toxicity of priority pollutants to bluegill (Lepomis macrochirus). Bulletin of
Environmental Contamination and Toxicology 26:446-452
Data Type:	Acute (0-96 hour); Aquatic; Fish
Hero ID:	18064
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 21: Statistical Methods
Medium x 1
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
Low
High
x 2
x 1
Not clear what method was used for TCE: "The
LC50s and 95 percent confidence intervals were cal-
culated, where possible, by the moving average an-
gle method (HARRIS 1959). The nominal test con-
centrations were transformed to logarithms and cor-
responding percentage mortalities to angles. Each
group of these successive angles was then averaged
and the LC50 was estimated by linear interpolation
between the successive concentrations whole aver-
age angles bracketed 45". When the test data did
not meet Harris' method requirements, the LC50s
were calculated by the log probit method, a modifi-
cation of the LITCHFIELD + WILCOXON (1949)
method."
The data for the static test were not presented in
full, and no information was reported for controls.
There were no unexpected outcomes for TCE in this
paper.
Overall Quality Determination"'"
Medium
2.0
Extracted
Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Geiger, D. L.,Northcott, C. E.,Call, D. J.,Brooke, L. T. eds. 1985. Acute toxicities of organic chemicals to fathead minnows (Pimephales
promelas): volume II.
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 32169
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Trichloroethylene identified by name, CAS, formula,
and molecular weight.
Metric 2:
Test Substance Source
High
X
1
1
Aldrich Chemical Co
Metric 3:
Test Substance Purity
Medium
X
1
2
Purity reported at 98 percent, no mention of what
impurities were.
Domain 2: Test Design
Metric 4: Negative Controls Medium X 2 4 Two controls reported. Unsure what kind (water or
solvent?)
Metric 5: Negative Control Response High x 1 1 Response of control was reported.
Metric 6: Randomized Allocation High X 1 1 At the start of a test, individual fish were removed
from the common pool of fish with a net and dis-
tributed at random among the exposure chambers.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
Metric 8: Consistency of Exposure Administration
x 2
Medium x 1
Metric 9: Measurement of Test Substance Concentra- High x 2 2
tion
Metric 10: Exposure Duration and Frequency High x 1 1
Flow through system used using cycling proportional
diluters with duplicate tanks for each test conc. It's
unclear exactly what system was used for TCE be-
cause the description at the beginning of the paper
is non-specific. It seems like the following system
was used: The electronic diluter was used for ex-
pensive and volatile chemicals or when acute toxicity
was very close to water solubility. Another form of
a liquid-liquid equilibrator was constructed from a
2.8 L culture flask atop a magnetic stirrer. A pump
forced lake water into this closed system which con-
tained a layer of the chemical.
Details of exposure administration was reported but
it's unclear what type of administration applies to
what chemicals.
Test concentrations were measured by gas-liquid
chromatography.
96 hour exposure using flow-through method for
TCE
Continued on next page

-------
. continued from previous page
Study Citation:
Data Type:
Hero ID:
Geiger, D. L.,Northcott, C. E.,Call, D. J.,Brooke, L. T. eds. 1985. Acute toxicities of organic chemicals to fathead minnows (Pimephales
promelas): volume II.
Acute (0-96 hour); Aquatic; Fish
32169
Domain

Metric
Rating^
MWF*
Score
Comments^

Metric 11:
Metric 12:
Number of Exposure Groups/Spacing of Ex-
posure Levels
Testing at or Below Solubility Limit
Medium
High
X 1
X 1
2
1
Five exposure concentrations were used, and mea-
sured concentrations were used, but it's unclear how
exactly an appropriate cone was determined. No
limit test is mentioned, only that test mean concen-
trations were usually >60 percent of saturation. The
toxicant concentrations were 100, 80, 60, 40, and 20
percent of the toxicant stock. In cases where test
chemical quantities were limited, toxicant concen-
trations were condensed to 100, 66, and 33 percent
of the toxicant stock.
Measured cone were reported and are below solubil-
ity.
Domain 4: Test Organism
Metric 13:
Test Organism Characteristics
High
x 2
2
Fathead minnows used in this test.
Continued on next page . . .

-------
. . . continued from previous page
Study Citation: Geiger, D. L.,Northcott, C. E.,Call, D. J.,Brooke, L. T. eds. 1985. Acute toxicities of organic chemicals to fathead minnows (Pimephales
promelas): volume II.
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 32169
Domain Metric Rating^ MWF* Score Comments^
Metric 14: Acclimitization and Pretreatment Conditions Medium x 1
Metric 15: Number of Organisms and Replicates per Low x 1 3
Group
Metric 16: Adequacy of Test Conditions Medium x 1 2
Only minor uncertainties. Study reports, "Fat-
head minnows used in the tests were cultured at
the U.S. EPA Environmental Research Laboratory-
Duluth and the University of Wisconsin-Superior
campus. Adults were held at 25" C in flowing wa-
ter with a 16 hr light-controlled photo-period and
fed frozen adult brine shrimp (Artemia sp.). They
were provided with asbestos pipes (cut in half longi-
tudinally) as spawning substrates, where naturally
spawned and fertilized embryos attached to the un-
derside. The substrates, with intact embryos, were
removed daily and placed in another 25C bath where
hatching occurred; however the spawning substrates
were removed just prior to hatching at the UW-
Superior culture unit, then placed in a rearing bath.
For tests conducted in 1977-1982, newly hatched lar-
vae from the stock culture unit were reared in a sys-
tem similar to the exposure systems at a temper-
ature of 25C. Tests conducted following 1982 used
fish that had been reared in flow-through tanks in
the lab Is culture unit. Larvae were fed 40-48 hr
old brine shrimp nauplii (Bio-Marine Research, Inc.,
Hawthorne, CA) in excess two times daily (once on
week-end days). Embryos and larvae were cultured
in water from the same source as used in the ex-
pos.ures to the test chemicals. Fish that were ap-
proximately 28-34 days old were used in the toxicity
tests." Note, it is acceptable to use asbestos pipes
for spawning purposes.
Number of test organisms was not reported for stud-
ies prior to 1982. The TCE test was in 1979.
Only minor uncertainties. The temperature is ap-
propriate for fathead minnows according to OECD
guidelines (OECD TG 203).
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High x 2 2 An LC50 was derived.
High X 1 1 Consistent assessment for outcomes in each study
group.
Continued on next page

-------
. .. continued from previous page
Study Citation: Geiger, D. L.,Northcott, C. E.,Call, D. J.,Brooke, L. T. eds. 1985. Acute toxicities of organic chemicals to fathead minnows (Pimephales
promelas): volume II.
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 32169
Domain Metric Rating^ MWF* Score Comments^
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure Medium x 1
2 No reported differences between study groups.
2 Data on attrition or health impacts unrelated to
exposure were not reported for each study group,
because only substantial differences among groups
were noted.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
High
x 1
Metric 22: Reporting of Data High x 2 2
Metric 23: Explanation of Unexpected Outcomes High x 1 1
The estimated LC50 (lethal concentration causing
50 percent mortality of thefish) and EC50 (effect
concentration causing 50 percent of the fish to show
aneffect) with corresponding 95 percent confidence
intervals were calculated using the corrected aver-
ages of the analyzed tank concentrations and the
Trimmed Spearman-Karber Method (Hamilton et
al., 1977). In cases of duplicate exposures, the effect
and mortaHty data were pooled, resulting in a sin-
gle estimate of LC50 and EC50 per test. The EC50s
are based upon loss of equilib.rium manifested by
the fish's inability to maintain an upright position
when swimming. Calculations were made for 96 hr of
exposure and also for intermediate exposure times.
The mean toxicant concentrations used in the cal-
culations were corrected for analytical recoveries of
spiked water samples. Some LC50 data may vary
slightly from previously published literature due to
the inclusion of a spike recovery factor in this vol-
ume.
Each exposure group results were reported.
No unexpected outcomes were reported.
Overall Quality Determination"'"
High
1.5
Extracted
Yes

Continued on next page

-------
. continued from previous page
Study Citation: Geiger, D. L.,Northcott, C. E.,Call, D. J.,Brooke, L. T. eds. 1985. Acute toxicities of organic chemicals to fathead minnows (Pimephales
promelas): volume II.
Data Type:	Acute (0-96 hour); Aquatic; Fish
Hero ID:	32169
Domain	Metric	Rating^ MWF* Score	Comments^
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Alexander, H. C.,McCarty, W. M.,Bartlett, E. A.. 1978. Toxicity of perchloroethylene, trichloroethylene, 1,1,1-trichloroethane, and
methylene chloride to fathead minnows. Bulletin of Environmental Contamination and Toxicology 20:344-352
Data Type: Acute (0-96 hour); Aquatic; Fish static
Hero ID: 58126
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1: Test Substance Identity High X 2 2 trichloroethylene identified by name
Metric 2: Test Substance Source Medium X 1 2 Authors work for chemical company that produces
TCE.
Metric 3: Test Substance Purity Low X 1 3 Not indicated, but because TCE is a common sol-
vent produced by the company, assuming at mini-
mum technical grade.
Domain 2: Test Design
Metric 4: Negative Controls High x 2 2
Metric 5: Negative Control Response Low x 1 3 Not indicated, only LC50 for TCE reported., but
no deviances from test guideline [COMMITTEE
ON METHODS FOR TOXICITY TESTS WITH-
AQUATIC ORGANISMS (1975)] indicated.
Metric 6: Randomized Allocation Low X 1 3 Randomization not indicated, but followed test
guidelines.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara-
tion
Metric 8: Consistency of Exposure Administration
Metric 9: Measurement of Test Substance Concentra-
tion
Metric 10: Exposure Duration and Frequency
Metric 11: Number of Exposure Groups/Spacing of Ex-
posure Levels
Metric 12: Testing at or Below Solubility Limit
Low
High
Low
High
Low
High
x 2
x 1
x 2
x 1
x 1
x 1
Covered with plastic wrap, but no indication that
headspace minimized.
No measurements were made in the static test and
this chemical is very volatile. It was mentioned that
containers were covered but there was no mention of
headspace minimizing. "In the static water fish toxi-
city tests the nominal concentration, or that amount
of chemical initially added to the test chambers, was
used to calculate the LC 10, LC SO, and LC 90 tox-
icity values."
Test concentrations not provided.
Continued on next page

-------
. continued from previous page
Study Citation: Alexander, H. C.,McCarty, W. M.,Bartlett, E. A.. 1978. Toxicity of perchloroethylene, trichloroethylene, 1,1,1-trichloroethane, and
methylene chloride to fathead minnows. Bulletin of Environmental Contamination and Toxicology 20:344-352
Data Type: Acute (0-96 hour); Aquatic; Fish static
Hero ID: 58126
Domain Metric Rating^ MWF* Score Comments^
Domain 4: Test Organism

Metric 13:
Test Organism Characteristics
Low
x 2
6
Fathead minnow is a well known lab species; but

from a bait shop, rather than cultured under con-

trolled conditions.
Metric 14:
Acclimitization and Pretreatment Conditions
High
x 1
1

Metric 15:
Number of Organisms and Replicates per
Low
x 1
3
Not indicated, but based on use of test guideline,

Group

suggests adequate number for statistical analysis.
Metric 16:
Adequacy of Test Conditions
High
x 1
1

Domain 5: Outcome Assessment

Metric 17:
Outcome Assessment Methodology
High
x 2
2

Metric 18:
Consistency of Outcome Assessment
High
x 1
1

Domain 6: Confounding / Variable Control

Metric 19:
Confounding Variables in Test Design and
High
x 2
2

Procedures

Metric 20:
Outcomes Unrelated to Exposure
High
x 1
1

Domain 7: Data Presentation and Analysis

Metric 21:
Statistical Methods
High
x 1
1

Metric 22:
Reporting of Data
High
x 2
2

Metric 23:
Explanation of Unexpected Outcomes
High
x 1
1

Overall Quality Determination"'"
High —
-s- Medium
1.7

Extracted Yes
* MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
* The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Alexander, H. C.,McCarty, W. M.,Bartlett, E. A.. 1978. Toxicity of perchloroethylene, trichloroethylene, 1,1,1-trichloroethane, and
methylene chloride to fathead minnows. Bulletin of Environmental Contamination and Toxicology 20:344-352
Data Type: Acute (0-96 hour); Aquatic; Fish flow-through
Hero ID: 58126
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
High x 2
Medium x 1
Metric 3: Test Substance Purity
Low
x 1
Authors work for chemical company that produces
TCE.
Not indicated, but because TCE is a common sol-
vent produced by the company, assuming at mini-
mum technical grade.
Domain 2: Test Design
Metric 4:
Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
High
Low
Low
x 2
x 1
x 1
Methyl or ethyl alcohol was used as the carrier sol-
vent for allcompounds except methylene chloride. A
control containing the sameamount of alcohol that
is present in the highest chemical concentration and
a Lake Huron water control were included in each
test series.
Not indicated, only LC50 for TCE reported., but
no deviances from test guideline [COMMITTEE
ON METHODS FOR TOXICITY TESTS WITH-
AQUATIC ORGANISMS (1975)] indicated.
Randomization not indicated, but followed test
guidelines.
Domain 3: Exposure Characterization
Metric 7:
Experimental System/Test Media Prepara- Medium x 2 4
tion
Metric 8: Consistency of Exposure Administration High x 1 1
Metric 9: Measurement of Test Substance Concentra- High x 2 2
tion
Metric 10: Exposure Duration and Frequency High x 1 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Low x 1 3
posure Levels
Metric 12: Testing at or Below Solubility Limit High x 1 1
Covered with plastic wrap, but no indication that
headspace minimized.
Gas chromatography used to analyze test concentra-
tions daily.
Test concentrations not provided.
Continued on next page

-------
. continued from previous page
Study Citation: Alexander, H. C.,McCarty, W. M.,Bartlett, E. A.. 1978. Toxicity of perchloroethylene, trichloroethylene, 1,1,1-trichloroethane, and
methylene chloride to fathead minnows. Bulletin of Environmental Contamination and Toxicology 20:344-352
Data Type:	Acute (0-96 hour); Aquatic; Fish flow-through
Hero ID:	58126
Domain	Metric	Rating^ MWF* Score	Comments^
Domain 4: Test Organism






Metric 13:
Test Organism Characteristics
Low
X
2
6
Fathead minnow is a well known lab species; but






from a bait shop, rather than cultured under con-






trolled conditions.
Metric 14:
Acclimitization and Pretreatment Conditions
High
X
1
1

Metric 15:
Number of Organisms and Replicates per
Low
X
1
3
Not indicated, but based on use of test guideline,

Group




suggests adequate number for statistical analysis.
Metric 16:
Adequacy of Test Conditions
High
X
1
1

Domain 5: Outcome Assessment





Metric 17:
Outcome Assessment Methodology
High
X
2
2

Metric 18:
Consistency of Outcome Assessment
High
X
1
1

Domain 6: Confounding / Variable Control





Metric 19:
Confounding Variables in Test Design and
High
X
2
2


Procedures





Metric 20:
Outcomes Unrelated to Exposure
High
X
1
1

Domain 7: Data Presentation and Analysis





Metric 21:
Statistical Methods
High
X
1
1

Metric 22:
Reporting of Data
High
X
2
2

Metric 23:
Explanation of Unexpected Outcomes
High
X
1
1

Overall Quality Determination^	High	1.5
Extracted	Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Fort, D. J.,Stover, E. L.,Rayburn, J. R.,Hull, M.,Bantle, J. A.. 1993. Evaluation of the developmental toxicity of trichloroethylene and
detoxification metabolites using Xenopus. Birth Defects Research, Part B: Developmental and Reproductive Toxicology 13:35-45
Data Type: Other; Aquatic; other South African clawed frog
Hero ID: 68271
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical identified by its name trichloroethylene.
High X 1 1 " Trichloroethylene and cyclohexene oxide were ob-
tained from the Aldrich Chemical Company, Mil-
waukee, Wisconsin."
Low X 1 3 Purity and grade were not reported.
Domain 2: Test Design
Metric 4: Negative Controls
High
Metric 5: Negative Control Response
High
Metric 6: Randomized Allocation
Low
x 2
x 1
x 1
"Ten to sixteen concentrations were tested in du-
plicate. Four separate dishes of 20 embryos each
were exposed to FETAX solution and designated
FETAX solution controls. Controls, including FET-
TAX solution, I percent v/v DMSO, uninhibited
MAS (with and without DMSO), each inhibited
MAS (with and without DMSO), activated acetyl-
hyclrazide (FETAX reference proteratogen), and un-
activated toxicant, were tested simultaneously with
each experiment. All control treatments received an-
tibiotics, as well."
"In each of the experiments conducted, the FETAX
solution, 1 percent v/v DMSO, andMAS or MAS
with 1 percent v/v DMSO, control mortality and
malformation rates were less than or equal to 5 per-
cent. Mortality and malformation rates for the in-
hibited MAS failed to exceed 6 percent. Embryos
exposed to 3. 0 g/1 acetylhydrazide and the in-
tact MAS exhibited mortality and deformity rates
of greater than or equal to 90 percent and 100 per-
cent, respectively."
Researchers did not report how organisms were al-
located to study groups.
Domain 3: Exposure Characterization
Continued on next page

-------
. . . continued from previous page
Study Citation: Fort, D. J.,Stover, E. L.,Rayburn, J. R.,Hull, M.,Bantle, J. A.. 1993. Evaluation of the developmental toxicity of trichloroethylene and
detoxification metabolites using Xenopus. Birth Defects Research, Part B: Developmental and Reproductive Toxicology 13:35-45
Data Type: Other; Aquatic; other South African clawed frog
Hero ID: 68271
Domain Metric Rating^ MWF* Score Comments^
Metric 7: Experimental System/Test Media Prepara- High x 2 2 A static renewal test was used for this assay and
•j-jQ-Q closed dishes were used which is important given
TCE's volatility. "For experiments conducted with-
out the MAS, groups of 20 embryos were placed in
covered 60 mm plastic Petri dishes (Fisher Scien-
tific, Houston, Texas) with varying constant con-
centrations of trichloroethylene, dichloroacetic acid,
trichloroacetic acid, trichloroethanol or oxalic acid."
Metric 8: Consistency of Exposure Administration High x 1 1 "Trichloroethylene was dissolved in 1 percent v/
v dimethylsulfoxide (DMSO) in FETAX solution.
Ten to sixteen concentrations were tested in du-
plicate. Four separate dishes of 20 embryos each
were exposed to FETAX solution and designated
FETAX solution controls. Each treatment vessel
contained a total of 8 ml of solution. Tests con-
ducted with the MAS or inhibited MAS were also
performed in duplicate with 20 embryos exposed
per replicate concentration. Each metabolically ac-
tivated treatment received O.A_nllits/dish of N-
nitrosodimethylamine activity 1 a NADPH generat-
ing system, and a penicillin-streptomycin mixture
to control bacterial contamination. For each treat-
ment, 8 to 14 concentrations were tested. Controls,
including FETAX solution, I percent v/v DMSO,
uninhibited MAS (with and without DMSO), each
inhibited MAS (with and without DMSO), acti-
vated acetylhyclrazide (FETAX reference proterato-
gen) [ 12], and unactivated toxicant, were tested
simultaneously with each experiment. All control
treatments received antibiotics, as well. One range-
finding and two definitive concentration-response ex-
periments were conducted with and without the
MAS or inhibited MAS. The pH of each of the stock
solutions was adjusted to 7 .0 with NaOH. Embryos
were cultured at 23 " 1 .CT'C. All solutions were
changed every 24 h of the 4-day test, dead embryos
removed, and fresh solutions added. Following 96 h
of exposure (stage 46 embryos), embryos were fixed
in 0. 7 percent formalin (pH7 7.0), and the number
of live malformed embryos were ascertained using a
dissecting microscope."
Continued on next page

-------
. continued from previous page
Study Citation: Fort, D. J.,Stover, E. L.,Rayburn, J. R.,Hull, M.,Bantle, J. A.. 1993. Evaluation of the developmental toxicity of trichloroethylene and
detoxification metabolites using Xenopus. Birth Defects Research, Part B: Developmental and Reproductive Toxicology 13:35-45
Data Type: Other; Aquatic; other South African clawed frog
Hero ID: 68271
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency
High
Metric 12: Testing at or Below Solubility Limit
High
x 2
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
x 1
Study does not report whether measurements were
taken, but it does say that it was a static renewal
test and that each petri dish had varying constant
concentrations of TCE.
96 hours of exposure and which is recommended for
African clawed frog tadpoles for acute tests and fresh
solution was added every 24 hours.
It was mentioned that a range finding test was con-
ducted prior to the assay, but not mentioned how
the spacing of the cone were decided on.
EC50s were far below the solubility for TCE.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 16: Adequacy of Test Conditions
Low
x 2
x 1
Metric 15: Number of Organisms and Replicates per Medium x 1
Group
x 1
Xenopus laevis was used and is the recommended
species for these tests according to EPA's test guid-
ance 890.2300.
Unable to fully determine from this study because
it reports that "Xenopus adult care, breeding, and
embryo collection were performed as described by
Dawson and Ban tie [ 17]."
20 animals per dish were tested which is recom-
mended in EPA guidelines, but only two replicates
were used where as guidelines recommend 3 for ex-
posure groups and 4 for controls.
EPA guidelines (OPPTS 890.1100) recommend 4 "
10 L (10 - 15 cm minimum water depth) /glass or
stainless steel, and study reports only 8 ml of solu-
tion.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High X 2 2 A 96 hour EC50 and LC50 were determined
Medium X 1 2 Incomplete reporting of minor details of outcome as-
sessment protocol across study groups.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2
Procedures
No reported differences among study groups in en-
vironmental conditions.
Continued on next page

-------
. continued from previous page
Study Citation: Fort, D. J.,Stover, E. L.,Rayburn, J. R.,Hull, M.,Bantle, J. A.. 1993. Evaluation of the developmental toxicity of trichloroethylene and
detoxification metabolites using Xenopus. Birth Defects Research, Part B: Developmental and Reproductive Toxicology 13:35-45
Data Type: Other; Aquatic; other South African clawed frog
Hero ID: 68271
Domain Metric Rating^ MWF* Score Comments^
Metric 20: Outcomes Unrelated to Exposure Medium x 1 2 Data on attrition and health outcomes unrelated to
exposure were not reported for each study group.
(Table 2 and 3 show percent responding out of the
total number of organisms for each exposure group,
but not attrition, or other health outcomes.)
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods High X 1 1 " Litchfield-Wilcoxon prob it analysis [ 19] was used
to determine the 96 h median lethal (LC50) and ter-
atogenic (EC50) concentrations of two pooled defini-
tive concentration-response tests. The 95 percent
confidence intervals were calculated as well. A ter-
atogenic index (TI) was calculated by taking the ra-
tio of the 96 h LC50 value to the 96 h EC50 (mal-
formation) value as a means of assessing teratogenic
potential [ 17]. Ninety-five percent fiducial inter-
vals for the TI values were calculated by the method
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High
High
x 2
x 1
2
1
of Finney [20]. Head-tail length of surviving em-
bryos was measured as an index of growth using an
IBM-AT compatible computer and Sigma Scan (Jan-
del Scientific, Corte Madra, CA) digitizing software.
The length data were then used to calculate a mini-
mum concentration to inhibit growth (MCIG) value
for each experiment using the t-test (P <0.05)."
Data on exposure related findings were presented for
each treatment and control group.
There was an explanation of why the authors
thought the controls died in the MAS activated
group.
Overall Quality Determination"'"
High

1.5

Extracted
Yes

Continued on next page . . .

-------
. continued from previous page
Study Citation: Fort, D. J.,Stover, E. L.,Rayburn, J. R.,Hull, M.,Bantle, J. A.. 1993. Evaluation of the developmental toxicity of trichloroethylene and
detoxification metabolites using Xenopus. Birth Defects Research, Part B: Developmental and Reproductive Toxicology 13:35-45
Data Type:	Other; Aquatic; other South African clawed frog
Hero ID:	68271
Domain	Metric	Rating^ MWF* Score	Comments^
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.
to
Cn

-------
Study Citation: Sanchez-Fortun, S.,Sanz, F.,Santa-Maria, A.,Ros, J. M.,De Vicente, M. L.,Encinas, M. T.,Vinagre, E.,Barahona, M. V.. 1997. Acute
sensitivity of three age classes of Artemia salina larvae to seven chlorinated solvents. Bulletin of Environmental Contamination and
Toxicology 59:445-451
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 200570
Domain
Metric
Rating"!" MWF* Score
Comments'^
Domain 1: Test Substance
Metric 1
Metric 2
Metric 3
Test Substance Identity
Test Substance Source
Test Substance Purity
High x 2
High x 1
Medium x 1
Purity not provided but authors note it is analytical
grade
Domain 2: Test Design
Metric 4:
Metric 5:
Negative Controls
Negative Control Response
High x 2
Medium x 1
Metric 6: Randomized Allocation
Low
x 1
Control response not reported but not expected to
affect results. Typically multi-chemical tests will
only report control results if significant (i.e. > 10
percent mortality).
Not reported
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low x 2
tion
Metric 8: Consistency of Exposure Administration High x 1
Metric 9: Measurement of Test Substance Concentra- Low x 2
tion
Metric 10: Exposure Duration and Frequency High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Low x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit High x 1
Nominal concentrations used without steps to reduce
volatilization of TCE.
Nominal concentrations with no analytical monitor-
ing reduces confidence in study results for methylene
chloride, but a trend is apparent when compared
across the solvents tested that informs the relative
toxicity of TCE.
24-72 hours.
Study does not provide exposure concentrations, but
paper indicates that "Each solvent concentration
was set in sextuplicate" suggesting six exposure con-
centrations were used for TCE. LC50/EC50s were
determined indicating exposure concentrations suf-
ficiently spaced.
Domain 4: Test Organism
Continued on next page

-------
. . . continued from previous page
Study Citation: Sanchez-Fortun, S.,Sanz, F.,Santa-Maria, A.,Ros, J. M.,De Vicente, M. L.,Encinas, M. T.,Vinagre, E.,Barahona, M. V.. 1997. Acute
sensitivity of three age classes of Artemia salina larvae to seven chlorinated solvents. Bulletin of Environmental Contamination and
Toxicology 59:445-451
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 200570
Domain Metric Rating"!" MWF* Score Comments"i"i"
Metric 13: Test Organism Characteristics
High
X
2
2

Metric 14: Acclimitization and Pretreatment Conditions
High
X
1
1

Metric 15: Number of Organisms and Replicates per
High
X
1
1
10 animals and four replicates per TCE concentra-
Group

tion tested.
Metric 16: Adequacy of Test Conditions
High
X
1
1

Domain 5: Outcome Assessment

Metric 17: Outcome Assessment Methodology
High
X
2
2

Metric 18: Consistency of Outcome Assessment
High
X
1
1

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test Design and
High
X
2
2

Procedures

Metric 20: Outcomes Unrelated to Exposure
Medium
X
1
2
Health outcomes unrelated to exposure (i.e. con-

trols) not reported, but not expected to affect inter-

pretation of results.
Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
High
X
1
1
"The 24 hr-LC50 values, with 95 percent confidence

limits, were calculated according to Litchfield and

Wilcoxon method (1949) implemented in the Phar-

macologicCalculation System (PCS version 4.0, New

York). These values were subjected to a two-way

analysis of variance with replication within the sub-

groups (ANOVA), followed by post hoc contrast with

Newman-Keuls Test."
Metric 22: Reporting of Data
Medium
X
2
4
Control results not provided, but unlikely to impact

results.
Metric 23: Explanation of Unexpected Outcomes
High
X
1
1

Overall Quality Determination"'"
High y Low 1.5 Nominal concentrations without analytical measure-
ment or measures to reduce volatilization of TCE
during testing.
Continued on next page

-------
. continued from previous page
Study Citation: Sanchez-Fortun, S.,Sanz, F.,Santa-Maria, A.,Ros, J. M.,De Vicente, M. L.,Encinas, M. T.,Vinagre, E.,Barahona, M. V.. 1997. Acute
sensitivity of three age classes of Artemia salina larvae to seven chlorinated solvents. Bulletin of Environmental Contamination and
Toxicology 59:445-451
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	200570
Domain
Metric
Rating"!" MWF* Score
Comments'r
Extracted

Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Brack, W.,Frank, H.. 1998. Chlorophyll a fluorescence: A tool for the investigation of toxic effects in the photosynthetic apparatus.
Ecotoxicology and Environmental Safety 40:34-41
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 660790
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Metric 3:
Test Substance Identity
Test Substance Source
Test Substance Purity
High
Low
Low
X 2 2 Test substance was
(Trichloroethylene)
X 1 3 Source was not identified.
X 1 3 Purity was not reported.
identified by name
Domain 2: Test Design
Metric 4:
Negative Controls
High
X
2
2
Negative controls in distilled water were used.
Metric 5:
Negative Control Response
Low
X
1
3
Control response not reported.
Metric 6:
Randomized Allocation
Low
X
1
3
It was not reported whether there was random alio-
cation to test groups
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High
tion
Metric 8: Consistency of Exposure Administration
Low
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency Medium
x 2
x 1
x 2
x 1
The volatility of chemicals was taken into consid-
eration. The tubes were gas-tight sealed by using
screw caps with Teflon-lined butyl rubber septa and
continuously shaken for 2 h at a temperature of 20
C.
Details of exposure for each study group were not re-
ported. the study did say that "Aliquots of 5mL of
the cell suspension were taken from the turbidostat
and diluted in 10-mL brown glass tubes with the
same volume of an aqueous solution of the chemi-
cal being tested. The tubes were gas-tight sealed
by using screw caps with Teflon-lined butyl rubber
septa and continuously shaken for 2 h at a temper-
ature of 20" C. With this procedure, nonvolatile and
volatile compounds could be tested. During incuba-
tion, light was excluded to prevent C02 consump-
tion by the algae and to avoid C02 deficiency during
incubation."
It was not reported whether exposure concentration
were measured or not.
Exposure duration is not standard (600 seconds),
but could be acceptable for what is being measured
(fluorescence).
Continued on next page

-------
. continued from previous page
Study Citation: Brack, W.,Frank, H.. 1998. Chlorophyll a fluorescence: A tool for the investigation of toxic effects in the photosynthetic apparatus.
Ecotoxicology and Environmental Safety 40:34-41
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	660790
Domain
Metric
Rating1
MWF* Score
Comments' t
Metric 11: Number of Exposure Groups/Spacing of Ex- Unacceptable x 1 4
posure Levels
Metric 12: Testing at or Below Solubility Limit	Low	x 1	3
Unclear how many exposure groups or what the ex-
posure levels were for TCE.
Unknown exactly what cone were tested but the tox-
icity threshold is well below the high solubility of
TCE.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics	High
Metric 14: Acclimitization and Pretreatment Conditions Medium
Metric 15: Number of Organisms and Replicates per Low
Group
Metric 16: Adequacy of Test Conditions	High
X 2	2 Green algae (Chlamydomonas reinhardtii)
X 1	2	Some acclimatization conducted with some mi-
nor uncertainties about pretreatment. The study
says, "Green algae of the species Chlamydomonas
reinhardtii [strain ll-32a SAG(#), according to
Schlosser (1982)] were cultivated in a nutrient solu-
tion for unicellular algae (Kuhl and Lorenzen, 1964)
in a turbiodstat; use of the turbidostat provides ex-
ponentially growing cell suspensions of a constant
density and physiological state by dilution with fresh
medium controlled by a photoelectric cell. The algae
were illuminated continuously by four cool white flu-
orescent tubes (4] 10 W) aerated, and maintained at
a temperature of 20" C. The cultures were kept at a
density of 2] 106 cells/mL for 2 weeks. The doubling
time in the turbidostat was about 13 h."
X 1	3 Not reported
X 1	1	Teflon containers that were sealed
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
Medium	X 2	4 For TCE a 600 second EC5 was reported of 13 (F'0/
F0)
Low	X 1	3 Details of the assessment protocol were not reported
for each study group
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Low
Procedures
x 2
Not enough information provided to allow a compar-
ison of environental conditions between study groups
for TCE.
Continued on next page

-------
. . . continued from previous page
Study Citation: Brack, W.,Frank, H.. 1998. Chlorophyll a fluorescence: A tool for the investigation of toxic effects in the photosynthetic apparatus.
Ecotoxicology and Environmental Safety 40:34-41
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 660790
Domain Metric Rating^ MWF* Score Comments^
Metric 20: Outcomes Unrelated to Exposure Medium x 1 2 Data on attrition from controls were not reported for
each chemical explicitly but it was mentioned that
the test concentrations were cored to the controls.
"Toxicity thresholds (TTs) are defined as concen-
trations that reduce or increase at least one of the
Fluorescence parameters for more than the three-
fold value of the maximum of standard deviations
of the controls. A TT is attained when the mea-
surement deviates by 3, 5, 10, or 20 percent from
the respective control value, depending on the re-
producibility of the particular parameter. The TTs
of the tested chemicals, calculated by linear extrap-
olation between the highest concentration without
significant effect and the lowest concentration with
it, are presented in Table 3."
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods High x 1 1
Metric 22: Reporting of Data Low x 2 6
Metric 23: Explanation of Unexpected Outcomes High x 1 1
Overall Quality Determination"'" Unacceptable 4.0
Extracted No
F'0/F0=EC05 and "Toxicity thresholds (TTs) are
defined as concentrations that reduce or increase at
least one of the Fluorescence parameters for more
than the threefold value of the maximum of stan-
dard deviations of the controls. A TT is attained
when the measurement deviates by 3, 5, 10, or 20
percent from the respective control value, depending
on the reproducibility of the particular parameter.
The TTs of the tested chemicals, calculated by lin-
ear extrapolation between the highest concentration
without significant effect and the lowest concentra-
tion with it, are presented in Table 3."
The EC05 was reported to TCE but not much other
detail was reported.
No unexplained outcomes
Continued on next page

-------
. continued from previous page
Study Citation: Brack, W.,Frank, H.. 1998. Chlorophyll a fluorescence: A tool for the investigation of toxic effects in the photosynthetic apparatus.
Ecotoxicology and Environmental Safety 40:34-41
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	660790
Domain	Metric	Rating^	MWF* Score	Comments^
** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Brack, W. ,Rottler, H.. 1994. Toxicity testing of highly volatile chemicals with green algae: A new assay. 1:223-228
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 661061
Domain
Metric
Rating^ MWF* Score
Comments^
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Metric 2:
Test Substance Source
High
X
1
1
Metric 3:
Test Substance Purity
High
X
1
1
Chemical was identified by name.
Authors identified Merck as the source of the test
substance.
Authors reported greater than 99 percent purity.
Domain 2: Test Design
Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
High
Low
Low
x 2
x 1
x 1
"Each test series contained three controls without
toxicant and two controls with 0.8 mg/L Cu2+
(CuS04). This concentration reduces algal growth
to50 percent and is used to check normal sensitivity
of the organisms."
The biological responses of the negative control
groups were not reported.
It was not reported whether there was random place-
ment of flasks.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High x 2 2 The purpose of the test was to determine a way
¦j-Jq-q of doing algae tests with volatile chemicals, as the
OECD guidelines recommends using a permeable
stopper in the flask to allow C02 to pass through
so as not to impede algae growth. However with
volatile chemicals this is not possible because of loss
of test substance through vitalization. Therefore in
test, they used a closed system that still provided
a source of C02 for the algae. Authors reported,
"Deviations between the duplicates, extracted from
the same test culture were less than 5 percent . To
estimate recovery of this analytical method, 20 mL
headspace vials were filled completely with water
or alga suspension. The vials were sealed gas" tight
with septa. Gravimetrically defined amounts of the
volatile chlorinated hydrocarbons were injected via
syringe through the Septa into the liquids and dis-
solved. From these solutions samples were taken
and extracted as explained above. Recovery of the
method amounted to 90 " S percent and was inde-
pendent from cell density."
Continued on next page

-------
. continued from previous page
Study Citation: Brack, W. ,Rottler, H.. 1994. Toxicity testing of highly volatile chemicals with green algae: A new assay. 1:223-228
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	661061
Domain
Metric
Ratingt MWF* Score
Commentstt
Metric 8: Consistency of Exposure Administration	High x 1
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency
High
Metric 11: Number of Exposure Groups/Spacing of Ex- High
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
x 1
x 1
1	Exposures were administered consistently across
study groups.
2	Concentrations were measured at the end of the test.
Authors stated, "Sampling for measurement of toxi-
cant concentration was possible without opening the
vessel through septa with screw caps (- Fig. 1)." and
"Measurements at the beginning and at the end of
the assays showed no significant differences in chem-
ical concentrations. Therefore, samples for analysis
were taken ac the end of the assay in order to avoid
leaks in the septa, which could allow evaporation
during the test period .
1	The test was 72 hours in duration, which is recom-
mended by OECD Guideline 201.
1	Test concentrations are reported in figure 3 and show
a dose response for growth inhibition. The figure
shows at least 5 concentrations tested which is rec-
ommended by OECD Guideline 201.
1	The test cone for TCE shown in figure 3 (highest
cone is <100 mg/1) are well below TCE's solubility
level of 999-1472 mg/1.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Medium x 2
Metric 14: Acclimitization and Pretreatment Conditions High x 1
This is not a commonly used algal species. Not a TG
species. Test used unicellular freshwater green alga
Chlamydomonas rehthardtii (strain number 11 -32a
SAG) from the University of Gottingen, Germany.
Pretreatment conditions included, "Precultures and
test cultures were grown in the medium for unicel-
lular algae according to KUflL (1962) (Table 2). In-
cubation of all cultures was done in a Orbital In-
cubator (Gallenkamp). The cultures were shaken
permanently with a frequency of 120 rpm. They
wereilluminated from above with 130 "E/m2s with-
out light dark cycle. The photosynthetically effec-
tive light was determined with a Quantum Sensor
from Licor Inc. The temperature was maintained at
20 " 10 -c."
Continued on next page

-------
. . . continued from previous page
Study Citation: Brack, W. ,Rottler, H.. 1994. Toxicity testing of highly volatile chemicals with green algae: A new assay. 1:223-228
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 661061
Domain Metric Ratingt MWF* Score Commentstt
Metric 15: Number of Organisms and Replicates per
Group
Medium x 1
Metric 16: Adequacy of Test Conditions
High
x 1
Two replicates per test concentration were reported.
OECD Guideline 201 states the test should include
three replicates, but if determination of a NOEC is
not required, the test may be altered to increase the
number of concentrations and reduce the number of
replicates per conc. There were more than 5 test
cone (the recommended number) used for TCE. The
cell density in the test cultures amounted to 5 " 103
cells/ml at the beginning of the assays.
Glass flasks which are recommended in OECD 201.
Temp and ph were within recommended ranges.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High X 2 2 EClOs and EC50s were determined and reported.
High X 1 1 Outcomes were assessed consistently across study
groups.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High
Procedures
Metric 20: Outcomes Unrelated to Exposure
x 2
Medium x 1
2 There were no reported differences among study
groups in environmental conditions or other factors
that would influence the outcome assessment.
2 Authors do not report attrition for each study group
or the control, but do say that the effective con-
centration was controlled analytically, leaving only
minor uncertainties. Authors report, "EC10 and
72" h EC50, which are the effective concentration of
a chemical by which algal growth is reduced by 10
percent or 50 percent compared to a control in 72 h,
respectively, and the 95 percent confidence intervals
of these values are shown in Table 3 (- p. 226)."
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
High X 1 1 Authors reported using the PROBIT routine of SAS.
Medium X 2 4 Figure 3 shows the results of the tests at each conc
for each chemical but it's difficult to determine the
exact concentrations from the figure, so some minor
uncertainties remain.
Continued on next page

-------
. continued from previous page
Study Citation:	Brack, W. ,Rottler, H.. 1994. Toxicity testing of highly volatile chemicals with green algae: A new assay. 1:223-228
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	661061
Domain	Metric	Ratingt MWF* Score	Commentstt
Metric 23: Explanation of Unexpected Outcomes	Medium x 1	2 SDs were provided, but it was unclear whether or not
there were any unexpected outcomes, leaving minor
uncertainties.
Overall Quality Determination^	High	1.4
Extracted	Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
^2 (Metric Score; x MWF,) / J] . MWF^
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Yoshioka, Y.,Ose, Y.,Sato, T.. 1985. Testing for the toxicity of chemicals with Tetrahymena pyriformis. Science of the Total Environ-
ment 43:149-157
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 676758
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Metric 3:
Test Substance Identity
Test Substance Source
Test Substance Purity
High
Low
Low
X 2 2 Test chemical was identified by name
X 1 3 Source was not reported
X 1 3 The purity was not reported.
Domain 2: Test Design
Metric 4:
Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
Low
N/A
Low
x 2 6 It is implied that they used a control because they
mention using a "blank" to calculate values and
show a growth curve for cells in the blank, but there
is not much information about what is in the blank.
N/A This is an acute study with lots of chemicals re-
ported, and they did not report on the control re-
sponse for each chemical.
X 1 3 There was no mention of random allocation.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Medium
tion
Metric 8: Consistency of Exposure Administration
Low
x 2
x 1
Metric 9: Measurement of Test Substance Concentra- Low x 2 6
tion
Metric 10: Exposure Duration and Frequency Medium x 1 2
Metric 11: Number of Exposure Groups/Spacing of Ex- Unacceptable x 1 4
posure Levels
Metric 12: Testing at or Below Solubility Limit High x 1 1
The system was closed for TCE which is volatile,
but TCE's system was not described in detail (no
info about headspace in glass, etc)
There were differences in how exposure was adminis-
tered but because the point of the study was to figure
out what housing conditions were best for this type
of protozoa. These differences could have effected
the EC50 reported. Authors report that some of
the temperatures, and amount of food changed the
growth rate of the protozoa.
Measurements were not reported
Various exposure durations were tested to determine
which duration was most effective.
no information was provided on number of exposure
groups or spacing of exposures.
Continued on next page

-------
. . . continued from previous page
Study Citation: Yoshioka, Y.,Ose, Y.,Sato, T.. 1985. Testing for the toxicity of chemicals with Tetrahymena pyriformis. Science of the Total Environ-
ment 43:149-157
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 676758
Domain Metric Rating^ MWF* Score Comments^
Domain 4: Test Organism
Metric 13:
Test Organism Characteristics
Medium
Metric 14:
Metric 15:
Acclimitization and Pretreatment Conditions
Number of Organisms and Replicates per
Group
Metric 16: Adequacy of Test Conditions
High
Low
Medium
x 2
x 1
x 1
x 1
Tetrahymena pyriformis was preserved in a sterile
medium of 2 percent proteasepeptone at 20" C which
was renewed at 2-4 week intervals. Unsure but it
sounds like they cultured their own animals in the
lab from descriptions of previous studies in this pa-
per. Acknowledgements say "Pr. Nozawa of Gifu
University for providing T. pyriformis in germ-free
condition"
Number of test organisms and replicates were not
reported for the test groups. It was reported that
20 cells per slide were counted using one method of
counting, but that was the only number provided.
In some of the housing conditions the animals did
not do as well, but that did not significantly change
the EC50 values. The point of the test was to try
out different housing conditions, it looks like for
testing conditions they used 24 hour test time and
conditions of no 4 for culturing (Pre-culture: temp
30, hours 24; test culture: medium PRO, temp 30
type VP (cultured in vertical vessel with a porous
silicone rubber stopper).
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology Medium x 2 4 They describe two different methods for counting
the cells but some uncertainties remain e.g. which
method they went with.
Metric 18: Consistency of Outcome Assessment Medium x 1 2 The assessment protocol was reported with minor
uncertainties.
Domain 6: Confounding / Variable Control
Continued on next page

-------
. continued from previous page
Study Citation: Yoshioka, Y.,Ose, Y.,Sato, T.. 1985. Testing for the toxicity of chemicals with Tetrahymena pyriformis. Science of the Total Environ-
ment 43:149-157
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	676758
Domain	Metric	Rating^	MWF* Score	Comments^
Metric 19: Confounding Variables in Test Design and
Procedures
Low
x 2
Metric 20: Outcomes Unrelated to Exposure
Medium
x 1
"In spite of the considerable influence of the compo-
sition of the medium on the growth curve, as shown
in Fig. 3 (compare No. 1 with No. 5 and No.
2 with No. 6), there was no significant difference
between the EC50 value in 2 percent proteose pep-
tone (No. 1) and that in 2 percent polypeptone (No.
5), which were both cultured at 30"C. However, the
EC50 value of No. 6 differs significantly from that
of No. 2, and the difference between them increased
as the test period was increased from 24 to 72 h."
They do mention, "When the culture was contami-
nated by the bacterium, the test was repeated." and
the effects on growth from test medium (food) was
not statistically significant for the EC50 values.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High
Low
High
x 1
x 2
x 1
The effective concentration 50 percent (EC50 ) val-
ues were obtained by plotting therelative growth
rates against the concentration of chemical on loga-
rithmic probability paper.
Data for exposure related findings were not shown
for each study group.
Unexpected outcomes were explained.
Overall Quality Determination^	Unacceptable	4.0
Extracted	No
** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
1 High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - wood frog and green frog
Hero ID: 700434
Domain
Metric
Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical was identified by name.
High X 1 1 Stock solutions were made from 95 percent pure,
analytical-grade PCE,TCE, and cis- and trans-DCE
(Sigma-Aldridge).
Medium x 1 2 "Stock solutions were made from 95 percent pure,
analytical-grade PCE, TCE, and cis- and trans-DCE
(Sigma-Aldridge)." Only minor uncertainties about
the purity being at 95 percent, analytical-grade.
Domain 2: Test Design
Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
High X 2 2 Three replicates of embryos were also raised in un-
contaminated groundwater as controls.
High X 1 1 Control mortality was reported in table 2 for each
species, and deformities in controls were reported in
figure 1. Control response for mortality for wood
frogs, green frogs and spotted salamanders were all
below 10 percent; for American toads it was about
10.1 percent with one of the replicates having a
very high 26 percent mortality rate, so there are
uncertainites for this species for this metric. Fig-
ure 1 shows that the negative control response for
all species for percent mortality is below 10 percent.
ASTM guidelines indcate "An acceptable clutch of
eggs has the capability of developing into Develop-
mental Stage 46 tadpoles with less than 10 percent
gross abnormalities and less than 10 percent mortal-
ity."
X 1 0 For the acute study it was not reported whether the
animals were distributed randomly.
Domain 3: Exposure Characterization
Continued on next page

-------
. continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type:	Other; Aquatic; other amphibian - wood frog and green frog
Hero ID:	700434
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 7: Experimental System/Test Media Prepara- High	X 2	2 Containers were covered and sealed and efforts were
•j-jQ-Q	made to minimize head space. Authors reported
that TCE solutions declined by 50 to 80 percent over
the 24 hour period between renewals. Authors also
mentioned, "Each egg mass was gently divided into
clusters of approximately 30 eggs (with the excep-
tion of spotted salamanders with 5 to 10 eggs) and
placed in a 1-L glass Mason jar containing 300 ml
of test solution. The lids on the jars were sealed to
reduce volatilization. Dissolved oxygen levels never
fell below 80 percent saturation. Three replicates of
embryos were also raised in uncontaminated ground-
water as controls. Temperature was maintained at
23 +-1"C using a water bath. All tests were con-
ducted under a 14L/10D light regime. An exhaust
hood over the water bath ensured the removal of
accidental gaseous PCE, TCE, and DCE volatilized
from the exposure vessels."
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - wood frog and green frog
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 8: Consistency of Exposure Administration Medium x 1 2 Authors indicate that there may have been some
losses of test chemical during decanting test solu-
tions and during the placing of eggs in test cham-
bers, and while testing a subsample at 1 hour of
exposure TCE cone were only within 70 percent of
nominal. The authors report, "Stock solutions of
TCE, PCE, and DCE were dissolved in local ground-
water in a dilution series. Groundwater was used as
the medium for acute tests to emulate conditions in
surface waters fed by chloroethylene- contaminated
groundwater. Nominal test concentrations were as
follows: PCE-2.5, 7.5, 12.5, and 20 mg/L; TCE-12.5,
20, 40, and60 mg/L; and cis- and trans-DCE-12.5,
60, and 100 mg/L. Based on the results of initial ex-
posures of American toad embryos, a second expo-
sure was conducted with elevated concentrations of
PCE and TCE as follows: PCE-15, 30, and 45 mg/
L; and TCE-35, 55, and 85 mg/L. Maximum expo-
sure concentrations of PCE and TCE were limited
by the compounds' solubility in groundwater. Con-
centrations of test solutions, including controls, were
measured at 24 h (just prior to solution renewal, see
below). Concentrations at t = 0 h were based on
dilutions of measured stock solutions. Some losses
occurred while decanting test solutions and during
the placing of eggs in test chambers. In a sub-
sample of test solutions measured at 1 h of expo-
sure,concentrations of PCE were within 99 percent
of nominal, while cis- and trans-DCE were within 90
percent. However, levels of TCE were only within 70
percent of nominal."
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - wood frog and green frog
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency
High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High x 1
Measured concentrations were taken, and used to
derive an EC50. . Authors reported "To prevent
volatilization, a glass flask (6 L) was filled to ca-
pacity with stock solution to reduce head space and
then stirred using a stir bar at low speed to pre-
vent disruption of the surface. Stock solutions were
stirred until the compound was dissolved or until
the solution had reached saturation (between 2 and
48 h, depending on the compound). Despite these
precautions,actual concentrations tended to be less
than nominal concentrations.For this reason, con-
centrations of stock solutions were measured priorto
mixing of the test solutions, using Purge and Trap
GC/MS accordingto U.S. EPA method 82 60B (U.S.
EPA 1996), to ensure that testsolutions were within
10 percent of the nominal concentration at lh of ex-
posure. The GS/MS was performed on a Hewlett
Packard 6890 gas chromatograph, using a Hewlett
Packard 5973 mass selective detector, a 3100 purge
and trap concentrator, and a PTA-30 purge and trap
autosampler. The method detection limit for all
chloroethylene analyses was 1 ppb.
ASTM guidelines for FETAX on American clawed
frog suggest 96 hours and a static renewal set up,
renewed every 24 hours, which this study does for
the acute test.
This study had four exposure groups for TCE and
ASTM FETAX Guidelines suggests the following
"At a minimum, five concentrations for each end-
point are used. However, additional concentrations
between the EC16 and EC84 are highly recom-
mended to ensure obtaining accurate 96-hour LC50
and EC50 values."
Solubility was taken into consideration, and expo-
sure concentrations kept below the solubility of TCE
in growndwater.
Domain 4: Test Organism
Continued on next page

-------
. continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - wood frog and green frog
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 13: Test Organism Characteristics
Medium x 2
Metric 14: Acclimitization and Pretreatment Conditions Low x 1
Metric 15: Number of Organisms and Replicates per High
Group
x 1
Wood frog and green frog: Test organisms seem to
be sufficiently sensitive to the exposures adminis-
tered to derive an EC50, but are not a suggested
species in the ASTM guideline or OEGD and EPA
guidelines for amphibian growth and development,
which suggest African clawed frog. This study in-
stead used these species to test sensitivity for North
American species. Only minor uncertainties because
they are not suggested species from a guideline.
Study authors did not report whether animals were
acclimatized or whether pretreatment conditions
were the the same for treatment and controls. They
authors do report, " In 2001 and 2002, egg masses of
wood frogs, spotted salamanders," American toads,
and green frogs were collected from a wetland not
contaminated with chloroethylenes in Flamborough
Township (Ontario,Canada). Water from wetlands
were tested for chloroethylenes in 2001 from each
site where eggs were collected. No chloroethylenes
were detected; the minimum detection limit for this
analysis was 1 ppb. Egg masses were less than 24 h
old when exposures were initiated. For each species,
three egg masses were used (with the exception of
the second exposure of American toads, where only
one egg mass was used). Each egg.mass was from a
different female and represented a replicate. Thus,
there were three replicate jars for each chemical by
concentration combination, for a total of 45 jars per
species. Eggs were not dejellied prior to exposure to
more accurately imitatenatural exposure conditions.
Each egg mass was gently divided into clusters of ap-
proximately 30 eggs (with the exception of spotted
salamanders with 5 to 10 eggs) and placed in a 1-L
glass Mason jar containing 300 ml of test solution."
ASTM guidelines suggest 20-25 and two replicates,
and the study authors reported, "there were three
replicate jars for each chemical" and, "Each egg
mass was gently divided into clusters of approxi-
mately 30 eggs (with the exception of spotted sala-
manders with 5 to 10 eggs) and placed in a 1-L glass
Mason jar containing 300 ml of test solution."
Continued on next page . . .

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - wood frog and green frog
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 16: Adequacy of Test Conditions Medium X 1 2 Animals were held in 1 L glass mason jars contain-
ing 300 ml of test solution. Jars were sealed and
temperature was maintined at 23+-1 degree C us-
ing a water bath. All tests were conducted under
14L/10D light regime. The ASTM guidelines rec-
ommend glass, and this temperature is approprate
for african clawed frog but unsure if this temperature
is also appropriate for these north american species.
Additionally the photoperiod is longer than the one
recommended in the ASTM Guidelines.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
x 2
Medium x 1
Outcome assessment methodology addressed the in-
tended outcomes of interest and was sensitive for
the outcomes of interest. LC50 and EC50 were
reported which are the recommended outcomes in
ASTM guidelines for FETAX.
All animals were assessed at the end of the 96 hour
period with minor uncertainties due to incomplete
reporting.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2
Procedures
Metric 20: Outcomes Unrelated to Exposure Medium x 1 2
No reported differences among study groups in en-
vironmental conidtions.
Controls for the wood frogs and green frogs were
under 10 percent mortality and deformities. Details
on attrition unrelated to exposure for each exposure
concentration were also reported as the average with
a range. There is a wide range of portailities between
the replicates.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
High X 1 1 A two-factor ANOVA was used. ASTM FETAX
Guidelines suggests either probit analysis, trimmed
Spearman-Karber analysis, or the two-point graphi-
cal method to estimate LC50 and EC50 values.
Continued on next page

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. continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type:	Other; Aquatic; other amphibian - wood frog and green frog
Hero ID:	700434
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 22: Reporting of Data	Medium x 2	4 Data was reported for each exposure group in ei-
ther table or graphical form. It's hard to tell the
exact numbers from the graphical representation of
the EC50 values for each exposure level, resulting in
minor uncertainites.
Metric 23: Explanation of Unexpected Outcomes	High	x 1	1 Unexpected outcomes were explained, (e.g. loss of
TCE through evaporation, damaged eggs in transit)
Overall Quality Determination^	High	1.4
Extracted	Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - american toad
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical was identified by name.
High X 1 1 Stock solutions were made from 95 percent pure,
analytical-grade PCE,TCE, and cis- and trans-DCE
(Sigma-Aldridge).
Medium x 1 2 "Stock solutions were made from 95 percent pure,
analytical-grade PCE, TCE, and cis- and trans-DCE
(Sigma-Aldridge)." Only minor uncertainties about
the purity being at 95 percent, analytical-grade.
Domain 2: Test Design
Metric 4:
Metric 5:
Negative Controls
Negative Control Response
Metric 6: Randomized Allocation
High X 2 2 Three replicates of embryos were also raised in un-
contaminated groundwater as controls.
Medium X 1 2 Control mortality was reported in table 2 for each
species, and deformities in controls were reported in
figure 1. Control response for mortality for wood
frogs, green frogs and spotted salamanders were all
below 10 percent; for American toads it was about
10.1 percent with one of the replicates having a very
high 26 percent mortality rate, so there are uncer-
tainites for this species for this metric. Authors
threw those numbers out and indicated that the high
mortality rate for that replicate could have been
due to damage the eggs recieved in transit. Fig-
ure 1 shows that the negative control response for
all species for percent mortality is below 10 percent.
ASTM guidelines indcate "An acceptable clutch of
eggs has the capability of developing into Develop-
mental Stage 46 tadpoles with less than 10 percent
gross abnormalities and less than 10 percent mortal-
ity."
X 1 3 For the acute study it was not reported whether the
animals were distributed randomly.
Low
Domain 3: Exposure Characterization
Continued on next page

-------
. continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type:	Other; Aquatic; other amphibian - american toad
Hero ID:	700434
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 7: Experimental System/Test Media Prepara- High	X 2	2 Containers were covered and sealed and efforts were
•j-jQ-Q	made to minimize head space. Authors reported
that TCE solutions declined by 50 to 80 percent over
the 24 hour period between renewals. Authors also
mentioned, "Each egg mass was gently divided into
clusters of approximately 30 eggs (with the excep-
tion of spotted salamanders with 5 to 10 eggs) and
placed in a 1-L glass Mason jar containing 300 ml
of test solution. The lids on the jars were sealed to
reduce volatilization. Dissolved oxygen levels never
fell below 80 percent saturation. Three replicates of
embryos were also raised in uncontaminated ground-
water as controls. Temperature was maintained at
23 +-1"C using a water bath. All tests were con-
ducted under a 14L/10D light regime. An exhaust
hood over the water bath ensured the removal of
accidental gaseous PCE, TCE, and DCE volatilized
from the exposure vessels."
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - american toad
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 8: Consistency of Exposure Administration Medium x 1 2 Authors indicate that there may have been some
losses of test chemical during decanting test solu-
tions and during the placing of eggs in test cham-
bers, and while testing a subsample at 1 hour of
exposure TCE cone were only within 70 percent of
nominal. The authors report, "Stock solutions of
TCE, PCE, and DCE were dissolved in local ground-
water in a dilution series. Groundwater was used as
the medium for acute tests to emulate conditions in
surface waters fed by chloroethylene- contaminated
groundwater. Nominal test concentrations were as
follows: PCE-2.5, 7.5, 12.5, and 20 mg/L; TCE-12.5,
20, 40, and60 mg/L; and cis- and trans-DCE-12.5,
60, and 100 mg/L. Based on the results of initial ex-
posures of American toad embryos, a second expo-
sure was conducted with elevated concentrations of
PCE and TCE as follows: PCE-15, 30, and 45 mg/
L; and TCE-35, 55, and 85 mg/L. Maximum expo-
sure concentrations of PCE and TCE were limited
by the compounds' solubility in groundwater. Con-
centrations of test solutions, including controls, were
measured at 24 h (just prior to solution renewal, see
below). Concentrations at t = 0 h were based on
dilutions of measured stock solutions. Some losses
occurred while decanting test solutions and during
the placing of eggs in test chambers. In a sub-
sample of test solutions measured at 1 h of expo-
sure,concentrations of PCE were within 99 percent
of nominal, while cis- and trans-DCE were within 90
percent. However, levels of TCE were only within 70
percent of nominal."
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - american toad
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency
High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Low x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High x 1
Measured concentrations were taken, and used to
derive an EC50. . Authors reported "To prevent
volatilization, a glass flask (6 L) was filled to ca-
pacity with stock solution to reduce head space and
then stirred using a stir bar at low speed to pre-
vent disruption of the surface. Stock solutions were
stirred until the compound was dissolved or until
the solution had reached saturation (between 2 and
48 h, depending on the compound). Despite these
precautions,actual concentrations tended to be less
than nominal concentrations.For this reason, con-
centrations of stock solutions were measured priorto
mixing of the test solutions, using Purge and Trap
GC/MS accordingto U.S. EPA method 82 60B (U.S.
EPA 1996), to ensure that testsolutions were within
10 percent of the nominal concentration at lh of ex-
posure. The GS/MS was performed on a Hewlett
Packard 6890 gas chromatograph, using a Hewlett
Packard 5973 mass selective detector, a 3100 purge
and trap concentrator, and a PTA-30 purge and trap
autosampler. The method detection limit for all
chloroethylene analyses was 1 ppb.
ASTM guidelines for FETAX on American clawed
frog suggest 96 hours and a static renewal set up,
renewed every 24 hours, which this study does for
the acute test.
This study had four exposure groups for TCE and
ASTM FETAX Guidelines suggests the following
"At a minimum, five concentrations for each end-
point are used. However, additional concentrations
between the EC16 and EC84 are highly recom-
mended to ensure obtaining accurate 96-hour LC50
and EC50 values." For American toads the concen-
trations were too low to generate either an LC50 or
an EC50.
Solubility was taken into consideration, and expo-
sure concentrations kept below the solubility of TCE
in growndwater.
Domain 4: Test Organism
Continued on next page

-------
. continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - american toad
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 13: Test Organism Characteristics
Medium x 2
Metric 14: Acclimitization and Pretreatment Conditions Low x 1
Metric 15: Number of Organisms and Replicates per High
Group
x 1
American toads: Test organisms seem to be suffi-
ciently sensitive to the exposures administered to
derive an EC50, but are not a suggested species in
the ASTM guideline or OECD and EPA guidelines
for amphibian growth and development which sug-
gest African clawed frog. This study instead used
these species to test sensitivity for North American
species. Only minor uncertainties because they are
not suggested species from a guideline.
Study authors did not report whether animals were
acclimatized or whether pretreatment conditions
were the the same for treatment and controls. They
authors do report, " In 2001 and 2002, egg masses of
wood frogs, spotted salamanders," American toads,
and green frogs were collected from a wetland not
contaminated with chloroethylenes in Flamborough
Township (Ontario,Canada). Water from wetlands
were tested for chloroethylenes in 2001 from each
site where eggs were collected. No chloroethylenes
were detected; the minimum detection limit for this
analysis was 1 ppb. Egg masses were less than 24 h
old when exposures were initiated. For each species,
three egg masses were used (with the exception of
the second exposure of American toads, where only
one egg mass was used). Each egg.mass was from a
different female and represented a replicate. Thus,
there were three replicate jars for each chemical by
concentration combination, for a total of 45 jars per
species. Eggs were not dejellied prior to exposure to
more accurately imitatenatural exposure conditions.
Each egg mass was gently divided into clusters of ap-
proximately 30 eggs (with the exception of spotted
salamanders with 5 to 10 eggs) and placed in a 1-L
glass Mason jar containing 300 ml of test solution."
ASTM guidelines suggest 20-25 and two replicates,
and the study authors reported, "there were three
replicate jars for each chemical" and, "Each egg
mass was gently divided into clusters of approxi-
mately 30 eggs (with the exception of spotted sala-
manders with 5 to 10 eggs) and placed in a 1-L glass
Mason jar containing 300 ml of test solution."
Continued on next page . . .

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - american toad
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 16: Adequacy of Test Conditions Medium X 1 2 Animals were held in 1 L glass mason jars contain-
ing 300 ml of test solution. Jars were sealed and
temperature was maintined at 23+-1 degree C us-
ing a water bath. All tests were conducted under
14L/10D light regime. The ASTM guidelines rec-
ommend glass, and this temperature is approprate
for african clawed frog but unsure if this temperature
is also appropriate for these north american species.
Additionally the photoperiod is longer than the one
recommended in the ASTM Guidelines.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
x 2
Medium x 1
Outcome assessment methodology addressed the in-
tended outcomes of interest and was sensitive for
the outcomes of interest. LC50 and EC50 were
reported which are the recommended outcomes in
ASTM guidelines for FETAX.
All animals were assessed at the end of the 96 hour
period with minor uncertainties due to incomplete
reporting.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2
Procedures
Metric 20: Outcomes Unrelated to Exposure Medium x 1 2
No reported differences among study groups in en-
vironmental conidtions.
One of the controls for the American toads had very
high mortality 26 percent, results from that clutch
were removed, authors suggest the egg mass may
have been damaged in transit. Details on attrition
unrelated to exposure for each exposure concentra-
tion were also reported as the average with a range.
There is a wide range of mortalities between the
replicates, and zero mortality at the highest concen-
tration.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
High X 1 1 A two-factor ANOVA was used. ASTM FETAX
Guidelines suggests either probit analysis, trimmed
Spearman-Karber analysis, or the two-point graphi-
cal method to estimate LC50 and EC50 values.
Continued on next page

-------
. continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type:	Other; Aquatic; other amphibian - american toad
Hero ID:	700434
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 22: Reporting of Data	Medium x 2	4 Data was reported for each exposure group in ei-
ther table or graphical form. It's hard to tell the
exact numbers from the graphical representation of
the EC50 values for each exposure level, resulting in
minor uncertainites.
Metric 23: Explanation of Unexpected Outcomes	High	x 1	1 Unexpected outcomes were explained, (e.g. loss of
TCE through evaporation, damaged eggs in transit)
Overall Quality Determination^	High	1.5
Extracted	Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - spotted salamder
Hero ID: 700434
Domain
Metric
Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical was identified by name.
High X 1 1 Stock solutions were made from 95 percent pure,
analytical-grade PCE,TCE, and cis- and trans-DCE
(Sigma-Aldridge).
Medium x 1 2 "Stock solutions were made from 95 percent pure,
analytical-grade PCE, TCE, and cis- and trans-DCE
(Sigma-Aldridge)." Only minor uncertainties about
the purity being at 95 percent, analytical-grade.
Domain 2: Test Design
Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
High X 2 2 Three replicates of embryos were also raised in un-
contaminated groundwater as controls.
High X 1 1 Control mortality was reported in table 2 for each
species, and deformities in controls were reported in
figure 1. Control response for mortality for wood
frogs, green frogs and spotted salamanders were all
below 10 percent; for American toads it was about
10.1 percent with one of the replicates having a
very high 26 percent mortality rate, so there are
uncertainites for this species for this metric. Fig-
ure 1 shows that the negative control response for
all species for percent mortality is below 10 percent.
ASTM guidelines indcate "An acceptable clutch of
eggs has the capability of developing into Develop-
mental Stage 46 tadpoles with less than 10 percent
gross abnormalities and less than 10 percent mortal-
ity."
Low X 1 3 For the acute study it was not reported whether the
animals were distributed randomly.
Domain 3: Exposure Characterization
Continued on next page

-------
. continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type:	Other; Aquatic; other amphibian - spotted salamder
Hero ID:	700434
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 7: Experimental System/Test Media Prepara- Low	X 2	6 Containers were covered and sealed but htere was
^jq-q	no mention of minimizing head space, and authors
mentioned that TCE solutions declined by 50 to 80
percent over the 24 hour period between renewals.
Authors also mentioned, "Each egg mass was gently
divided into clusters of approximately 30 eggs (with
the exception of spotted salamanders with 5 to 10
eggs) and placed in a 1-L glass Mason jar contain-
ing 300 ml of test solution. The lids on the jars were
sealed to reduce volatilization. Dissolved oxygen lev-
els never fell below 80 percent saturation. Three
replicates of embryos were also raised in uncontam-
inated groundwater as controls. Temperature was
maintained at 23 +-1"C using a water bath. All
tests were conducted under a 14L/10D light regime.
An exhaust hood over the water bath ensured the
removal of accidental gaseous PCE, TCE, and DCE
volatilized from the exposure vessels."
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - spotted salamder
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 8: Consistency of Exposure Administration Medium x 1 2 Authors indicate that there may have been some
losses of test chemical during decanting test solu-
tions and during the placing of eggs in test cham-
bers, and while testing a subsample at 1 hour of
exposure TCE cone were only within 70 percent of
nominal. The authors report, "Stock solutions of
TCE, PCE, and DCE were dissolved in local ground-
water in a dilution series. Groundwater was used as
the medium for acute tests to emulate conditions in
surface waters fed by chloroethylene- contaminated
groundwater. Nominal test concentrations were as
follows: PCE-2.5, 7.5, 12.5, and 20 mg/L; TCE-12.5,
20, 40, and60 mg/L; and cis- and trans-DCE-12.5,
60, and 100 mg/L. Based on the results of initial ex-
posures of American toad embryos, a second expo-
sure was conducted with elevated concentrations of
PCE and TCE as follows: PCE-15, 30, and 45 mg/
L; and TCE-35, 55, and 85 mg/L. Maximum expo-
sure concentrations of PCE and TCE were limited
by the compounds' solubility in groundwater. Con-
centrations of test solutions, including controls, were
measured at 24 h (just prior to solution renewal, see
below). Concentrations at t = 0 h were based on
dilutions of measured stock solutions. Some losses
occurred while decanting test solutions and during
the placing of eggs in test chambers. In a sub-
sample of test solutions measured at 1 h of expo-
sure,concentrations of PCE were within 99 percent
of nominal, while cis- and trans-DCE were within 90
percent. However, levels of TCE were only within 70
percent of nominal."
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type:	Other; Aquatic; other amphibian - spotted salamder
Hero ID:	700434
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 9: Measurement of Test Substance Concentra- High	x 2	2 Measured concentrations were taken, and used to
•j-jQ-Q	derive an EC50. Authors reported "To prevent
volatilization, a glass flask (6 L) was filled to ca-
pacity with stock solution to reduce head space and
then stirred using a stir bar at low speed to pre-
vent disruption of the surface. Stock solutions were
stirred until the compound was dissolved or until
the solution had reached saturation (between 2 and
48 h, depending on the compound). Despite these
precautions,actual concentrations tended to be less
than nominal concentrations.For this reason, con-
centrations of stock solutions were measured priorto
mixing of the test solutions, using Purge and Trap
GC/MS accordingto U.S. EPA method 82 60B (U.S.
EPA 1996), to ensure that testsolutions were within
10 percent of the nominal concentration at lh of ex-
posure. The GS/MS was performed on a Hewlett
Packard 6890 gas chromatograph, using a Hewlett
Packard 5973 mass selective detector, a 3100 purge
and trap concentrator, and a PTA-30 purge and trap
autosampler. The method detection limit for all
chloroethylene analyses was 1 ppb.
Metric 10: Exposure Duration and Frequency	Low	x 1	3 ASTM guidelines for FETAX on American clawed
frog suggest 96 hours and a static renewal set up, re-
newed every 24 hours, which this study does for the
acute test. However, it appears that 96 hours is not
enough time for salamanders. Authors state, "Ex-
posures followed a 96-h static renewal process with
test solutions refreshed daily. Most eggs hatched
during the 96-h exposure period with the exception
of spotted sal anders. After 96 h, survivorship was
assessed; larvae were then euthanized with a solution
of clove oil. Spotted salamanders had not hatched
by the end of the 96-h period since they take up to a
week longer to develop to hatching than the anuran
species chosen. The developing salamander embryos
were placed in clean groundwater until hatching was
complete. Anuran embryos were staged at 96 h ac-
cording to Gosner (1960) to test for effects of ex-
posures on developmental rates. Salamander larvae
were staged at 192 h according to Harrison (1969).
Larvae were examined for developmental deformities
according to the Atlas of Abnonnalities (Bantle et al.
1998) for Xenopus laevis tadpoles.
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type:	Other; Aquatic; other amphibian - spotted salamder
Hero ID:	700434
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 11: Number of Exposure Groups/Spacing of Ex-
posure Levels
Metric 12: Testing at or Below Solubility Limit
Medium x 1
High
x 1
This study had four exposure groups for TCE and
ASTM FETAX Guidelines suggests the following
"At a minimum, five concentrations for each end-
point are used. However, additional concentrations
between the EC16 and EC84 are highly recom-
mended to ensure obtaining accurate 96-hour LC50
and EC50 values."
Solubility was taken into consideration, and expo-
sure concentrations kept below the solubility of TCE
in growndwater.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics	Medium X 2	4 Spotted salamander: Test organisms seem to be suf-
ficiently sensitive to the exposures administered to
derive an EC50, but are not a suggested species in
the ASTM guideline or OECD and EPA guidelines
for amphibian growth and development which sug-
gest African clawed frog. This study instead used
these species to test sensitivity for North American
species. Only minor uncertainties because they are
not suggested species from a guideline.
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - spotted salamder
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 14: Acclimitization and Pretreatment Conditions Low x 1 3 Study authors did not report whether animals were
acclimatized or whether pretreatment conditions
were the the same for treatment and controls. They
authors do report, " In 2001 and 2002, egg masses of
wood frogs, spotted salamanders," American toads,
and green frogs were collected from a wetland not
contaminated with chloroethylenes in Flamborough
Township (Ontario,Canada). Water from wetlands
were tested for chloroethylenes in 2001 from each
site where eggs were collected. No chloroethylenes
were detected; the minimum detection limit for this
analysis was 1 ppb. Egg masses were less than 24 h
old when exposures were initiated. For each species,
three egg masses were used (with the exception of
the second exposure of American toads, where only
one egg mass was used). Each egg.mass was from a
different female and represented a replicate. Thus,
there were three replicate jars for each chemical by
concentration combination, for a total of 45 jars per
species. Eggs were not dejellied prior to exposure to
more accurately imitatenatural exposure conditions.
Each egg mass was gently divided into clusters of ap-
proximately 30 eggs (with the exception of spotted
salamanders with 5 to 10 eggs) and placed in a 1-L
glass Mason jar containing 300 ml of test solution."
Metric 15: Number of Organisms and Replicates per Low x 1 3 ASTM guidelines suggest 20-25 and two replicates,
Group and study authors reported, "there were three
replicate jars for each chemical" and, "Each egg
mass was gently divided into clusters of approxi-
mately 30 eggs (with the exception of spotted sala-
manders with 5 to 10 eggs) and placed in a 1-L
glass Mason jar containing 300 ml of test solution."
This resulted in the nominal and measured cone for
TCE not having an adequate sample size to gener-
ate confidence intervals. Because salamanders are
difficult to rear in the lab in high numbers, this was
taken into consideration in the scoring for this met-
ric (given a low instead of an unacceptable). Addi-
tionally the number of organisms suggested in the
ASTM guidelines are based on another species.
Continued on next page

-------
. . . continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type: Other; Aquatic; other amphibian - spotted salamder
Hero ID: 700434
Domain Metric Rating^ MWF* Score Comments^
Metric 16: Adequacy of Test Conditions Medium X 1 2 Animals were held in 1 L glass mason jars contain-
ing 300 ml of test solution. Jars were sealed and
temperature was maintined at 23+-1 degree C us-
ing a water bath. All tests were conducted under
14L/10D light regime. The ASTM guidelines rec-
ommend glass, and this temperature is approprate
for african clawed frog but unsure if this temperature
is also appropriate for these north american species.
Additionally the photoperiod is longer than the one
recommended in the ASTM Guidelines.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
x 2
Medium x 1
Outcome assessment methodology addressed the in-
tended outcomes of interest and was sensitive for
the outcomes of interest. LC50 and EC50 were
reported which are the recommended outcomes in
ASTM guidelines for FETAX. Dead individuals were
counted and removed at the end of each 24 hours.
All animals were assessed at the end of the 96 hour
period with minor uncertainties due to incomplete
reporting.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure Medium x 1
2 No reported differences among study groups in en-
vironmental conidtions.
2 Controls for the spotted salamanders were under 10
percent mortality and deformities. Details on attri-
tion unrelated to exposure for each exposure con-
centration were also reported as the average with a
range. There is a wide range of portailities between
the replicates.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Medium X 1 2 A two-factor ANOVA was used. ASTM FETAX
Guidelines suggests either probit analysis, trimmed
Spearman-Karber analysis, or the two-point graphi-
cal method to estimate LC50 and EC50 values. How-
ever due to sample size authors were not able to gen-
erate confidence intervals.
Continued on next page

-------
. continued from previous page
Study Citation: McDaniel, T.,Martin, P.,Ross, N.,Brown, S.,Lesage, S.,Pauli, B.. 2004. Effects of chforinated sofvents on four species of North American
amphibians. Archives of Environmental Contamination and Toxicology 47:101-109
Data Type:	Other; Aquatic; other amphibian - spotted salamder
Hero ID:	700434
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 22: Reporting of Data	Medium x 2	4 Data was reported for each exposure group in ei-
ther table or graphical form. It's hard to tell the
exact numbers from the graphical representation of
the EC50 values for each exposure level, resulting in
minor uncertainites.
Metric 23: Explanation of Unexpected Outcomes	High	x 1	1 Unexpected outcomes were explained, (e.g. loss of
TCE through evaporation, damaged eggs in transit)
Overall Quality Determination^	Medium	1.8
Extracted	Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Fort, D.,Rayburn, J.,Deyoung, D.,Bantle, J.. 1991. Assessing the efficacy of an Arocior 1254-induced exogenous metaboiic activation
system for FETAX. Drug and Chemicaf Toxicoiogy 14:143-160
Data Type: Other; Aquatic; other amphibian - frog
Hero ID: 701994
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Identified by name trichloroethylene
Metric 2:
Test Substance Source
High
X
1
1
Aldrich, Milwaukee, WI
Metric 3:
Test Substance Purity
Low
X
1
3
Not reported
Domain 2: Test Design
Metric 4:
Negative Controls
High
x 2
Metric 5: Negative Control Response
High
x 1
Metric 6: Randomized Allocation
Low
x 1
Four separate dishes of 20 embryos were exposed
to FETAX solution alone and designated FETAX
solution controls. Controls including FETAX solu-
tion,each MAS , CO-MAS + toxicant (negative con-
trol), cyclophosphamide (FETAX reference Arocior
1254 MAS proteratogen7 " 9 - 11) and acetylhy-
drazide (additional mixed MAS positive control7),
and unactivated toxicant were tested concurrently
with each experiment .
In this study, FETAX solution control embryo mor-
tality and malformation rates were <2 . 0 percent
and <5 percent, respectively . Mortality and malfor-
mation rates for each MAS control were <4 percent
and <9 percent, respectively. Acceptable rates of
FETAX solution a nd MAS control mortality and
malformation are generally 1 0 percent . No less
than 86 percent of those embryos subjected to 4.0
mg/ml activated cyclophosphamide and e ither the
Arocior 1254 or the mixed MAS died . Survivors
of the previous treatments were severely deformed
. Exposure of embryos to 3 . 0 mg/ml activated
acetylhydrazide and the INH MAS or mixed MAS
produced mortality and malformation rates >43 per-
cent and 100 percent, respectively .
Not reported how allocation was conducted
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low x 2 6 Covered petri dishes were used but there was no
¦j-Jq-q mention of headspace or other considerations for
volatility.
Continued on next page

-------
. continued from previous page
Study Citation: Fort, D.,Rayburn, J.,Deyoung, D.,Bantle, J.. 1991. Assessing the efficacy of an Arocior 1254-induced exogenous metaboiic activation
system for FETAX. Drug and Chemicai Toxicoiogy 14:143-160
Data Type: Other; Aquatic; other amphibian - frog
Hero ID: 701994
Domain Metric Rating^ MWF* Score Comments^
Metric 8: Consistency of Exposure Administration
Low
x 1
Metric 9: Measurement of Test Substance Concentra- Low x 2
tion
Metric 10: Exposure Duration and Frequency
High x 1
Only general details of exposure administration were
given. All solutions were renewed every 24 hours in
a static renewal system for 96 hours.
Not reported how test concentrations were deter-
mined, and because substance is volatile could have
substantial impacts on results.
Published protocol for FETAX suggest a static-
renewal, 96 hour test, which is what the authors
of this paper conducted in this study. While it's
not an OECD or EPA guideline study, other peer
reviewed publications have used FETAX repeatedly,
and the ASTM guidelines methods were reviewed
by NTP at the request of EPA, and determined
that FETAX data can be used to identify and pri-
oritize sites with increased developmental toxicity
risks. "Due to varying susceptibilities among ani-
mals, testing in multiple species is considered neces-
sary to protect the environment. For each species,
it is a combination of toxicants, water quality, and
the organism itself that defines the hazard for a spe-
cific concentration of a toxicant within defined water
quality conditions. Ecotoxicological standards are
generally based on the susceptibility of the adult
animal, which may not provide adequate protec-
tion for embryonic development and reproduction in
many species. It is inherently impossible to eval-
uate developmental toxicity without exposing ani-
mals throughout development and assessing for ad-
verse effects in multiple life stages, and for Early
embryonic and juvenile stages are often the most
susceptible periods for the toxic effects of many en-
vironmental contaminants. Embryonic development
in amphibians is sensitive to water quality. Be-
cause ofthis, FETAX has been used in ecotoxicolog-
ical studies to evaluate the potential developmental
hazard of contaminated surface waters, sediments,
waste site soils, and industrial wastewater and to
evaluate the efficacy of wastewater treatment pro-
cedures. In this context, the resulting data can be
used to identify and prioritize sites with increased
developmental toxicity risks. "
Continued on next page . . .

-------
. continued from previous page
Study Citation: Fort, D.,Rayburn, J.,Deyoung, D.,Bantle, J.. 1991. Assessing the efficacy of an Arocior 1254-induced exogenous metaboiic activation
system for FETAX. Drug and Chemicai Toxicoiogy 14:143-160
Data Type: Other; Aquatic; other amphibian - frog
Hero ID: 701994
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 11: Number of Exposure Groups/Spacing of Ex- High x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
For each compound, 12-18 concentrations were
tested with replicates. FETAX publications suggest
at least 5 test cone are appropriate. Also the study
reports "For each compound, one range-finding and
two definitive dose-response assays were conducted
with and without the MAS."
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Low
x 2
x 1
Metric 15: Number of Organisms and Replicates per Medium x 1
Group
2 Xenopus laevis was used in this study and is the
recommended species in ASTM guidelines.
3 Uncertainties remain because the acclimatization
was not fully described in this paper. Authors write,
"Xenopus adult care, breeding, and embryo collec-
tion were performed as described by Dawson and
Bantle."
2 20 embryos were used in replicate. Guidelines for
ASTM FETAX state "Each of the three definitive
tests is conducted using embryos from a different
male/female pair of X. laevis. Each test consists
of several different concentrations of the test sub-
stance with two replicate dishes at each test con-
centration and four replicate dishes for each control.
Each plastic or glass Petri dish contains 20 or 25
embryos, respectively. The number of embryos per
dish, the number of replicate dishes per test sub-
stance concentration, and the number of replicate
tests per study were not based on a formal scien-
tific analysis. Rather, selection was based on the
best scientific judgement of the developers/users of
the assay at the time the ASTM FETAX Guideline
(1991, 1998) was prepared (J. Bantle and D. Fort,
personal communication)."
Continued on next page

-------
. . . continued from previous page
Study Citation: Fort, D.,Rayburn, J.,Deyoung, D.,Bantle, J.. 1991. Assessing the efficacy of an Arocior 1254-induced exogenous metaboiic activation
system for FETAX. Drug and Chemicai Toxicoiogy 14:143-160
Data Type: Other; Aquatic; other amphibian - frog
Hero ID: 701994
Domain Metric Rating^ MWF* Score Comments^
Metric 16: Adequacy of Test Conditions Medium X 1 2 Minor uncertainties about housing. Temp and ph
are appropriate according to published recommen-
dations. "For experiments conducted without the
MAS, groups of 20 embryos were placed in 60 mm
covered plastic Petri dishes with varying concen-
trations of the appropriate test compound." "Each
treatment dish contained a total o f 8 ml o f solu-
tion." "The pH of all stock solutions was 7.0. Em-
bryos were cultured at 23"l"c. All solutions were
renewed every 24 h. Dead embryos were removed at
this time ." The ASTM guidance states " Glass, Type
316 stainless steel, nylon, and fluorocarbon plastic
should be used whenever possible to minimize dis-
solution, leaching, and sorption." It is unclear what
kind of plastic these petri dishes are made of.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
High
x 2
Metric 18: Consistency of Outcome Assessment
Medium x 1
ASTM guidance states that "Only those biological
responses related to mortality and malformations
are considered in this analysis" and an LC50 and
EC50 for malformations were established.The study
states, "96-h median lethal concentrations (LC50)
and the concentrations inducing gross terata in 50
percent of the surviving larvae (EC50) along with
respective 95 percent confidence limits were deter-
mined using Litchfield Wilcoxon probit analysis .
20 A Teratogenic Index (TI=LC50/EC50) was used
as a meth6d of assessing teratogenic potential . 5
18- 11, 21 Head-tail length of surviving embryos was
measured as an index of embryo growth. Minimum
concentrations to inhibit growth (MCIG) were de-
termined with t he t-Test (p<0.05)."
Incomplete reporting of minor details of outcome as-
sessment protocol
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Medium x 2 4 Incomplete data across study groups, and there
Procedures could be confounding due to the volatility of the
chemical.
Continued on next page

-------
. . . continued from previous page
Study Citation: Fort, D.,Rayburn, J.,Deyoung, D.,Bantle, J.. 1991. Assessing the efficacy of an Arocior 1254-induced exogenous metaboiic activation
system for FETAX. Drug and Chemicai Toxicoiogy 14:143-160
Data Type: Other; Aquatic; other amphibian - frog
Hero ID: 701994
Domain Metric Rating^ MWF* Score Comments^
Metric 20: Outcomes Unrelated to Exposure Medium x 1 2 There was some discussion of health outcomes un-
related to exposure with only minor uncertainties
about why there were differences between the vari-
ous controls. "In this study, FETAX solut ion con-
trol embryo mortality and malformation rates were
<2 . 0 percent and <5 percent, respectively . Mor-
tality and malformation rates for each MAS contro 1
were <4 percent and <9 percent, res pectively. Ac-
ceptable rates of FETAX solution a nd MAS con-
trol morta 1 ity and malformation are generally 1
0 percent . No less than 86 percent of those em-
bryos subjected to 4.0 mg/ml activated cyclophos-
phamide and e ither the Arocior 1254 or the mixed
MAS died . Survivors of the previous treatments
were s everely deformed . Exposure of embryos to
3 . 0 mg/ml activated acetylhydrazide and the INH
MAS or mixed MAS produced mortality and malfor-
mation rates >43 percent and 100 percent, respec-
tively."
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
High
x 1
Metric 22: Reporting of Data
Medium x 2
ASTM guidance says that either probit analysis,
trimmed Spearman-Karber analysis or the two-point
graphical methode are used to estimate the LC50
and EC50 values. This study states that the "96-h
median lethal concentrations (LC50) and the con-
centrations inducing gross terata in 50 percent of
the surviving larvae (EC50) along with respective
95 percent confidence limits were determined using
Litchfield Wilcoxon probit analysis. A Teratogenic
Index (TI=LC50/EC50) was used as a method of
assessing teratogenic potential. Head-tail length of
surviving embryos was measured as an index of em-
bryo growth. Minimum concentrations to inhibit
growth (MCIG) were determined with t he t-Test
(p<0.05)."
Some information is given for each treatment group,
but it's incomplete, information was given for TCE
alone, TCE and MAS, etc but not for each exposure
group
Continued on next page

-------
. continued from previous page
Study Citation: Fort, D.,Rayburn, J.,Deyoung, D.,Bantle, J.. 1991. Assessing the efficacy of an Arocior 1254-induced exogenous metaboiic activation
system for FETAX. Drug and Chemicai Toxicoiogy 14:143-160
Data Type:	Other; Aquatic; other amphibian - frog
Hero ID:	701994
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 23: Explanation of Unexpected Outcomes	High	x 1	1 Unexpected outcomes were discussed. "Several of
the TCE-induced malformations including cardiac
effects observed in the avian modelare similar to
those recorded with Xenopus. Differences in the
ability of TCE to induce terata in mammals, birds,
and frog embryos may be the result ofdifferences
in embryonic susceptibility across the phylogenetic
range, route of exposure, as well as, pharmaco-
logical nature of the test system. Themajority of
mammalian studies utilized pulmonary exposure,
whereas, the avian system and FETAX provide di-
rect embryonic exposure to toxicants. Positive re-
sults in FETAX may, in fact, be due to highly
embryotoxic intermediates or metabolites of TCE.
The formation of a reactive epoxide intermediate
(TCEoxide) has been identified in rat, rabbit, and
mouse."
Overall Quality Determination^	Medium	1.8
Extracted	Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Fort, D.,Rogers, R.,Stover, E.,Finch, R.. 2001. Optimization of an exogenous metabolic activation system for FETAX. I. Post-isolation
rat liver microsome mixtures. Drug and Chemical Toxicology 24:103-115
Data Type: Other; Aquatic; other amphibian - frog
Hero ID: 701995
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Chemical identified by name
Metric 2:
Test Substance Source
High
X
1
1
Signma St Louise Missouri
Metric 3:
Test Substance Purity
Low
X
1
3
Purity not reported
Domain 2: Test Design

Metric 4:
Negative Controls
High
X
2
2
Four separate dishes of 20 embryos were exposed FE-

TAX solution alone and designated FETAX solution

controls.
Metric 5:
Negative Control Response
High
X
1
1
In this study, FETAX solution control embryo mor-

tality and malformation rates were 5.0 percent and

6.3 percent, respectively. Mortality and malforma-

tion rates for each MAS control were 7.5 percent and

7.6 percent, respectively.
Metric 6:
Randomized Allocation
Low
X
1
3
It was not reported how animals were allocated
Domain 3: Exposure Characterization

Metric 7:
Experimental System/Test Media Prepara-
Low
X
2
6
Dishes were covered but no mention of headspace

tion

minimizing was given. TCE is very volatile, and it

was not indicated whether concentrations were con-

firmed through measurement.
Metric 8:
Consistency of Exposure Administration
Medium
X
1
2
Only general details of exposure administration were
Metric 9: Measurement of Test Substance Concentra- Low
tion
x 2
given. This study reports that solutions were re-
newed every 24 hours in a static renewal system
for 96 hours, and "For each compound, 12" 18 con-
centrations were tested in duplicate. Four separate
dishes of 20 embryos were exposed FETAX solu-
tion alone and designated FETAX solution controls.
Each treatment dish contained a total of lOmL of
solution."
It was not reported whether test concentrations were
measured, and based on professional judgment of ex-
periment design and nature of test substance, actual
concentration cannot be expected to be similar to
nominal concentration.
Continued on next page

-------
. continued from previous page
Study Citation: Fort, D.,Rogers, R.,Stover, E.,Finch, R.. 2001. Optimization of an exogenous metabolic activation system for FETAX. I. Post-isolation
rat liver microsome mixtures. Drug and Chemical Toxicology 24:103-115
Data Type: Other; Aquatic; other amphibian - frog
Hero ID: 701995
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 10: Exposure Duration and Frequency
High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- High
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
x 1
FETAX is a static-renewal bioassay, and in accor-
dance with other published FETAX assays (and the
ASTM guidelines which were reviewed by NTP), the
study authors did a 96 hour test with the solutions
renewed every 24 hours.
"For each compound, one range-finding and two
definitive dose-responseassays were conducted with
and without the MASs." and "For each com-
pound,12" 18 concentrations were tested in dupli-
cate." FETAX publications suggest at least 5 test
cone are appropriate.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Low
x 2
x 1
Metric 15: Number of Organisms and Replicates per Medium x 1
Group
2 Xenopus laevis are used in this study are the recom-
mended species for FETAX in ASTM guidelines
3 The study reports, "X. laevis adult care, breeding,
and embryo collection were preformed as described
in ASTM E1439-98" and it is not clear from this
paper whether animals were acclimatized.
2 This study used 20 in replicate in plastic dishes.
Guidelines for ASTM FETAX state "Each of the
three definitive tests is conducted using embryos
from a different male/female pair of X. laevis. Each
test consists of several different concentrations of the
test substance with two replicate dishes at each test
concentration and four replicate dishes for each con-
trol. Each plastic or glass Petri dish contains 20
or 25 embryos, respectively. The number of embryos
per dish, the number of replicate dishes per test sub-
stance concentration, and the number of replicate
tests per study were not based on a formal scien-
tific analysis. Rather, selection was based on the
best scientific judgement of the developers/users of
the assay at the time the ASTM FETAX Guideline
(1991, 1998) was prepared (J. Bantle and D. Fort,
personal communication)."
Continued on next page . . .

-------
. . . continued from previous page
Study Citation: Fort, D.,Rogers, R.,Stover, E.,Finch, R.. 2001. Optimization of an exogenous metabolic activation system for FETAX. I. Post-isolation
rat liver microsome mixtures. Drug and Chemical Toxicology 24:103-115
Data Type: Other; Aquatic; other amphibian - frog
Hero ID: 701995
Domain Metric Rating^ MWF* Score Comments^
Metric 16: Adequacy of Test Conditions Medium x 1 2 Minor uncertainties about housing. The ASTM
guidance on FETAX suggest 23 degrees C +-3 which
was maintained in this study, and ph was maintained
between the suggested 6.5 and 9.0 range with 7.7 be-
ing optimal. Each treatment dish contained a total
of lOmL of solution for 20 embryos each which is
enough according to ASTM Guidance. This guid-
ance also states "Glass, Type 316 stainless steel, ny-
lon, and fluorocarbon plastic should be used when-
ever possible to minimize dissolution, leaching, and
sorption." It is unclear what kind of plastic these
petri dishes are made of.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
x 2
Medium x 1
Outcome assessment methodology addressed the in-
tended outcomes of interest. ASTM guidance states
that "Only those biological responses related to mor-
tality and malformations are considered in this anal-
ysis" and an LC50 and EC50 for malformations were
established.
Incomplete reporting of minor details of outcome as-
sessment protocol
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Medium x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure
High
x 1
Incomplete data across study groups, and there
could be confounding due to the volatility of the
chemical.
The study reports "In this study, FETAX solu-
tion control embryo mortality and malformation
rates were 5.0 percent and 6.3 percent, respectively.
Mortality and malformation rates for each MAS
control were 7.5 percent and 7.6 percent, respec-
tively.Acceptable rates of FETAX solution and MAS
control mortality andmalformation are generally 10
percent."
Domain 7: Data Presentation and Analysis
Continued on next page

-------
. continued from previous page
Study Citation: Fort, D.,Rogers, R.,Stover, E.,Finch, R.. 2001. Optimization of an exogenous metabolic activation system for FETAX. I. Post-isolation
rat liver microsome mixtures. Drug and Chemical Toxicology 24:103-115
Data Type:	Other; Aquatic; other amphibian - frog
Hero ID:	701995
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 21: Statistical Methods
High
x 1
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
Low	x 2
High x 1
ASTM guidance says that either probit analysis,
trimmed Spearman-Karber analysis or the two-point
graphical methode are used to estimate the LC50
and EC50 values. The study reports, "96 h median
lethal concentrations (LC50) and the concentrations
inducing gross terata in 50 percent of the surviv-
ing larvae (EC50) along with respective 95 per-
cent fiducial limits were determined using trimmed
Spearman-Karber analysis. Head-tail length of sur-
viving embryos was measured as an index of embryo
growth. Minimum concentrations to inhibit growth
(MCIG) were determined using ANOVA [Bonferroni
t-test (p<0.05)]."
Data for exposure related findings were not shown
for each study group but results were described in
the text.
No unexplained outcomes.
Overall Quality Determination"'"
Medium
1.8
Extracted
Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 707209
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 TCE identified by name
High X 1 1 Test compounds (Table 1) were obtained from
AldrichChemical (Milwaukee, Wisconsin). All
were widely employedindustrial chemicals and have
a mode-of-actionclassified as nonpolar narcosis
(Schultz et al., 1990; VanLeeuwen et al., 1992; Rus-
som et al., 1997).
Medium X 1 2 Test substance purity is reported as 99.5 percent as
labeled but not independently verified.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
Low X 2 6 Authors reported using negative controls but did not
report details of the negative control group.
High X 1 1 Negative control response for mortality for 2-day
study reported in table 4.
Low X 1 3 Not randomly allocated
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High
tion
Metric 8: Consistency of Exposure Administration
x 2
Medium x 1
The test was conducted in a closed container and
filled to capacity to limit volitization. "25-ml
borosilicate glass vials filled to capacity and closed
tightly using teflon PTFE-lined silicon septa held in
place by polypropylene screw-on caps. These vials
are sold by scientific suppliers as 'EPA vials' forstor-
age of water samples. Masten et al. (1994) found
that static-renewal tests with these vials maintained
concentrations of volatile chemicals more success-
fully than flowthrough test designs."
Only minor uncertainties about exposure adminis-
tration
Continued on next page

-------
. continued from previous page
Study Citation: Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 707209
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 9: Measurement of Test Substance Concentra- High
tion
x 2
Metric 10: Exposure Duration and Frequency High
Metric 11: Number of Exposure Groups/Spacing of Ex- High
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
x 1
x 1
"In tests with individual components, toxicant con-
centrations were measured at the beginning and end
of the experiment. Newly prepared test solution
and 24-h-old test solution composited from three
replicates from each treatment level were analyzed.
Acute toxicological results are based on the geomet-
ric mean of the two measured concentrations for each
treatment level measured at the beginning of the
test."
48 hour test for daphnia
EPA effluent guidelines for C. dubia recommends 5
test concentrations and there were 5 in this acute
test
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Low
Group
X 2 2 Ceriodaphnia dubia is suitable and recom-
mended in EPA effluent test guidelines. https:/
/www.epa.gov/sites/production/files/2015-12/
documents/method_1002_2002.pdf
X 1 3 The study does not report how long test organisms
were acclimatized.
X 1 3 The study says that "Responses are based on a sam-
ple size of 10" but it's unclear if that means 10 in-
dividuals or 10 brood cups (10 brood cups is recom-
mended in the EPA effluent guidelines for C. dubia.)
Elsewhere in the study it states "Newly prepared
test solution and 24-h-old test solution composited
from three replicates from each treatment level were
analyzed." The methods say they follow the guide-
lines, but the description isn't explicit about how
many animals were actually used.
Continued on next page

-------
. continued from previous page
Study Citation: Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 707209
Domain Metric Rating^ MWF* Score Comments^
Metric 16: Adequacy of Test Conditions Medium X 1 2 Only minor uncertainties about housing. "Thestan-
dard, short-term, chronic toxicity test method de-
veloped for U.S. EPA's Whole Effluent Testing Pro-
gram (U.S. EPA, 1994) was followed with modifi-
cations to minimize volatilization of test chemicals.
Instead of 30-ml beakers, individual organisms were
tested in 25-ml borosilicate glass vials filled to capac-
ity and closed tightly using teflon PTFE-lined sili-
con" septa held in place by polypropylene screw-on
caps. These vials are sold by scientific suppliers as
"EPA vials" for storage of water samples. Masten et
al. (1994) found that static-renewal tests with these
vials maintained concentrations of volatile chemi-
cals more successfully than flowthrough test designs.
Tests were conducted in artificial moderately hard
water (U.S. EPA, 1994; Table 2). Light was pro-
vided by full spectrum fluorescent bulbs with a color
rendering index >90 at an intensity of 20 mE/m 2/S
and a photoperiod of 16L: 8D. Daphnids were feel an
algae and cereal leaf mix containing equal numbers
of cells of Se/enastrwn capricornutum and Ch/ore/
la uu/garis mixed with a rye grass infusion (ASTivI,
1994). This mixture was added to diluted stock so-
lutions to yield a final concentration of 3 x 105 algal
cells/ml and 0.03 mg/ml solids from cereal grass in
each test vial. Component algae were cultured indi-
vidually in modified Bold's basal medium (ASTM,
1994). Solutions were renewed daily. Dissolved oxy-
gen was monitored on 24-h-old solutions and always
remained above 7.0 ppm."
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
LC50 measured for acute toxicity over 48 hours
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High
Procedures
x 2
No reported differences between study groups in en-
vironmental conditions or other factors that could
influence the outcome assessment.
Continued on next page . . .

-------
. continued from previous page
Study Citation: Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	707209
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 20: Outcomes Unrelated to Exposure
High
x 1
Table 4 indicates sample size for each test concen-
tration, and shows if the sample size dropped to 9
rather than 10. All test cone for TCE had a sample
size of 10.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
High
x 1
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High
High
x 2
x 1
"Toxicity tests with individual chemicals were an-
alyzed to determine criterion effect levels for mor-
tality and reproduction following standard methods
(U.S. EPA, 1994). Both 2- and 7-clay LC50s (con-
centrations lethal to 50'Yo of the organisms) were
calculated using binomial or trimmed Spearman-
Karber tests, as appropriate. After appropriate tests
of normality, effects on reproduction (number of
young per female through the end of the lest) were
analyzed withANOV A, followed by one-sided Dun-
nett's comparison of means to the control to deter-
mine NOELs (no-observed effect levels) and LOELs
(lowest-observed-effect levels). In addition, IC50s
(concentrations inhibiting reproduction by50 per-
cent relative to mean control performance) were cal-
culated from smoothed data using bootstrap analy-
sis (Norberg-King, 1993)."
Results were reported for each exposure group.
No unexpected outcomes for TCE.
Overall Quality Determination"'"
High
1.4
Extracted
Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Data Type: Other; Aquatic; Invertebrates
Hero ID: 707209
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 TCE identified by name
High X 1 1 Test compounds were obtained from Aldrich Chem-
ical (Milwaukee, Wisconsin). All were widely em-
ployed industrial chemicals and have a mode-of-
action classified as nonpolar narcosis (Schultz et al.,
1990; Van Leeuwen et al., 1992; Russom et al., 1997).
Medium X 1 2 Test substance purity is reported as 99.5 percent as
labeled but not independently verified.
Domain 2: Test Design
Metric 4:
Metric 5:
Negative Controls
Negative Control Response
Metric 6: Randomized Allocation
Low X 2 6 Authors reported using negative controls but did not
report details of the negative control group.
High X 1 1 Negative control response was reported in Table 4.
No mortality was observed, and mean young for re-
pro was reported.
Low X 1 3 Not randomly allocated
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High
tion
Metric 8: Consistency of Exposure Administration
x 2
Medium x 1
The test was conducted in a closed container and
filled to capacity to limit volitization. "25-ml
borosilicate glass vials filled lo capacity and closed
tightly using teflon PTFE-lined silicon septa held in
place by polypropylene screw-on caps. These vials
are sold by scientific suppliers as "EPA vials" for
storage of water samples. Masten et al. (1994) found
that static-renewal tests with these vials maintained
concentrations of volatile chemicals more success-
fully than flow through test designs."
Only minor uncertainties about exposure adminis-
tration.
Continued on next page

-------
. continued from previous page
Study Citation: Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Data Type: Other; Aquatic; Invertebrates
Hero ID: 707209
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency
High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- High
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
x 1
"In tests with individual components, toxicant con-
centrations were measured at the beginning and end
of the experiment. Newly prepared test solution and
24-h-old test solution composited from three repli-
cates from each treatment level were analyzed. [. . .
] Chronic toxicological results are based on the ge-
ometric "mean of the four measured concentrations
for each treatment level measured during the dura-
tion of the experiment."
7 days recommended for EPA effluent guidelines for
C. dubia. https://www.epa.gov/sites/production/
files/2015-12/documents/method_1002_2002.pdf
EPA effluent guidelines recommend 5 test concen-
trations and 5 were used in this study. https:/
/www.epa.gov/sites/production/files/2015-12/
documents/method_1002_2002.pdf
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Low
Group
X 2 2 Ceriodaphnia dubia is suitable and recom-
mended in EPA effluent test guidelines. https:/
/www.epa.gov/sites/production/files/2015-12/
documents/method_1002_2002.pdf
X 1 3 The study not report how long test organisms were
acclimatized.
X 1 3 The study says that "Responses are based on a sam-
ple size of 10" but it's unclear if that means 10 in-
dividuals or 10 brood cups (10 brood cups is recom-
mended in the EPA effluent guidelines for C. dubia.)
Elsewhere in the study it states "Newly prepared
test solution and 24-h-old test solution composited
from three replicates from each treatment level were
analyzed." The methods say they follow the guide-
lines, but the description isn't explicit about how
many animals were actually used.
Continued on next page

-------
. continued from previous page
Study Citation: Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Data Type: Other; Aquatic; Invertebrates
Hero ID: 707209
Domain Metric Rating^ MWF* Score Comments^
Metric 16: Adequacy of Test Conditions Medium X 1 2 Only minor uncertainties about housing. "Thestan-
dard, short-term, chronic toxicity test method de-
veloped for U.S. EPA's Whole Effluent Testing Pro-
gram (U.S. EPA, 1994) was followed with modifi-
cations to minimize volatilization of test chemicals.
Instead of 30-ml beakers, individual organisms were
tested in 25-ml borosilicate glass vials filled to capac-
ity and closed tightly using teflon PTFE-lined sili-
con" septa held in place by polypropylene screw-on
caps. These vials are sold by scientific suppliers as
"EPA vials" for storage of water samples. Masten et
al. (1994) found that static-renewal tests with these
vials maintained concentrations of volatile chemi-
cals more successfully than flowthrough test designs.
Tests were conducted in artificial moderately hard
water (U.S. EPA, 1994; Table 2). Light was pro-
vided by full spectrum fluorescent bulbs with a color
rendering index >90 at an intensity of 20 mE/m 2/S
and a photoperiod of 16L: 8D. Daphnids were feel an
algae and cereal leaf mix containing equal numbers
of cells of Se/enastrwn capricornutum and Ch/ore/
la uu/garis mixed with a rye grass infusion (ASTivI,
1994). This mixture was added to diluted stock so-
lutions to yield a final concentration of 3 x 105 algal
cells/ml and 0.03 mg/ml solids from cereal grass in
each test vial. Component algae were cultured indi-
vidually in modified Bold's basal medium (ASTM,
1994). Solutions were renewed daily. Dissolved oxy-
gen was monitored on 24-h-old solutions and always
remained above 7.0 ppm."
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology High x 2 2 LC50, NOEC, LOEC, and IC50 (for repro)
were measured after 7 days which is in accor-
dance with EPA effluent guidelines for C. du-
bia. https: / / www.epa.gov/sites/production/files/
2015-12/documents/method_1002_2002.pdf
Metric 18: Consistency of Outcome Assessment High x 1 1
Domain 6: Confounding / Variable Control
Continued on next page

-------
. continued from previous page
Study Citation:
Data Type:
Hero ID:
Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Other; Aquatic; Invertebrates
707209
Domain
Metric Rating"!" MWF*
Score
Comments^

Metric 19: Confounding Variables in Test Design and High x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure High x 1
2
1
No reported differences between study groups in en-
vironmental conditions or other factors that could
influence the outcome assessment.
Table 4 indicates sample size for each test concen-
tration, and shows if the sample size dropped to 9
rather than 10. All test cone for TCE had a sample
size of 10.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods High x 1
Metric 22: Reporting of Data High x 2
Metric 23: Explanation of Unexpected Outcomes High x 1
1
2
1
"Toxicity tests with individual chemicals were an-
alyzed to determine criterion effect levels for mor-
tality and reproduction following standard methods
(U.S. EPA, 1994). Both 2- and 7-day LC50s (con-
centrations lethal to 50'Yo of theorganisms) were
calculated using binomial or trimmed Spearman-
Karber tests, as appropriate. After appropriate tests
of normality, effects on reproduction (number of
young per female through the end of the lest) were
analyzed withANOVA, followed by one-sided Dun-
nett's comparison of means to the control to deter-
mine NOELs (no-observed effect levels) and LOELs
(lowest-observed-effect levels). In addition, IC50s
(concentrations inhibiting reproduction by 50 per-
cent relative to mean control performance) were cal-
culated from smoothed data using bootstrap analy-
sis (Norberg-King, 1993)."
Results were reported for each exposure group.
No unexpected outcomes for TCE.
Overall Quality Determination"'" High
1.4

Extracted
Yes

Continued on next page

-------
. continued from previous page
Study Citation: Niederlehner, B.,Cairns, J.,Smith, E.. 1998. Modeling acute and chronic toxicity of nonpolar narcotic chemicals and mixtures to
Ceriodaphnia dubia. Ecotoxicology and Environmental Safety 39:136-146
Data Type:	Other; Aquatic; Invertebrates
Hero ID:	707209
Domain	Metric	Rating^ MWF* Score	Comments^
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.
oo
o

-------
Study Citation: Labra, M.,Mattia, F.,Bernasconi, M.,Bertacchi, D.,Grassi, F.,Bruni, I.,Citterio, S.. 2010. The Combined Toxic and Genotoxic Effects
of Chromium and Voiatiie Organic Contaminants to Pseudokirchnerieiia subcapitata. Water, Air, and Soii Poiiution 213:57-70
Data Type: Acute (0-96 hour); Aquatic; Piants
Hero ID: 1059985
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Metric 3:
Test Substance Identity
Test Substance Source
Test Substance Purity
High X 2 2 Test substance was identified by name,
trichloroethylene.
High X 1 1 Authors report that TCE is from Applichem, Ger-
many.
Low X 1 3 Purity not reported.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
Low X 2 6 There was a control, but authors reported only lim-
ited details.
Low X 1 3 The biological responses of the negative control
groups were not reported.
Low X 1 3 Authors did not comment on randomized allocation
of test species.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
Metric 8: Consistency of Exposure Administration
Metric 12: Testing at or Below Solubility Limit
Low
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency High
Metric 11: Number of Exposure Groups/Spacing of Ex- High
posure Levels
High
X 2 6 There was no mention of covering or sealing Erlen-
meyer flasks, and it was not mentioned whether mea-
surements were taken.
X 1 3 Exposure administration seems consistent but not
confirmed with measured concentrations. Only nom-
inal concentrations used and there was no mention
of flasks being covered.
X 2 6 TCE is volatile and authors did not appear to mea-
sure the concentration in water.
X 1 1 The study had a duration of 72 hours with a sin-
gle exposure in Erlenmeyer flasks, which is in accor-
dance with OECD 201 test guidelines.
X 1 1 Authors report 5 exposure groups, and at least 5 are
suggested in OECD 201 test guidelines. Addition-
ally, spacing of exposure groups were able to mea-
sure an effect.
X 1 1 Concentrations of TCE in this study were below
TCE's solubility level.
Continued on next page

-------
. . . continued from previous page
Study Citation: Labra, M.,Mattia, F.,Bernasconi, M.,Bertacchi, D.,Grassi, F.,Bruni, I.,Citterio, S.. 2010. The Combined Toxic and Genotoxic Effects
of Chromium and Voiatiie Organic Contaminants to Pseudokirchnerieiia subcapitata. Water, Air, and Soii Poiiution 213:57-70
Data Type: Acute (0-96 hour); Aquatic; Piants
Hero ID: 1059985
Domain
Metric
Rating^
MWF* Score
Comments^1

Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 16: Adequacy of Test Conditions
x 2
Metric 14: Acclimitization and Pretreatment Conditions High x 1
Metric 15: Number of Organisms and Replicates per High x 1
Group
Medium x 1
Pseudokirchnerieiia subcapitata was used in this
test, and is a recommended species in OECD 201
test guidelines.
Authors reported that the algae were acclimatized.
" The P. subcapitata strain (SAG 61.81) used in this
study was obtained from the Collection of Algal Cul-
tures, G"ttingen, Germany (SAG). The algae were
cultured in static and axenic condition in Bristol"s
medium solution (Nichols 1979). Algae were period-
ically transferred in a new medium (every 7 days)
to obtain viable cell in log phase. These cultures
were used to inoculate Erlenmeyer flasks containing
100 mL of solution to a final concentration of about
1" 105 cell mL" 1."
Algal cell concentrations were reported at about
1" 105 cell/mL which is within OECD 201 recom-
mendations, and the test included two replicates.
Authors did not report all test conditions however
controls seemed to do well, so only minor uncertain-
ties remained.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
The authors described the outcome assessment
methodology, reported results in the figures and the
text, and the test was sensitive for the outcome of
interest.
Study protocol was reported and outcomes were as-
sessed consistently across study groups using the
same protocol in all study groups.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High
Procedures
Metric 20: Outcomes Unrelated to Exposure
High
x 2
x 1
There were no reported differences among the study
groups in environmental conditions of other factors
that could influence the outcome of the assessment.
There were no reported differences among study
groups that could influence the outcome of the as-
sessment.
Continued on next page

-------
. continued from previous page
Study Citation: Labra, M.,Mattia, F.,Bernasconi, M.,Bertacchi, D.,Grassi, F.,Bruni, I.,Citterio, S.. 2010. The Combined Toxic and Genotoxic Effects
of Chromium and Voiatiie Organic Contaminants to Pseudokirchnerieiia subcapitata. Water, Air, and Soii Poiiution 213:57-70
Data Type:	Acute (0-96 hour); Aquatic; Piants
Hero ID:	1059985
Domain	Metric	Rating^ MWF* Score	Comments^
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
High
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
x 1
Medium x 2
High x 1
Authors reported acceptable statistical methods,
and results of the analysis were reported for each
study group. "Data were used to calculate mean
values " standard deviations. Data were statistically
analyzed by Statistica for Windows v. 6.0 (StatSoft
Italia srl 2001) and StatGraphics programme for-
Windows (version 5.0, Manugistic, MD, USA). The
Duncan test for a multiple sample comparison was
applied when normality and homogeneity of variance
were satisfied."
Some of the key data points were reported, but au-
thors did not explicitly report a NOEC and a LOEC.
There were no unexplained outcomes reported.
Overall Quality Determination"'"
High —
-s- Medium 1.7
Extracted
Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
4	if any metric is Unacceptable
Overall rating =
^2 (Metric Score; x MWF,) / J] . MWF^
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Abernethy, S.,Bobra, A. M.,Shiu, W. Y.,Wells, P. G.,Mackay, D.. 1986. ACUTE LETHAL TOXICITY OF HYDROCARBONS
AND CHLORINATED HYDROCARBONS TO TWO PLANKTONIC CRUSTACEANS THE KEY ROLE OF ORGANISM-WATER
PARTITIONING. Aquatic Toxicology
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 1486051
Domain Metric Rating"!" MWF* Score Comments"*1"!"
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Chemical identified by name (trichloroethylene).
Metric 2:
Test Substance Source
Low
X
1
3
The source of the chemicals were not identified.
Metric 3:
Test Substance Purity
Medium
X
1
2
Test purity was reported at at least 97 percent, but
it is unclear exactly what purity TCE.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls Medium X 2 4 Study authors reported using a negative control but
didn't say whether it was a water or solvent control.
Negative Control Response High X 1 1 Negative control response was reported at always
less than or equal to 10 percent.
Randomized Allocation Low X 1 3 Researchers did not report how organisms were as-
signed to study groups
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High x 2
tion
Metric 8: Consistency of Exposure Administration Medium x 1
Metric 9: Measurement of Test Substance Concentra- Medium x 2
tion
Closed system was used for TCE and maintenance
of consistent exposure concentration was considered
prime importance. Air spaces in exposure chambers
were eliminated to minimize volatilization loss of the
toxicant from water, a serious uncontrolled variable
in many previous toxicity studies with hydrocarbons
in static systems. This limited available oxygen to
that dissolved iii the water at the start of each test,
typically at a concentration of 8-9 mg/1.
Briefly, saturated aqueous solutions of single com-
pounds were prepared and diluted to provide at least
5 exposure concentrations plus a control for each
toxicity test. Test chambers were filled and sealed
in 33 ml glass vials with tefllon-lined screw caps.
Only nominal cone were used but measures were
taken to limit test chemical loss in the test cham-
bers.
Continued on next page

-------
. . . continued from previous page
Study Citation: Abernethy, S.,Bobra, A. M.,Shiu, W. Y.,Wells, P. G.,Mackay, D.. 1986. ACUTE LETHAL TOXICITY OF HYDROCARBONS
AND CHLORINATED HYDROCARBONS TO TWO PLANKTONIC CRUSTACEANS THE KEY ROLE OF ORGANISM-WATER
PARTITIONING. Aquatic Toxicology
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 1486051
Domain Metric Rating"!" MWF* Score Comments"i"i"
Metric 10: Exposure Duration and Frequency
High
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1 2
posure Levels
Metric 12: Testing at or Below Solubility Limit High x 1 1
Static exposure for 24 hour duration for Artemia.
Handbook of Ecotoxicology ed Calow (1998) says an
LC50 can be obtained using Artemia after a 24 hour
exposure.
Five exposure cone plus controls but unsure of the
spacing of exposure levels.
LC50 was well below TCE's solubility.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Low
Group
Metric 16: Adequacy of Test Conditions Low
X 2 2 Artemia used
X 1 3 Study did not report whether organisms were accli-
matized.
X 1 3 Number of organisms was not reported.
X 1 3 Handbook of Ecotoxicology ed Calow recommends
25 degrees in darkness for test procedures for this
species. Temperature in this study may be too low.
Study reports, Artemia nauplii were hatched from
cysts under controlled environmental conditions to
provide a large number of test organisms of exactly
the same age and physiological condition for each
test (Wells et al., 1982). Bioassays were conducted
in the dark at 20 " 1 "C for Artemia tests. Animals
were not feed, nor were toxicant solutions aerated
during the 48 h exposure period. Checks on animal
condition were made at 24 h and 48 h. Each animal
was classified as alive/swimming or dead. Death was
defined by the lack of visible internal or external
movements after gently rotating the test chamber
under a Stercostar zoom microscope.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology High x 2 2 Outcome of interest was reported (LC50s).
Metric 18: Consistency of Outcome Assessment Medium x 1 2 Incomplete details of test protocol was reported.
Domain 6: Confounding / Variable Control
Continued on next page

-------
. continued from previous page
Study Citation: Abernethy, S.,Bobra, A. M.,Shiu, W. Y.,Wells, P. G.,Mackay, D.. 1986. ACUTE LETHAL TOXICITY OF HYDROCARBONS
AND CHLORINATED HYDROCARBONS TO TWO PLANKTONIC CRUSTACEANS THE KEY ROLE OF ORGANISM-WATER
PARTITIONING. Aquatic Toxicology
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	1486051
Domain
Metric
Rating"!" MWF* Score
Comments'r
Metric 19: Confounding Variables in Test Design and Medium x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure
Medium x 1
Study did not provide enough information to allow
a comparison of environmental conditions or other
non-treatment-related factors across study groups.
Unlikely to have major impacts on results.
Data on attrition for each study group was not re-
ported, but unlikely to have major impacts on re-
sults.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
Medium x 1
Low	x 2
High x 1
Percent mortality at each concentration was cor-
rected for control mortality(always < or = to 10 per-
cent) (Abbott, 1925) and nominal medial lethal con-
centrations (LC50 values), slope factors, and 95 per-
cent confidence limits were calculated by the graph-
ical method of Litchfield and Wilcoxon (1948).
Data for exposure-related findings were not shown
for each study group, but results were described in
the test. Data were only reported for some out-
comes.
No unexpected outcomes reported.
Overall Quality Determination^	Medium	1.8
Extracted	Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: . 1987. ASSESSMENTS OF THE FEASIBILITY AND EVIDENCE OF TEST METHODS OF LEVELS I AND II OF THE CHEM-
ICALS ACT ON THIOUREA.
Data Type: Other; Aquatic; Fish
Hero ID: 1745587
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Metric 3:
Test Substance Identity
Test Substance Source
Test Substance Purity
High
High
Low
X 2 2 Trichloroethylene identified by name
X 1 1 Source is Merck 11872 p.a., lot no 8594665
X 1 3 Purity was not reported for TCE.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
Low
Low
Low
x 2
x 1
x 1
A control group was not reported but the authors
say they did the test in accordance with OECD 305
which calls for a control.
Response was not reported
Not reported
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
Metric 8: Consistency of Exposure Administration
x 2
Unacceptable x 1
Metric 9: Measurement of Test Substance Concentra- Medium x 2 4
tion
Metric 10: Exposure Duration and Frequency High x 1 1
It was a semi-static closed system that did have
some head space. Authors admitted that a flow
through design would be preferable with minimized
headspace for volatile chemicals. The authors say
that TCE in particular had considerable cone fluc-
tuations in the various aquaria, and the guideline
could not be followed in a meaningful way.
The authors say that TCE in particular had consid-
erable cone fluctuations in the various aquaria, and
the guideline could not be followed in a meaningful
way.
TCE was analyzed by gas chromatography with EC
detector.
The exposure was for 2 weeks according to OECD
guideline 305 B. there were 8 aquaria each with 25
L capacity arranged and filled with 20 L deionized
water. Stock solutions of test substances were pre-
pared in water or acetone. Test conditions were semi
static.
Continued on next page

-------
. . . continued from previous page
Study Citation: . 1987. ASSESSMENTS OF THE FEASIBILITY AND EVIDENCE OF TEST METHODS OF LEVELS I AND II OF THE CHEM-
ICALS ACT ON THIOUREA.
Data Type: Other; Aquatic; Fish
Hero ID: 1745587
Domain Metric Rating^ MWF* Score Comments^
Metric 11: Number of Exposure Groups/Spacing of Ex- Low
posure Levels
x 1
Only one exposure group was used. The authors
say, "The number of 7 samples per aquarium of 7
fish each sample, as proposed in the Guideline, could
not be complied with, since, the oxygen content in
the basins dropped exceedingly over 48 hours. The
number of fish in the aquaria, was reduced to 28
or: 32 fish and, in general, a parallel experiment
has been carried out. In the case of 28 fish on 7
consecutive days, in the case, of 32 fish on 8 consec-
utive days, 4 fish were sampled. In accordance with
the Guideline, the work was carried out with two
or three test concentrations, which differed by the
factor of 10, whereby, as far as possible, the higher
concentration was 1/50 below the EC-50 determined
in this Institute."
Metric 12: Testing at or Below Solubility Limit
High
x 1
GO
GO
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Medium
Metric 15: Number of Organisms and Replicates per High
Group
X 2 2 Zebra fish (Branchydanio rerio) were used which are
a recommended species according to EPA test guide-
lines 850.1075.
X 1 2 Fish were acclimatized but uncertainties remain.
Authors report, "Prior to test begin, the test fish
were acclimatized in an 80 L aquarium. Unlike the
test aquaria, this was continuously aerated. The wa-
ter was reconditioned like in the test aquaria. The
fish were fed several times daily with Tetra Min, us-
ing an automatic feeding device."
X 1 1 The study says, "The number of 7 samples per
aquarium of 7 fish each sample, as proposed in the
Guideline, could not be complied with, since, the
oxygen content in the basins dropped exceedingly
over 48 hours. The number of fish in the aquaria,
was reduced to 28 or: 32 fish and, in 9eneral, a par-
allel experiment has been carried out. In the case
of 28 fish on 7 consecutive days, in the case, of 32
fish on 8 consecutive days, 4 fish were sampled. In
accordance with the Guideline, the work was carried
out with two or three test concentrations, which dif-
fered by the factor of 10, whereby, as far as possible,
the higher concentration was 1/50 below the EC50
determined in this Institute."
Continued on next page . . .

Metric 17: Outcome Assessment Methodology
High
X
2
2

Metric 18: Consistency of Outcome Assessment
High
X
1
1

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test Design and
High
X
2
2

Procedures

Metric 20: Outcomes Unrelated to Exposure
Medium
X
1
2
Data on attrition and outcomes unrelated to expo-

sure were not reported for each study group.
Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
High
X
1
1

Metric 22: Reporting of Data
High
X
2
2
Average cone in water, average cone in fish, BCFs

and duration were reported for each test conc. no

information was provided for a control.
Metric 23: Explanation of Unexpected Outcomes
High
X
1
1
Unexpected outcomes were addressed.
Overall Quality Determination"'"
Unacceptable

4.0

Extracted
No

Continued on next page

-------
. continued from previous page
Study Citation: . 1987. ASSESSMENTS OF THE FEASIBILITY AND EVIDENCE OF TEST METHODS OF LEVELS I AND II OF THE CHEM-
ICALS ACT ON THIOUREA.
Data Type:	Other; Aquatic; Fish
Hero ID:	1745587
Domain	Metric	Rating^	MWF* Score	Comments^
** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: . 1987. ASSESSMENTS OF THE FEASIBILITY AND EVIDENCE OF TEST METHODS OF LEVELS I AND II OF THE CHEM-
ICALS ACT ON THIOUREA.
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 1745587
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Trichloroethylene identified by name
Metric 2:
Test Substance Source
High
X
1
1
Source is Merck 11872 p.a., lot no 8594665
Metric 3:
Test Substance Purity
Low
X
1
3
Purity not reported
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
used.
Negative Controls
Negative Control Response
Randomized Allocation
Medium x 2 4 Some information was given about a negative con-
trol, but minor uncertainties remain.
Medium X 1 2 A biological response in the control was alluded to
when authors referred to the fact that growth for
algae in this test was very slow after 3 days, so test
results cannot be used.
Low X 1 3 It was not reported whether a random allocation was
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Unacceptable x 2 8 Growth of algae was very slow, so test criteria could
not be satisfied, and tests were discontinued. "Since,
in the case of low growth inhibition (approx. 1-15
percent), the growth curves for each concentration
indicate very flat course, the EC10 values for all the
chemicals tested could not be very accurately de-
termine. Also the EC10 and EC50 values relating
to the volatile substances, e.q. 1,1-dichlocoethylene,
trichloroethylene and benzene are uncertain. Since
a large part of these chemicals volatilize, the EC10
and EC50 values could be lower than the values de-
termined according to the guideline in Erlenmeyer
flasks fitted with Kapsenberg caps. Therefore we
have also carried out experiments in enclosed sys-
tem on the basis of work published by Galassi and
Vighi (7). Nevertheless, our experiments in 300 ml
Erlenmeyer flasks fitted with ground-glass stopper
indicated that the growth of algae was very slow and
after 3 days stagnation set in. Since the test criteria
could not be satisfied, these experiments were then
discontinued."
Continued on next page

-------
. continued from previous page
Study Citation: . 1987. ASSESSMENTS OF THE FEASIBILITY AND EVIDENCE OF TEST METHODS OF LEVELS I AND II OF THE CHEM-
ICALS ACT ON THIOUREA.
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 1745587
Domain
Metric
Rating1
MWF* Score
Comments' t
Metric 8: Consistency of Exposure Administration High
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency
High
Metric 11: Number of Exposure Groups/Spacing of Ex- Low
posure Levels
Metric 12: Testing at or Below Solubility Limit High
x 1
x 2
x 1
x 1
x 1
It is not clear whether measurements were taken
but authors mention that volatile chemicals were at
lower cone at the end of the test, alluding that they,
the exposure was for 96 hours and in a static system,
which is in accordance with EPA guidelines
This was not reported and results were not reliable
according to study authors.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Low
Group
Metric 16: Adequacy of Test Conditions
x 2
x 1
x 1
Unacceptable x 1
2 Fresh-water algae (Scenedesmus subsplcatus) were
used, which is in agreement with EPA test guideline
850.5400.
3 Pretreatment was not recorded.
3 It is unclear how many organisms per exposure
group.
4 Authors reported very slow growth in the algae,
leading to authors discontinuing the test. "Since,
in the case of low growth inhibition (approx. 1-15
percent), the growth curves for each concentration
indicate very flat course, the EC10 values for all the
chemicals tested could not be very accurately de-
termine. Also the EC10 and EC50 values relating
to the volatile substances, e.q. 1,1-dichlocoethylene,
trichloroethylene and benzene are uncertain. Since
a large part of these chemicals volatilize, the EC10
and EC50 values could be lower than the values de-
termined according to the guideline in Erlenmeyer
flasks fitted with Kapsenberg caps. Therefore we
have also carried out experiments in enclosed sys-
tem on the basis of work published by Galassi and
Vighi (7). Nevertheless, our experiments in 300 ml
Erlenmeyer flasks fitted with ground-glass stopper
indicated that the growth of algae was very slow and
after 3 days stagnation set in. Since the test criteria
could not be satisfied, these experiments were then
discontinued."
Continued on next page

-------
. . . continued from previous page
Study Citation: . 1987. ASSESSMENTS OF THE FEASIBILITY AND EVIDENCE OF TEST METHODS OF LEVELS I AND II OF THE CHEM-
ICALS ACT ON THIOUREA.
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 1745587
Domain Metric Rating^ MWF* Score Comments^
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology High x 2 2
Metric 18: Consistency of Outcome Assessment High x 1 1
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Unacceptable x 2 8
Procedures
It seems that non-treatment-related factors may
have influenced he results of the test. "Since, in
the case of low growth inhibition (approx. 1-15
percent), the growth curves for each concentration
indicate very flat course, the EC10 values for all the
chemicals tested could not be very accurately de-
termine. Also the EC10 and EC50 values relating
to the volatile substances, e.q. 1,1-dichlocoethylene,
trichloroethylene and benzene are uncertain. Since
a large part of these chemicals volatilize, the EC10
and EC50 values could be lower than the values de-
termined according to the guideline in Erlenmeyer
flasks fitted with Kapsenberg caps. Therefore we
have also carried out experiments in enclosed sys-
tem on the basis of work published by Galassi and
Vighi (7). Nevertheless, our experiments in 300 ml
Erlenmeyer flasks fitted with ground-glass stopper
indicated that the growth of algae was very slow and
after 3 days stagnation set in. Since the test criteria
could not be satisfied, these experiments were then
discontinued."
Continued on next page

-------
. continued from previous page
Study Citation: . 1987. ASSESSMENTS OF THE FEASIBILITY AND EVIDENCE OF TEST METHODS OF LEVELS I AND II OF THE CHEM-
ICALS ACT ON THIOUREA.
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	1745587
Domain	Metric	Rating^	MWF* Score	Comments^
Metric 20: Outcomes Unrelated to Exposure	Unacceptable x 1	4 "Since, in the case of low growth inhibition (approx.
1-15 percent), the growth curves for each concen-
tration indicate very flat course, the EC10 values for
all the chemicals tested could not be very accurately
determine. Also the EC10 and EC50 values relating
to the volatile substances, e.q. 1,1-dichlocoethylene,
trichloroethylene and benzene are uncertain. Since
a large part of these chemicals volatilize, the EC10
and EC50 values could be lower than the values de-
termined according to the guideline in Erlenmeyer
flasks fitted with Kapsenberg caps. Therefore we
have also carried out experiments in enclosed sys-
tem on the basis of work published by Galassi and
Vighi (7). Nevertheless, our experiments in 300 ml
Erlenmeyer flasks fitted with ground-glass stopper
indicated that the growth of algae wa3s very slow
and after 3 days stagnation set in. Since the test cri-
teria could not be satisfied, these experiments were
then discontinued."
Domain 7: Data Presentation and Analysis
Metric 21
Metric 22
Metric 23
Statistical Methods	High	x 1	1
Reporting of Data	Medium	x 2 4
Explanation of Unexpected Outcomes	High	x 1	1
Only EC10 and EC50 were reported
Unexpected outcomes were addressed
Overall Quality Determination^	Unacceptable	4.0
Extracted	No
** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, four of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
I V. (Metric Score.- x MWF.-') I V ..
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Bacsi, I.,Toeroek, T.,B-E>eres, V.,Toeroek, P.,Tothmeresz, B.,Nagy, A. S.,Vasas, G.. 2013. Laboratory and microcosm experiments
testing the toxicity of chlorinated hydrocarbons on a cyanobacterium strain (Synechococcus PCC 6301) and on natural phytoplankton
assemblages. Hydrobiologia 710:189-203
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	2127844
Domain
Metric
Rating1
MWF* Score
Comments' t
Domain 1: Test Substance
Metric 1:	Test Substance Identity
Metric 2:	Test Substance Source
Metric 3:	Test Substance Purity
High
Low
Low
x 2
x 1
x 1
TCE was identified by name and described. Authors
reported, "trichloroethylene (TCE)"one of the most
widely produced chlorinated hydrocarbons"
not reported
not reported
Domain 2: Test Design
Metric 4:
Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
Medium
Low
Low
x 2
x 1
x 1
controls were used but details about what exactly
controls included were not given. Authors reported,
"The growth of the control cultures (without addi-
tion of chlorinated hydrocarbons) and treated cul-
tures was monitored bymeasuring chlorophyll-a con-
tent and by counting cell numbers."
control response was given but only until 25 hours,
not reported
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Unacceptable x 2
tion
Metric 8:
Metric 9:
Consistency of Exposure Administration
Low
Measurement of Test Substance Concentra- Low
tion
x 1
x 2
8	the laboratory system was open and measurements
were not taken, and flasks were open and aerated
which can lead to rapid volatilization of TCE, how-
ever this was by design in order to better compare
results in the lab to a microcosm experiment also
preformed.
3	details not given about exposure administration for
each exposure level.
6	measured concentrations were not taken and cannot
be expected to be close to nominal concentration
due to the volatility of the chemical. However, this
experiment measured effects in just the few hours
after exposure.
Continued on next page

-------
. . . continued from previous page
Study Citation: Bacsi, I.,Toeroek, T.,B-Beres, V.,Toeroek, P.,Tothmeresz, B.,Nagy, A. S.,Vasas, G.. 2013. Laboratory and microcosm experiments
testing the toxicity of chlorinated hydrocarbons on a cyanobacterium strain (Synechococcus PCC 6301) and on natural phytoplankton
assemblages. Hydrobiologia 710:189-203
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 2127844
Domain Metric Rating^ MWF* Score Comments"^
Metric 10: Exposure Duration and Frequency
Low
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Unacceptable x 1 4
posure Levels
Metric 12: Testing at or Below Solubility Limit Low x 1 3
exposure happened once and was measured in the
few hours after exposure. This is not in accordance
with any guidelines, but was designed to mimic con-
ditions that were carried out in microcosm experi-
ment for comparison purposes.
it appears only one exposure group was used to
mimic the conditions in the microcosm
unsure what the actual exposure concentration was
from the author's reporting.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Medium
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Medium
Group
Metric 16: Adequacy of Test Conditions
Low
x 2
x 1
x 1
x 1
4 cyanobacterium Synecococcus elongatus (PCC
6301). not a recommended test species in OECD
201 but in the same genus as a recommended test
species for cyanobacteria
3 not reported
2 the initial cell density is outside the range for this
genus in OECD201 (synechococcus leopoliensis rec-
ommended cell density is 5x104-105). This experi-
ment starts at about 100x106. Each study was done
in triplicate which is recommended.
3 limited reporting of housing conditions
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Low
x 2
Metric 18: Consistency of Outcome Assessment
Low
x 1
Outcome assessment methodology is described for
changes in growth and enzyme activity. Growth
measures are appropriate but some uncertainties re-
main for how enzyme activity was measured (with
incomplete methodology described). Uncertainties
also exist for when measures were taken. Measure-
ments were taken for growth every second hour, and
for enzyme activity at hour 0, 4, 8, 12, 24.
details regarding execution of study protocol across
study groups was not reported.
Domain 6: Confounding / Variable Control
Continued on next page

-------
. continued from previous page
Study Citation: Bacsi, I.,Toeroek, T.,B-Beres, V.,Toeroek, P.,Tothmeresz, B.,Nagy, A. S.,Vasas, G.. 2013. Laboratory and microcosm experiments
testing the toxicity of chlorinated hydrocarbons on a cyanobacterium strain (Synechococcus PCC 6301) and on natural phytoplankton
assemblages. Hydrobiologia 710:189-203
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 2127844
Domain
Metric
Rating^ MWF* Score
Comments^
Metric 19: Confounding Variables in Test Design and Medium x 2 4
Procedures
Metric 20: Outcomes Unrelated to Exposure Low x 1 3
study did not provide enough information about env
conditions across study groups.
authors did not report data on health outcomes un-
related to exposure
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High
Low
High
X 1 1 Significance was determined trough ANOVA and
Tukey test. All experiments were done in triplicate.
X 2 6 data was reported in figures, but not very well in
text and the exact concentrations at which algae was
exposed is not reported.
X 1 1 Unexpected outcomes were explained e.g. "In labo-
ratory cultures, chlorophyll content of the cells sig-
nificantly changed only at the end ofthe experiments
(Fig. lc). Thus, the changes in the concentration
of chlorophyll-a were because of the decrease in cell
number. It is not clear if the toxicity of tetrachloro
derivatives is higher. The membrane damagingef-
fects of these lipophilic compounds, i.e., because of
their metabolism and oxidative stress can cause re-
duced chlorophyll-a levels. Such chlorophyllloss (de-
composition of antenna pigments) was shown under
oxidative stress in the green alga, Chlorella vulgaris
(Qian et al., 2009)."
Overall Quality Determination"'"
Unacceptable
4.0
Extracted
No

** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, two of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
* MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
* The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Dobaradaran, S.,Mahvi, A. H.,Nabizadeh, R.,Ramavandi, B.,Nazmara, S.,Zarei, S.. 2012. BIOASSAY COMPARISON OF
TRICHLOROETHYLENE (TCE) TOXICITY ON DAPHNIA MAGNA (D. MAGNA) BEFORE AND AFTER ULTRASOUND AND
PHOTOLYSIS PROCESSES. Fresenius Environmental Bulletin 21:1533-1538
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 2127941
Domain Metric Rating"!" MWF* Score Comments"*1"!"
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
TCE is identified by name and uses described
Metric 2:
Test Substance Source
High
X
1
1
Test source reported as Merck company, Germany.
Metric 3:
Test Substance Purity
High
X
1
1
The solutions were prepared by diluting the pure
TCE (>99 percent), which was produced by Merck
company, Germany.
Domain 2: Test Design

Metric 4:
Negative Controls
High
X
2
2
For bioassay experiments, 10 infants (age <24 h)
were exposedto a volume of 100 ml in a 250-ml glass
beaker (8 glass beakers for 8 different concentrations,
and 1 beaker was considered as control in which TCE
was not added).
Metric 5:
Negative Control Response
Low
X
1
3
Not reported
Metric 6:
Randomized Allocation
Low
X
1
3
Not reported
Domain 3: Exposure Characterization

Metric 7:
Experimental System/Test Media Prepara-
Low
X
2
6
There was no mention that containers were covered,

tion

and no measurements were reported that would con-

firm exposure concentrations.
Metric 8:
Consistency of Exposure Administration
Low
X
1
3
Details of how exposures were administered were not
reported, and it is important to know given TCE is
volatile
Metric 9:
Measurement of Test Substance Concentra-
tion
Low
X
2
6
It was not reported whether measurements were
taken
Metric 10:
Exposure Duration and Frequency
Low
X
1
3
There is no mention of renewing the TCE treated
beakers with test chemical. Especially with volatile
chemicals it would be better to have static renewal
or flow through systems.
Continued on next page

-------
. . . continued from previous page
Study Citation: Dobaradaran, S.,Mahvi, A. H.,Nabizadeh, R.,Ramavandi, B.,Nazmara, S.,Zarei, S.. 2012. BIOASSAY COMPARISON OF
TRICHLOROETHYLENE (TCE) TOXICITY ON DAPHNIA MAGNA (D. MAGNA) BEFORE AND AFTER ULTRASOUND AND
PHOTOLYSIS PROCESSES. Fresenius Environmental Bulletin 21:1533-1538
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 2127941
Domain Metric Rating"!" MWF* Score Comments"i"i"
Metric 11: Number of Exposure Groups/Spacing of Ex- High x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit High x 1
There were 8 different exposure levels, and OECD
guideline 202 recommends at least 5. Authors re-
port, "The initial concentration of TCE was 100 mg
L-l, experimental concentrations tested were 100,
75, 50, 40, 30, 20, 10 and 5 percent of effluents from
processes diluted with dilution water (ultrapure wa-
ter, resistivity 18.2 M" cm)."
Highest test cone was 100 mg/L far below the solu-
bility of TCE
Domain 4: Test Organism
Metric 13:
Metric 14:
Test Organism Characteristics High
Acclimitization and Pretreatment Conditions High
Metric 15: Number of Organisms and Replicates per High
Group
Metric 16: Adequacy of Test Conditions High
X 2 2 Daphnia magna used in this test
X 1 1 Authors report, "Firstly, D. magna was caught from
its living site, then cultured lonely, and infants of
this primary D. magna were used for culture in large
amounts. Dilution water was made up of tap water,
which was dechlorinated by sodium thiosulfate, and
also passed through a column of activated carbon.
The quality parameters of dilution water are shown
in Table 2. D. magna was maintained in a 10-L glass
vessel containing medium for culture in a 12/12-h
light-dark cycle, with temperature-control at 21" 1
"C. Sheep manure was used as culture medium, and
D. magna was fed with yeast every two days at a
concentration of 100 mg L-l."
X 1 1 10 animals were placed in each beaker and the ex-
periment was done in triplicate.
X 1 1 10 L glass housing was used, with ph, hardness and
temperature within the recommended ranges
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
Medium x 2
High
x 1
Methodology for counting lethality is good, however
the recommended outcome to measure is immobi-
lization which was not measured. Lethality was sen-
sitive enough to see an effect for this experiment.
Consistent execution of the study.
Continued on next page

-------
. continued from previous page
Study Citation: Dobaradaran, S.,Mahvi, A. H.,Nabizadeh, R.,Ramavandi, B.,Nazmara, S.,Zarei, S.. 2012. BIOASSAY COMPARISON OF
TRICHLOROETHYLENE (TCE) TOXICITY ON DAPHNIA MAGNA (D. MAGNA) BEFORE AND AFTER ULTRASOUND AND
PHOTOLYSIS PROCESSES. Fresenius Environmental Bulletin 21:1533-1538
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	2127941
Domain	Metric	Rating"!" MWF* Score	Comments"i"i"
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2
Procedures
Metric 20: Outcomes Unrelated to Exposure	High x 1	1
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High
Low
High
x 1
x 2
x 1
1	Probit analysis was used to determine an LC50
6	Reporting of effects at each exposure level was great,
but the actual exposure cone was reported in per-
centv/v, and measurements were not reported.
Overall Quality Determination"'"
Medium
1.7
Extracted
Yes
* MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
^ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; X MWF;) / J] . MWF,
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
U Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation:
Lukavsky, J.,Furnadzhieva, S.,Dittrt, F.. 2011. Toxicity of Trichloroethylene (TCE)
on Some Algae and Cyanobacteria. Bulletin of

Environmental Contamination and Toxicology 86:226-231

Data Type:
Acute (0-96 hour); Aquatic; Plants

Hero ID:
2128165

Domain
Metric Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance
Metric 1:	Test Substance Identity	High
Metric 2:	Test Substance Source	Low
Metric 3:	Test Substance Purity	Low
X 2	2 Chemical is identified by name and its uses are de-
scribed.
X 1	3 The source was not reported.
X 1	3 Purity was not reported.
Domain 2: Test Design
Metric 4: Negative Controls
Medium x 2
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
High
Low
x 1
x 1
Authors report using a control with methanol. Au-
thors report, "Nutrient solution "Z", after Zehnder
in Staub (1961), was spiked with TCE: 100 mg was
dissolved in 2 mL of methanol (Lachema, CZ), and
then added to 100 mL of sterile Z medium. Controls
received an equal volume of methanol. This solu-
tion was diluted to the desired concentrations by an
identical nutrient solution, but without either TCE
or methanol."
Negative control response was reported in figures
and in the text.
Random allocation was not reported.
Domain 3: Exposure Characterization
Continued on next page

-------
. continued from previous page
Study Citation: Lukavsky, J.,Furnadzhieva, S.,Dittrt, F.. 2011. Toxicity of Trichloroethylene (TCE) on Some Algae and Cyanobacteria. Bulletin of
Environmental Contamination and Toxicology 86:226-231
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 2128165
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 7: Experimental System/Test Media Prepara- High
tion
o
to
Metric 8: Consistency of Exposure Administration
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency
Metric 11: Number of Exposure Groups/Spacing of Ex- High x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
X 2 2 Authors took the volatility of TCE into considera-
tion. Authors reported, "Assay in glass enclosures
were performed according to ISO/DIS 14442 (1998)
the glass enclosures (volume of 1.5 mL), were sealed
with silicone stoppers coated with Teflon on the in-
ner side (screw top vials, Supelco, USA). An identi-
cal nutrient solution was used, as described above,
but also spiked with 3 g/L KHC03 (source of in-
organic carbon). The inoculum of 0.04"0.06 g/L of
dry weight was higher because of a shorter exposi-
tion time. The enclosures were exposed horizontally
under the same light intensity and temperature as
the plates and shaken 10 x/day. Every day, 0.2 mL
was measured into 6 replicate wells in an immuno-
logical FB plate, and the OD 750 nm was measured
identically as in the previous protocol. 02 concen-
trations were measured by a polarographic Clark-
type oxygen sensor, and pH by a miniaturized com-
bined electrode, both joined to a MEM 102 multime-
ter (Chemoprojekt Satalice, CZ), the measurements
were performed with no replicates."
Medium X 1 2 Some species were tested in plates and some were
tested in glass enclosures.
X 2 6 Measurements of TCE were not taken, however, au-
thors reported measuring other conditions includ-
ing "Every day, 0.2 mL was measured into 6 repli-
catewells in an immunological FB plate, and the OD
750 nm was measured identically as in the previ-
ous protocol. 02 concentrations were measured by
a polarographic Clarktype oxygen sensor, and pH
by a miniaturized combinedelectrode, both joined
to a MEM 102 multimeter (Chemoprojekt Satal-
ice, CZ), the measurements were performed with no
replicates."
X 1 3 The duration of the experiment were acceptable for
algae, however authors did not indicate whether the
experiment was static or static renewal.
1 There were 5 exposure groups which is OECD 201
recommended, and the concentrations were sensitive
enough to derive an EC50.
X 1 1 Cone were far below TCE's solubility level.
Low
High
Continued on next page

-------
. . . continued from previous page
Study Citation:
Lukavsky, J.,Furnadzhieva, S.,Dittrt, F.. 2011. Toxicity of Trichloroethylene (TCE)
on Some Algae and Cyanobacteria. Bulletin of

Environmental Contamination and Toxicology 86:226-231

Data Type:
Acute (0-96 hour); Aquatic; Plants

Hero ID:
2128165

Domain
Metric Rating"!" MWF* Score
Comments^
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Low
Group
Metric 16: Adequacy of Test Conditions Low
x 2
x 1
x 1
x 1
2 At least one of the cyanobacteria species used are
a recommended species in OECD201, Desmodesmus
subspicatus. Other species used include Raphido-
celis subcapitata and Chlorella kessleri.
3 Acclimatization conditions were not reported.
3 6 replicates were reported but no initial cell density
was reported.
3 Temperature reported in study (30 degrees C) is
higher than recommended temperature in OECD201
(21-24 +-2 degrees C). No temperature was given for
the glass enclosures. It appears some of the health
outcomes (related to growth) could have been due to
housing conditions. However, part of the experiment
was to determine whether the housing was adequate
or not and they used two different housing condi-
tions to compare.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
High
x 2
Metric 18: Consistency of Outcome Assessment
Medium x 1
Authors measured growth using dry weight. Au-
thors indicate "OD in plates were evaluated under an
iEMS reader (Labscale, SF) at wavelength 750 nm,
and the optical density was converted to dry weight
after individual conversion curves for each species.
EC50 (the concentration of a tested substance at
which the cell density, biomass, 02 production or
pH increment is 50 percent of that of the untreated
algae) was determined graphically according to stan-
dard ISO (2004) omitting."
There are minor uncertainties about consistency be-
tween outcome assessment between the dish and
glass enclosure studies. Consider this minor because
part of the purpose of the experiment was to de-
termine which housing was more conducive to the
experiment.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Medium x 2 4 Only minor uncertainties. There was no mention
Procedures temPerature in the glass enclosures. Both test
setups were enclosed and sealed.
Continued on next page

-------
. continued from previous page
Study Citation: Lukavsky, J.,Furnadzhieva, S.,Dittrt, F.. 2011. Toxicity of Trichloroethylene (TCE) on Some Algae and Cyanobacteria. Bulletin of
Environmental Contamination and Toxicology 86:226-231
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	2128165
Domain Metric
Rating^
MWF*
Score
Comments^
Metric 20: Outcomes Unrelated to Exposure
Low
X 1
3
It seemed some of the species were affected by the
housing conditions.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
High
X 1
1
Authors reported, "EC50 (the concentration of a
tested substance at which the cell density, biomass,
02 production or pH increment is 50 percent of that
of the untreated algae) was determined graphically
according to standard ISO (2004) omitting the stim-
ulation peaks. In addition, growth curves plotted as
time" dry weight or OD 750, can indicate EC50 as
concentration when cell number is constant, and sta-
bilized. Six replicates were measured, for OD eval-
uation, error bars are plotted as " sx = H (nRx2 -
(Rx)2/n2)."
Metric 22: Reporting of Data	Medium X 2	4 Some results were reported in detail for each expo-
sure group. However, not all results were reported
in this way
Metric 23: Explanation of Unexpected Outcomes	Medium x 1	2 Some of the unexpected outcomes were explained.
Overall Quality Determination^	Medium	1.9
Extracted	Yes
* MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
$ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

matography was used to verify identity of chemical.

"The determination of the test compounds in wa-

ter samples was accomplished by solvent extraction

followed by gas chromatography analysis."
Metric 3:
Test Substance Purity
Low
X
1
3
Purity of the test substance was not reported.
Domain 2: Test Design

Metric 4:
Negative Controls
High
X
2
2
Both water and acetone controls were used in acute

static and flow-through tests
Metric 5:
Negative Control Response
Low
X
1
3
Control response was not reported
Metric 6:
Randomized Allocation
Low
X
1
3
Researchers did not report the method for how or-

ganisms were allocated to study groups, or their de-

ficiencies regarding allocation method.
Domain 3: Exposure Characterization

Metric 7:
Experimental System/Test Media Prepara-
High
X
2
2
Static system with minimal headspace for volatile

tion

substance was used.
Metric 8:
Consistency of Exposure Administration
Low
X
1
3
Exposure concentrations were not reported in the

static test
Metric 9:
Measurement of Test Substance Concentra-
Medium
X
2
4
Static acute test didn't test concentrations and only

tion

relied on nominal concentrations.
Metric 10:
Exposure Duration and Frequency
High
X
1
1
Static test was renewed every 24 hours over the

course of 96 hours.
Metric 11:
Number of Exposure Groups/Spacing of Ex-
Medium
X
1
2
Concentrations were prepared in a logarithmic series

posure Levels

but the method used to determine an appropriate

range was not mentioned.
Metric 12:
Testing at or Below Solubility Limit
High
X
1
1

Domain 4: Test Organism
Continued on next page

laboratory raised. Not an OECD or EPA recom-

mended species. Also had minor uncertainties about

where the fish were obtained.
Metric 14:
Acclimitization and Pretreatment Conditions
Medium
X
1
2
Minor uncertainties about details provided. Authors

report, "Laboratory-reared juvenile (2-4 month)

flagfish were used. Fish were raised in the dilu-

ent water and fed freshly-hatched and adult brine

shrimp. Fish were not fed during the tests."
Metric 15:
Number of Organisms and Replicates per
Low
X
1
3
Only 5 juvenile flagfish were used per aquarium

Group

when OECD recommends at least 7.
Metric 16:
Adequacy of Test Conditions
High
X
1
1

Domain 5: Outcome Assessment

Metric 17:
Outcome Assessment Methodology
High
X
2
2

Metric 18:
Consistency of Outcome Assessment
High
X
1
1

Domain 6: Confounding / Variable Control

Metric 19:
Confounding Variables in Test Design and
High
X
2
2

Procedures

Metric 20:
Outcomes Unrelated to Exposure
Low
X
1
3
No adverse outcomes were reported for TCE, and

control response was not reported.
Domain 7: Data Presentation and Analysis

Metric 21:
Statistical Methods
High
X
1
1

Metric 22:
Reporting of Data
Low
X
2
6
The data for the static test were not presented in

full, and no information was reported for controls.
Metric 23:
Explanation of Unexpected Outcomes
High
X
1
1
There were no unexpected outcomes for TCE in this

paper.
Overall Quality Determination"'"
Medium

1.7

Extracted

Yes

Continued on next page

-------
. . . continued from previous page
Study Citation: Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J. A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute
and chronic toxicity of 10 chlorinated organic-compounds to the american flagfish (jordanella-floridae). Archives of Environmental
Contamination and Toxicology 20:94-102
Data Type:	Acute (0-96 hour); Aquatic; Fish static
Hero ID:	2298399
Domain	Metric	Rating"!" MWF* Score	CommentsII
* MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
^ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; X MWF;) / ]T\ MWF,
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
II Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J. A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute
and chronic toxicity of 10 chlorinated organic-compounds to the american flagfish (jordanella-floridae). Archives of Environmental
Contamination and Toxicology 20:94-102
Data Type: Acute (0-96 hour); Aquatic; Fish flow-through
Hero ID: 2298399
Domain
Metric
Rating"!" MWF* Score
Comments'^
Domain 1: Test Substance
Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical was identified by name.
Medium x 1 2 The source of TCE was not reported, but gas chro-
matography was used to verify identity of chemical.
"The determination of the test compounds in wa-
ter samples was accomplished by solvent extraction
followed by gas chromatography analysis."
Low X 1 3 Purity of the test substance was not reported.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
High X 2 2 Both water and acetone controls were used in acute
static and flow-through tests.
Low X 1 3 Control response was not reported.
Low X 1 3 Researchers did not report the method for how or-
ganisms were allocated to study groups, or their de-
ficiencies regarding allocation method.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High x 2
tion
Metric 8: Consistency of Exposure Administration Low x 1
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency
High x 1
" Fresh solutions were added at a rate of 6 L/hr.
Each aquarium wassampled at least three times to
determine the concentrations of thetest solutions."
Exposure concentrations were not reported in the
flow-through test. Five or six duplicate, logarithmi-
cally distributed concentrations of the test solutions
were used in 30-L aquaria.
Flow-through acute test measured test concentra-
tions and Fresh solutions were added at a rate of
6 L/hr. Each aquarium was sampled at least three
times to determine the concentrations of the test so-
lutions.
Flow-through test was renewed every
the course of 96 hours.
hours over
Continued on next page

-------
. . . continued from previous page
Study Citation: Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J. A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute
and chronic toxicity of 10 chlorinated organic-compounds to the american flagfish (jordanella-floridae). Archives of Environmental
Contamination and Toxicology 20:94-102
Data Type: Acute (0-96 hour); Aquatic; Fish flow-through
Hero ID: 2298399
Domain Metric Rating"!" MWF* Score Comments"i"i"
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
Concentrations were prepared in a logarithmic series
but the method used to determine an appropriate
range was not mentioned.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Medium x 2
Metric 14: Acclimitization and Pretreatment Conditions Medium x 1
Metric 15: Number of Organisms and Replicates per High x 1 1
Group
Metric 16: Adequacy of Test Conditions High x 1 1
Juvenile flagfish (2-4 months) were used, and were
laboratory raised. Not an OECD or EPA recom-
mended species. Also had minor uncertainties about
where the fish were obtained.
Minor uncertainties about details provided. Authors
report, "Laboratory-reared juvenile (2-4 month)
flagfish were used. Fish were raised in the dilu-
ent water and fed freshly-hatched and adult brine
shrimp. Fish were not fed during the tests."
10 juvenile flagfish were used per aquarium, and
OECD recommends at least 7.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2
Procedures
Metric 20: Outcomes Unrelated to Exposure Low x 1 3
No adverse outcomes were reported for TCE, and
control response was not reported.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
High
Low
x 1
x 2
The data for the static test were not presented in
full, and no information was reported for controls.
Continued on next page

-------
. . . continued from previous page
Study Citation: Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J. A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute
and chronic toxicity of 10 chlorinated organic-compounds to the american flagfish (jordanella-floridae). Archives of Environmental
Contamination and Toxicology 20:94-102
Data Type:	Acute (0-96 hour); Aquatic; Fish flow-through
Hero ID:	2298399
Domain	Metric	Rating"!" MWF* Score	Comments"i"i"
Metric 23: Explanation of Unexpected Outcomes	High	x 1	1 There were no unexpected outcomes for TCE in this
paper.
Overall Quality Determination"'"
High
1.6
Extracted
Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
4	if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J. A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute
and chronic toxicity of 10 chlorinated organic-compounds to the american flagfish (jordanella-floridae). Archives of Environmental
Contamination and Toxicology 20:94-102
Data Type: Chronic (>21 days); Aquatic; Fish
Hero ID: 2298399
Domain
Metric
Rating"!" MWF* Score
Comments'^
Domain 1: Test Substance
Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical was identified by name.
Medium x 1 2 The source of TCE was not reported, but gas chro-
matography was used to verify identity of chemical.
"The determination of the test compounds in wa-
ter samples was accomplished by solvent extraction
followed by gas chromatography analysis."
Low X 1 3 Purity of the test substance was not reported.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
High
High
x 2
x 1
Medium x 1
2 Both water and acetone controls were used in chronic
test
1 Negative control response was reported for chronic
test.
2 Researchers reported allocating fish randomly to the
exposure apparatus. Did not specifically say if they
were randomly allocated to control, but it is as-
sumed, so only minor uncertainty.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High
tion
Metric 8: Consistency of Exposure Administration High
Metric 9: Measurement of Test Substance Concentra- High
tion
Metric 10: Exposure Duration and Frequency
X 2 2 The flow-through diluter was the same unit used in
the acute
X 1 1 Exposure concentrations were reported for the
chronic test. Five duplicate, logarithmically dis-
tributed concentrations of the test solutions, were
used in 30-L aquaria.
X 2 2 Water samples were analyzed 5 days per week
throughout the 28-day exposure period; sample col-
lection was from the mid-point of the tanks. De-
termination of the test compounds in water samples
was accomplished by solvent extraction followed by
gas chromatography (GC) analysis.
High X 1 1 Flow-through exposure with fresh solutions added at
a rate of 6 L/hr.
Continued on next page

-------
. . . continued from previous page
Study Citation: Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J. A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute
and chronic toxicity of 10 chlorinated organic-compounds to the american flagfish (jordanella-floridae). Archives of Environmental
Contamination and Toxicology 20:94-102
Data Type: Chronic (>21 days); Aquatic; Fish
Hero ID: 2298399
Domain Metric Rating"!" MWF* Score Comments"i"i"
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High x 1
"Concentrations were prepared in a logarithmic se-
ries and the 96-hrLC50's calculated from the acute
flagfish data were used to establishthe exposure gra-
dients employed in these chronic tests."
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Medium x 2
Metric 14: Acclimitization and Pretreatment Conditions Medium x 1
Metric 15: Number of Organisms and Replicates per Medium x 1
Group
Metric 16: Adequacy of Test Conditions
High
x 1
Embryo/larval flagfish were used, and were labora-
tory raised. Not an OECD or EPA recommended
species. Also had minor uncertainties about where
the fish were obtained.
Minor uncertainties about details provided. Authors
report, "Laboratory-reared juvenile (2-4 month)
flagfish were used. Fish were raised in the dilu-
ent water and fed freshly-hatched and adult brine
shrimp. Fish were not fed during the tests."
50 fry (one week old) per test level and the controls.
Duplicate exposures were used, but OECD recom-
mends 4 or 5.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
A 28-day outcome was measured which is what is
recommended by OECD, but a 10-day outcome was
also reported.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2
Procedures
Metric 20: Outcomes Unrelated to Exposure High x 1 1
No adverse outcomes were reported for TCE, and
controls had 87 percent survival for a 28-day survival
Domain 7: Data Presentation and Analysis
Continued on next page

-------
. . . continued from previous page
Study Citation: Smith, A. D.,Bharath, A.,Mallard, C.,Orr, D.,Smith, K.,Sutton, J. A.,Vukmanich, J.,McCarty, L. S.,Ozburn, G. W.. 1991. The acute
and chronic toxicity of 10 chlorinated organic-compounds to the american flagfish (jordanella-floridae). Archives of Environmental
Contamination and Toxicology 20:94-102
Data Type:	Chronic (>21 days); Aquatic; Fish
Hero ID:	2298399
Domain	Metric	Rating"!" MWF* Score	Comments"i"i"
Metric 21:
Statistical Methods
High
X
1
1

Metric 22:
Reporting of Data
High
X
2
2
Control information was reported for outcomes.
Metric 23:
Explanation of Unexpected Outcomes
High
X
1
1
There were no unexpected outcomes for TCE in this






paper.
Overall Quality Determination"'"

High


1.3

Extracted
Yes
* MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
^ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; X MWF;) / ]T\ MWF,
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Rayburn, D. J. Deyoung, J. A. Bantle, D. J. Fort, R. Mcnew. 1991. Altered developmental toxicity caused by three carrier solvents.
Journal of Applied Toxicology 11:253-260
Data Type: Other; Aquatic; other
Hero ID: 2307041
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity
N/A
Medium x 1
Low
x 1
N/A chemical identified by name, trichloroethylene, and
by cas 79-01-6
2 Source was reported, Aldrich chemical co, but
batch/lot number was not reported and it was not
reported whether the manufacturer verified the iden-
tity of the chemical or whether authors verified the
identiry through analytical methods.
3 purity and or grade were not reported.
Domain 2: Test Design
Metric 4:
Metric 5:
Negative Controls
Negative Control Response
Metric 6: Randomized Allocation
High x 2 2 "Four groups of 25 embryos were exposed to FETAX
solution and used as controls for each test."
Medium X 1 2 negative control response is repoted in the text and
in several tables. In at least one of the experiments
the percent mortaility was just over 10 percent.
Low x 1 3 it was not reported whether animals were allocated
randomly
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Unacceptable x 2
tion
Metric 8: Consistency of Exposure Administration Medium x 1
Metric 9: Measurement of Test Substance Concentra- Low x 2
tion
Metric 10: Exposure Duration and Frequency High x 1
The study does not say whether petri dishes were
covered or sealed in some way, and there were no
concentrations measureed to verify what the animals
were exposed to.
exposure concentrations were not reported.
duration was 96 hour and every 24 hours solutions
were renewed, which is the recommended duration
and frequency from the ASTM Guidance.
Continued on next page

-------
. continued from previous page
Study Citation: Rayburn, D. J. Deyoung, J. A. Bantle, D. J. Fort, R. Mcnew. 1991. Altered developmental toxicity caused by three carrier solvents.
Journal of Applied Toxicology 11:253-260
Data Type: Other; Aquatic; other
Hero ID: 2307041
Domain
Metric
Rating1
MWF* Score
Comments' t
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium
posure Levels
x 1
Metric 12: Testing at or Below Solubility Limit
Medium
x 1
There were 6 exposure groups for TCE, but it is
unclear how the spacing of exposure levels was de-
termined, although it does appear as though they
did a range finding test as they state, "One range
and three definitive experiments were performed to
determine the 96-h LC50, 96-h EC50 (malforma-
tion), the Teratogenic Index (TI) (96-h LCsc/96-h
EC50) and the Minimum Concentration to Inhibit
Growth (MCIG) for the three solvents and two ter-
atogens. The 96-h LC25 and LCsci, and 96-h EC25
and EC50 (malformation) were determined using
Litchfield-Wilcoxon probit analysis."
authors do not mention having trouble testing TCE
at the levels they did, but with the way they re-
ported the exposure levels percent(v/v) it's hard to
tell whether the cones were below the solubility of
TCE.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics High
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per High
Group
Metric 16: Adequacy of Test Conditions Low
X 2 2 Xenopus embryos were used in these experiments
which are the suggested species in ASTM guidance.
X 1 3 study did not report whether the organisms were
acclimatized. it only states, " Animal care and
breeding were performed according toBantle et al.
(1989)."
X 1 1 two replicates per group with 20-25 embrayos used,
which is according to the ASTM guidance.
X 1 3 Some information was given about housing, but not
many details. Authors report adequate volume of so-
lution in the Petri dishes which are plastic. Authors
also say that each experiment folloed standard meth-
ods of test opteration and embryo evaluation from
two papers including Bantle et al (1989)and Dawson
and Bangle (1987). However no specifics were given
about temperaure, ph etc.
Domain 5: Outcome Assessment
Continued on next page

-------
. . . continued from previous page
Study Citation: Rayburn, D. J. Deyoung, J. A. Bantle, D. J. Fort, R. Mcnew. 1991. Altered developmental toxicity caused by three carrier solvents.
Journal of Applied Toxicology 11:253-260
Data Type: Other; Aquatic; other
Hero ID: 2307041
Domain Metric Rating^ MWF* Score Comments^
Metric 17: Outcome Assessment Methodology High x 2 2 LC50 and EC50s were measured which were the sug-
gested outcomes measured in the ASTM guidance,
and the authors did measured NOELs and LOELs at
the for mortality and malformations at the exposure
levels tested.
Metric 18: Consistency of Outcome Assessment Medium x 1 2 incomplete reporting of minor details
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Low
Procedures
Metric 20: Outcomes Unrelated to Exposure
Medium
x 2
x 1
study did not report enough details to allow a com-
parison of environmental conditions across study
groups.
authors reported that at least one of the controls
had a greater than 10 percent mortality and malfor-
mation rate, but it was just over 10 percent.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods High x 1 1
Metric 22: Reporting of Data Unacceptable x 2 8
Metric 23: Explanation of Unexpected Outcomes Medium x 1 2
Overall Quality Determination"'" Unacceptable 4.0
Extracted No
"The 96-h LC25 and LCsci, and 96-h EC25
and EC50 (malformation) were determined us-
ing Litchfield-Wilcoxon probit analysis.20 Dunnett's
testwas used to determine the No-Observable-Effect
Concentrations (NOEC) for malformation and mor-
tality. The MCIG was determined by comparing
head-tail lengths between control and experimental
groups using the Hest for grouped observations."
results were reported in percent v/v which is not a
concetration.
explainaitons were given for unexpected outcomes.
Continued on next page

-------
. continued from previous page
Study Citation: Rayburn, D. J. Deyoung, J. A. Bantle, D. J. Fort, R. Mcnew. 1991. Altered developmental toxicity caused by three carrier solvents.
Journal of Applied Toxicology 11:253-260
Data Type:	Other; Aquatic; other
Hero ID:	2307041
Domain	Metric	Rating^	MWF* Score	Comments^
** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, two of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Hayashi, M.,Ueda, T.,Uyeno, K.,Wada, K.,Kinae, N.,Saotome, K.,Tanaka, N.,Takai, A.,Sasaki, Y. F.,Asano, N.,Sofuni, T.,Ojima, Y.
1998. Development of genotoxicity assay systems that use aquatic organisms. Mutation Research 399:125-133
Data Type: Other; Aquatic; Fish
Hero ID: 2800252
Domain
Metric
Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical was identified by name, trichloroethylene
High X 1 1 Source was identified by Wako Pure Chemicals, Os-
aka, Japan.
Low X 1 3 Not reported
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
Medium x 2
High
Low
x 1
x 1
Authors report a concentration of 0 for TCE in their
tables of results. No control is mentioned in the
text otherwise. Unsure if the control is just water or
contains a solvent.
Control response is noted in the tables
Not reported
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
Metric 8: Consistency of Exposure Administration
Low
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency Low
Metric 11: Number of Exposure Groups/Spacing of Ex- High
posure Levels
X 2 6 Very little information is given about the actual ex-
posure of the cells to TCE in this paper. It is un-
clear whether the volatile nature of TCE was taken
into consideration during in the test setup to re-
duce volatilization. It also doesn't say anything
about whether the source of the fish (collected peri-
odicallyat Mochimune Harbor, Shizuoka prefecture,
Japan) was contaminated or now.
X 1 3 Very little information was given about how cells
were exposed to TCE
X 2 6 The test substance was not measured and the test
chemical is very volatile.
X 1 3 Duration was 48 hours for this study. OECD guid-
ance on genotoxicity assays say that 24 hours should
be enough for assays without metabolic activation.
It appears that there was only a one time exposure
but it's unclear and no measurements were reported
to determine the actual cone cells were exposed to.
X 1 1 6 exposure groups were used for this experiment
Continued on next page

-------
. . . continued from previous page
Study Citation: Hayashi, M.,Ueda, T.,Uyeno, K.,Wada, K.,Kinae, N.,Saotome, K.,Tanaka, N.,Takai, A.,Sasaki, Y. F.,Asano, N.,Sofuni, T.,Ojima, Y..
1998. Development of genotoxicity assay systems that use aquatic organisms. Mutation Research 399:125-133
Data Type: Other; Aquatic; Fish
Hero ID: 2800252
Domain Metric Rating^ MWF* Score Comments^
Metric 12: Testing at or Below Solubility Limit Medium x 1 2 A subset of the exposure cone exceeded the water
solubility limit but a sufficient range of exposure
cone was tested to characterize a conc-response rela-
tionship. Just one cone (3000mg/L) was above the
solubility level and there were 5 other cone levels
below the solubility level.
Domain 4: Test Organism
R. ocellatus ocellatus embryos were used for the
chromosomal aberration. There are no guidelines for
this type of study for fish. This species is suitable
for metaphase analysis.
Odontobutis obscura obscura were collected from
the Tomio River, Nara prefecture, Japan and then
cultured in R. laboratory aquariums. Aquarium con-
ditions were not reported.
Guidelines say that there should be enough cells and
replicates at each exposure group to have statistical
power. There were about 30 cells in each exposure
level. However some cone levels had no replicates,
some had duplicates and some had triplicates.
Housing was not described.
Metric 13: Test Organism Characteristics Medium x 2 4
Metric 14: Acclimitization and Pretreatment Conditions Medium x 1
Metric 15: Number of Organisms and Replicates per Low x 1 3
Group
Metric 16: Adequacy of Test Conditions Low x 1 3
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology Medium x 2 4 Outcome assessment methodology was partially re-
ported by the authors but minor uncertainties re-
main.
Metric 18: Consistency of Outcome Assessment High x 1 1 It appears that the outcomes were assessed consis-
tently across study groups.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure
High
x 1
No reported differences among the study groups in
environmental conditions that could influence the
outcomes assessment.
Cell attrition reported for each study group and are
acceptable.
Domain 7: Data Presentation and Analysis
Continued on next page

-------
. continued from previous page
Study Citation: Hayashi, M.,Ueda, T.,Uyeno, K.,Wada, K.,Kinae, N.,Saotome, K.,Tanaka, N.,Takai, A.,Sasaki, Y. F.,Asano, N.,Sofuni, T.,Ojima, Y.
1998. Development of genotoxicity assay systems that use aquatic organisms. Mutation Research 399:125-133
Data Type:	Other; Aquatic; Fish
Hero ID:	2800252
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
Medium x 1
Medium x 2
High x 1
Fisher exact tests was used. The OECD genetic toxi-
cology test guidelines state "the result is outside the
distribution of the historical negative control data
(e.g. Poisson-based 95 percent control limits)."
Most data was presented in tables for each exposure
level. Only minor uncertainties remain about how
many replicates there were at higher exposure levels.
Overall Quality Determination^
Medium
2.0
Extracted
Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF TRICHLOROETHYLENE TO SALTWATER ORGAN-
ISMS. Bulletin of Environmental Contamination and Toxicology 37:830-836
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 2801609
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1: Test Substance Identity High x 2 2 TCE is identified by name.
Metric 2: Test Substance Source Medium X 1 2 Source was not reported but actual TCE test concen-
trations were analytically determined at the initia-
tion and termination of the test or when 100 percent
mortality occurred in a treatment.
Metric 3: Test Substance Purity Low x 1 3 Test substance purity was not reported.
Domain 2: Test Design
Metric 4: Negative Controls High X 2 2 Included a negative control group using just seawa-
ter.
Metric 5: Negative Control Response Low x 1 3 Negative control response not reported.
Metric 6: Randomized Allocation Low X 1 3 Researchers did not report how organisms were al-
located to study groups.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
x 2
Metric 8: Consistency of Exposure Administration
High
x 1
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
The alga, S. costatum, was tested in 125-mL flasks
containing 50 mL of testt solution or control wa-
ter. Each flask was inoculated with approximately
2.0 x 104 cells/mL. The cultures were incubated at
20+l"C under 4,300 lux illumination. Test concen-
trations and c"5ni:rols were triplicated. Measure-
ments of in vivo chlorophyll a were made using a
Turner Model III fluorometer after 24, 48, 72, and
96 h of exposure. Cell counts were made after 96 h of
exposure using a hemacytometer and Zeiss Standard
14-compound microscope. There was no mention of
containers being covered, but the study did report
test concentrations measured at the beginning and
end of the test.
Details of exposure administration were reported
and exposures were administered consistently across
study groups.
Test concentrations were measured at the beginning
and end of the test.
Continued on next page

-------
. continued from previous page
Study Citation: Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF TRICHLOROETHYLENE TO SALTWATER ORGAN-
ISMS. Bulletin of Environmental Contamination and Toxicology 37:830-836
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 2801609
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 10: Exposure Duration and Frequency
High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High x 1
The duration of exposure and frequency was re-
ported as ok for algae. Could be 72 or 96 hours.
In this case it was 96 hours.
There were 5 exposure groups with a duplicate at
each exposure level, no range finding test was con-
ducted, and instead concentrations were spaced by
6.25, 12.5, 25, 50 and 100 percent of the water solu-
ble fraction.
Test concentrations were spaced by 6.25, 12.5, 25,
50 and 100 percent of the water soluble fraction.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics High x 2
Metric 14: Acclimitization and Pretreatment Conditions High x 1
Metric 15: Number of Organisms and Replicates per Medium x 1
Group
Metric 16: Adequacy of Test Conditions
Medium x 1
2 Skeletonema costatum is one of the recommended
species for saltwater from EPA guidelines.
1 "The alga tested was the chain-forming diatom;
Skeletonemacostatum. The culture was obtained
from the EPA EnvironmentalResearch Laboratory,
Gulf Breeze, Fla., and maintained in stockcul-
ture at Bionomics Marine Research Laboratory
(BMRL),Pensacola, Fla, according to procedures in
U.S. EPA (1978)."
2 Each flask was inoculated with approximately 2.0
x 104 cells/mL (1.5 x 106 is recommended by EPA
guidelines for Skeletonema), and tests were done in
triplicate which is recommended by EPA guidelines.
2 The alga, S. costatum, was tested in 125-mL flasks
containing 50 mL of test solution or control wa-
ter. The cultures were incubated at 20+l"C un-
der 4,300 lux illumination. This is recommended by
EPA guidelines. Only minor uncertainties remain.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High X 2 2 Outcomes of interest were measured appropriately.
Medium X 1 2 Incomplete reporting of minor details of outcome as-
sessment protocol execution.
Domain 6: Confounding / Variable Control
Continued on next page

-------
. continued from previous page
Study Citation: Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF TRICHLOROETHYLENE TO SALTWATER ORGAN-
ISMS. Bulletin of Environmental Contamination and Toxicology 37:830-836
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	2801609
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 19: Confounding Variables in Test Design and Low	x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure
Low
x 1
The study did not provide enough information to
allow a comparison of conditions or other non-
treatment related factors across study groups.
Data on attrition and/or health outcomes unrelated
to exposure were not reported for each study group.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High
Low
High
x 1
x 2
x 1
Based on the results of the tests, 24-, 48-, 72-, and
96-h LC50s or EC50s and 95 percent confidence lim-
its were calculated, where possible. The computer
program generated the LC- or EC50 values using the
following statistical methods: moving average angle,
probit, and binomial probability (Stephan 1977).
Data for exposure related finding were not shown for
each study group.
No unexplained outcomes reported.
Overall Quality Determination^
Medium
1.8
Extracted
Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF TRICHLOROETHYLENE TO SALTWATER ORGAN-
ISMS. Bulletin of Environmental Contamination and Toxicology 37:830-836
Data Type:	Acute (0-96 hour); Aquatic; Fish
Hero ID:	2801609
Domain	Metric	Rating^ MWF* Score	Comments^
Domain 1: Test Substance
Metric 1: Test Substance Identity	High	x 2	2 TCE is identified by name.
Metric 2: Test Substance Source	Medium X 1	2 Source was not reported but actual TCE test concen-
trations were analytically determined at the initia-
tion and termination of the test or when 100 percent
mortality occurred in a treatment.
Metric 3: Test Substance Purity	Low	x 1	3 Test substance purity was not reported.
Domain 2: Test Design
Metric 4: Negative Controls	High	X 2	2 Included a negative control group using just seawa-
ter.
Metric 5: Negative Control Response	Low	x 1	3 Negative control response not reported.
Metric 6: Randomized Allocation	Low	X 1	3 Researchers did not report how organisms were al-
located to study groups
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Medium x 2
tion
Metric 8: Consistency of Exposure Administration	High x 1
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency	High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
4	The study used covered glass containers but voliti-
zation still occurred in the static system. The study
did report test concentrations measured at the be-
ginning and end of the test.
1	Details of exposure administration were reported
and exposures were administered consistently across
study groups.
2	Test concentrations were measured at the beginning
and end of the test.
1	The duration of exposure and frequency was re-
ported as is appropriate for fish. 96 hour static test.
2	There were 5 exposure groups with a duplicate at
each exposure level. No range finding test was con-
ducted, and instead concentrations were spaced by
6.25, 12.5, 25, 50 and 100 percent of the water solu-
ble fraction.
1 Test concentrations were spaced by 6.25, 12.5, 25,
50 and 100 percent of the water soluble fraction.
Continued on next page

Domain 4: Test Organism

Metric 13:
Test Organism Characteristics
High
X
2
2
Sheepshead minnow is one of the recommended
species for saltwater from EPA guidelines (EPA TG
850.1085).
Metric 14:
Acclimitization and Pretreatment Conditions
Medium
X
1
2
Some uncertainty around how acclimatization oc-
curred. Study reports, "Sheepshead minnows,
Cyprinodon variegatus, were hatched and reared for
4 to 6 days at BMRL. Sheepshead minnow eggs were
spawned naturally in the laboratory in natural sea
water basically following procedures in U.S. EPA
(1978)."
Metric 15:
Number of Organisms and Replicates per
Group
High
X
1
1
At least 7 fish per exposure group is recommended
by OECD, and this test had 10.
Metric 16:
Adequacy of Test Conditions
Medium
X
1
2
Mysids and fish were tested in 1.6-L covered-glass
dishes containing 1.0 L of test solution or control
seawater. With 10 fish per container, and OECD
recommended 1 liter per g of fish, and the average
mass of fish were 1.4 mg we weight. Only small un-
certainties about housing remain.
Domain 5: Outcome Assessment

Metric 17:
Outcome Assessment Methodology
High
X
2
2
Outcomes of interest were measured appropriately
Metric 18:
Consistency of Outcome Assessment
Medium
X
1
2
Incomplete reporting of minor details of outcome as-
sessment protocol execution.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Low x 2 6 The study did not provide enough information to
Procedures allow a comparison of conditions or other non-
treatment related factors across study groups
Metric 20: Outcomes Unrelated to Exposure Low X 1 3 Data on attrition and/or health outcomes unrelated
to exposure were not reported for each study group.
Domain 7: Data Presentation and Analysis
Continued on next page . . .

-------
. continued from previous page
Study Citation: Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF TRICHLOROETHYLENE TO SALTWATER ORGAN-
ISMS. Bulletin of Environmental Contamination and Toxicology 37:830-836
Data Type:	Acute (0-96 hour); Aquatic; Fish
Hero ID:	2801609
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High	X 1	1 Based on the results of the tests, 24-, 48-, 72-, and
96-h LC50s or EC50s and 95 percent confidence lim-
its were calculated, where possible. The computer
program generated the LC- or EC50 values using the
following statistical methods: moving average angle,
probit, and binomial probability (Stephan 1977).
Low	X 2	6 Data for exposure related finding were not shown for
each study group.
High	X 1	1	Unexpected outcome was explained, "Differences be-
tween TCE test concentrations at which mild intoxi-
cation symptoms were observed in test organisms in
this study and by Borthwick (1977) may possibly be
explained on the basis of carrier solvent synergism
and/or temperature effects."
Overall Quality Determination"'"
Medium
1.7
Extracted
Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF TRICHLOROETHYLENE TO SALTWATER ORGAN-
ISMS. Bulletin of Environmental Contamination and Toxicology 37:830-836
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	2801609
Domain
Metric
Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance






Metric 1:
Test Substance Identity
High
X
2
2
TCE is identified by name.
Metric 2:
Test Substance Source
Medium
X
1
2
Source was not reported but actual TCE test concen-
trations were analytically determined at the initia-
tion and termination of the test or when 100 percent
mortality occurred in a treatment.
Metric 3:
Test Substance Purity
Low
X
1
3
Test substance purity was not reported.
Domain 2: Test Design






Metric 4:
Negative Controls
High
X
2
2
Reported using a negative control group with just
seawater.
Metric 5:
Negative Control Response
Low
X
1
3
Negative control response was not reported.
Metric 6:
Randomized Allocation
Low
X
1
3
Researchers did not report how organisms were al-
located to study groups.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Medium x 2
tion
Metric 8: Consistency of Exposure Administration
High
Metric 9: Measurement of Test Substance Concentra- High
tion
Metric 10: Exposure Duration and Frequency	High
x 1
x 2
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
4	The study used covered glass containers but voliti-
zation still occurred in the static system. The study
did report test concentrations measured at the be-
ginning and end of the test.
1	Details of exposure administration were reported
and exposures were administered consistently across
study groups.
2	Test concentrations were measured at the beginning
and end of the test.
1	The duration of exposure and frequency was re-
ported as is appropriate for mysids. 96 hour static
test
2	There were 5 exposure groups with a duplicate at
each exposure level, no range finding test was con-
ducted, and instead concentrations were spaced by
6.25, 12.5, 25, 50 and 100 percent of the water solu-
ble fraction.
Continued on next page

Metric 13:
Test Organism Characteristics
High
X
2
2
Mysid shrimp is one of the recommended species for
saltwater from EPA guidelines.
Metric 14:
Acclimitization and Pretreatment Conditions
High
X
1
1
Study reports, "Mysid shrimp, Mysidopsis bahia,
were born in culture at BMRL and maintained for
3 days before testing. Mysids were reared in natu-
ral sea water generally following procedures in U.S.
EPA (1978). During holding, temperature was main-
tained at 22+l"C and salinity at 19 ppt."
Metric 15:
Number of Organisms and Replicates per
High
X
1
1
Ten mysids for each container and each concentra-

Group

tion had a duplicate. EPA recommends 20 animals

at each cone level (EPA TG 850.1035).
Metric 16:
Adequacy of Test Conditions
Medium
X
1
2
Mysids and fish were tested in 1.6-L covered-glass
dishes containing 1.0 L of test solution or control
seawater. Shrimp were fed live (48-h old) brine
shrimp nauplii on Days 0 and 2 during the test. Only
small uncertainties about housing remain.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology High X 2 2 Outcomes of interest were measured appropriately.
Metric 18: Consistency of Outcome Assessment Medium x 1 2 Incomplete reporting of minor details of outcome as-
sessment protocol execution.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Low x 2 6 The study did not provide enough information to
Procedures allow a comparison of conditions or other non-
treatment related factors across study groups
Metric 20: Outcomes Unrelated to Exposure Low X 1 3 Data on attrition and/or health outcomes unrelated
to exposure were not reported for each study group
Domain 7: Data Presentation and Analysis
Continued on next page . . .

-------
. continued from previous page
Study Citation: Ward, G. S.,Tolmsoff, A. J.,Petrocelli, S. R.. 1986. ACUTE TOXICITY OF TRICHLOROETHYLENE TO SALTWATER ORGAN-
ISMS. Bulletin of Environmental Contamination and Toxicology 37:830-836
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	2801609
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High
Low
High
x 1
x 2
x 1
Based on the results of the tests, 24-, 48-, 72-, and
96-h LC50s or EC50s and 95 percent confidence lim-
its were calculated, where possible. The computer
program generated the LC- or EC50 values using the
following statistical methods: moving average angle,
probit, and binomial probability (Stephan 1977).
Data for exposure related finding were not shown for
each study group.
No unexplained outcomes were reported.
Overall Quality Determination"'"
Medium
1.7
Extracted
Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Dierickx, P. J.. 1993. Comparison between fish lethality data and the in vitro cytotoxicity of lipophilic solvents to cultured fish cells
in a two-compartment model. Chemosphere 27:1511-1518
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 2803221
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Chemical identified by name, trichloroethylene
Metric 2:
Test Substance Source
Low
X
1
3
Not reported
Metric 3:
Test Substance Purity
Low
X
1
3
Purity, grade not reported
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
Medium x 2
Low
Low
x 1
x 1
A control was used, but not much detail is given.
Authors report, "During this period the cells, re-
maining in culture medium, are in contact with the
test chemical, solubilized in paraffin, through the
microporous membrane. Pure paraffin was used as
the control."
Not reported
Allocation method not reported
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
Metric 8: Consistency of Exposure Administration High
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency
Metric 11: Number of Exposure Groups/Spacing of Ex- Low
posure Levels
x 2
x 1
x 2
Medium x 1
x 1
TCE is very volatile, and there was no mention of
covering the wells or preventing loss of test sub-
stance. However the test was only 24 hours long
so some of the TCE may have been in the wells by
the end of the test.
Exposure cone was not measured or not reported
The exposure duration was 24 hours, which was
enough time to record an effect of total protein inhi-
bition, so it appears to be a sufficient duration. Only
minor uncertainties remain. It was a static exposure
in wells.
Number of exposure groups and spacing were not re-
ported; however the authors were able to determine
an EC50 for total protein expression.
Continued on next page

-------
. . . continued from previous page
Study Citation: Dierickx, P. J.. 1993. Comparison between fish lethality data and the in vitro cytotoxicity of lipophilic solvents to cultured fish cells
in a two-compartment model. Chemosphere 27:1511-1518
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 2803221
Domain
Metric
Rating^
MWF*
Score
Comments^
Metric 12:
Testing at or Below Solubility Limit
Medium
X
1
2
Because exposure levels were not reported, it is un-
clear if they were all below the solubility limit, how-
ever the EC50 was far below the solubility for TCE.
Domain 4: Test Organism

Metric 13:
Test Organism Characteristics
Medium
X
2
4
Cultured fathead minnow fish cells were used for this
study. While this is a well studies species, minor un-
certainties remain about whether this cell line (FHM
cells American Type Culture Collection N" CCI42
established fishcell line, derived from tissue posterior
to the anus from fathead minnow) are appropriate
for this type of in vitro test.
Metric 14:
Acclimitization and Pretreatment Conditions
High
X
1
1
Cells were cultured in the lab and pretreated before
the test.
Metric 15:
Number of Organisms and Replicates per
Medium
X
1
2
Authors report, "6.105 cells in 0.2 mL culture

Group

medium were seeded in each of the 12 transwells."

Minor uncertainties about what cell density is ap-
propriate for fish cells.
Metric 16:
Adequacy of Test Conditions
Low
X
1
3
Control response was not reported for TCE so it's
difficult to tell whether the housing conditions were
adequate for this cell line's normal functioning
Domain 5: Outcome Assessment
Continued on next page . . .

-------
. continued from previous page
Study Citation: Dierickx, P. J.. 1993. Comparison between fish lethality data and the in vitro cytotoxicity of lipophilic solvents to cultured fish cells
in a two-compartment model. Chemosphere 27:1511-1518
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 2803221
Domain Metric Rating^ MWF* Score Comments^
Metric 17: Outcome Assessment Methodology High X 2 2 EC50s (for total protein inhibition) were determined
to measure cytotoxicity using the following method-
ology, "Each well was rinsed once with 0.5 mL
Hank's balanced salt solution on the outer mem-
branic side and twice on the inner side, and placed
in an empty well of a new 24well tissue culture plate.
The remaining cells were lysed with 0.2 mL 2 per-
cent Na2C03 in 0.1 N NaoH per transwell for 15
min at 34 "C. This solution was then replaced 2
times with the same solution, at room temperature,
and the combined fractions were used for a protein
determination according to Lowry et al.. (1951)
as previously described for Hep G2 cells (Dierickx,
1989). The results are expressed as percentages of
control cultures, which typically contained 85-105
"g protein/transwell. The relative toxicity of the
test chemicals is established by the determination of
the EC50. This is the concentration of test chem-
ical in the lower compartment required to induce
a 50 percent inhibition of the total protein content
in the upper compartment (transwell). In order to
determine the reproducibility of EC50 determina-
tions, the EC50 values of cyclopentanol, cyclohex-
anone and 2-chlorotoluene were measured in three
independent assays. A standard deviation of less
than 6 percent was found in these independent as-
says. Therefore, the EC50 values of the other com-
pounds were determined in single experiments."
Metric 18: Consistency of Outcome Assessment High x 1 1
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Low x 2 6 The study did not provide enough information to
Procedures allow a comparison of the environmental conditions
for each study group.
Metric 20: Outcomes Unrelated to Exposure Low x 1 3 Cell attrition was not reported for TCE for each
study group.
Domain 7: Data Presentation and Analysis
Continued on next page . . .

-------
. continued from previous page
Study Citation: Dierickx, P. J.. 1993. Comparison between fish lethality data and the in vitro cytotoxicity of lipophilic solvents to cultured fish cells
in a two-compartment model. Chemosphere 27:1511-1518
Data Type:	Acute (0-96 hour); Aquatic; Fish
Hero ID:	2803221
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
Low	X 1	3 The statistical method used to derive the EC50 was
not reported in detail. Authors simply state, "In-
terpolation of these curves gives the EC50 values,by
computing the concentration needed to reduce the
total protein content by 50 percent."
Low	X 2	6 Data for exposure related findings were not reported
for each study group.
Low	X 1	3 Authors acknowledge the discrepancy between this
study (which did not find a linear correlation be-
tween the published LC50 levels and the EC50 val-
ues they produced in this test) and previous studies
which did, but the reasons are unclear.
Overall Quality Determination"'"
Low
2.3
Extracted
Yes


*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Bacsi, I.,Gonda, S.,B-Beres, V.,Novak, Z.,Nagy, S. A.,Vasas, G.. 2015. Alterations of phytoplankton assemblages treated with chlori-
nated hydrocarbons: effects of dominant species sensitivity and initial diversity. Ecotoxicology 24:823-834
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 3298076
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Metric 3:
Test Substance Identity
Test Substance Source
Test Substance Purity
High
Low
Low
X 2 2 Test substance identified by name, trichloroethylene,
and use was described.
X 1 3 Not reported
X 1 3 Not reported
Domain 2: Test Design
Metric 4:
Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
Medium
Medium
N/A
x 2
x 1
N/A
Controls in beakers and pond sample controls were
used but details about what exactly controls in-
cluded were not given. Authors reported, "12 1
water sample from the pond was filled into 4 plas-
tic (polimethylpenthene" PMP) beakers (3 1 to each
one)."
Control response was reported in figures, until 3
days. Some uncertainties remain about exact num-
bers for control response, but an approximation can
be seen in the figures.
Not applicable to allocate individual algae to study
groups randomly.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Unacceptable x 2
tion
Metric 8: Consistency of Exposure Administration
Low
Metric 9: Measurement of Test Substance Concentra- Low
tion
x 1
x 2
o Beakers were used for the pond experiment and the
authors allude the fact that the beakers are "en-
closed" , but it is unclear whether enough precau-
tions are taken to avoid volatilization of the test
chemicals and no measurements of test chemical
were taken. No nominal concentrations were given
either.
3 Details not given about exposure administration for
each exposure level.
6 Measured concentrations were not taken and cannot
be expected to be close to nominal concentration due
to the volatility of the chemical. Additionally this
experiment measured effects 24 hours, 48 hours and
96 hours after exposure, giving this substance plenty
of time to volatilize.
Continued on next page

-------
. continued from previous page
Study Citation: Bacsi, I.,Gonda, S.,B-Beres, V.,Novak, Z.,Nagy, S. A.,Vasas, G.. 2015. Alterations of phytoplankton assemblages treated with chlori-
nated hydrocarbons: effects of dominant species sensitivity and initial diversity. Ecotoxicology 24:823-834
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 3298076
Domain
Metric
Rating1
MWF* Score
Comments' t
Metric 10: Exposure Duration and Frequency
Low
Metric 11: Number of Exposure Groups/Spacing of Ex- N/A
posure Levels
Metric 12: Testing at or Below Solubility Limit Low
X 1 3 96 hours is an acceptable amount of time to measure
effects in algae however the exposure only occurred
once at time 0, and this chemical volatilizes quickly,
so multiple exposures are necessary to maintain test
concentrations.
N/A it appears only one exposure group was used how-
ever, with a microcosm experiment this may be ac-
ceptable.
X 1 3 Unsure what the actual exposure concentration was
from the author's reporting. No measurements were
taken to confirm, but authors report, "Treated as-
semblages were theoretically saturated solvents at
the beginning of the experiments."
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Medium
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Medium
Group
Metric 16: Adequacy of Test Conditions
Low
x 2
x 1
x 1
x 1
4 Test organisms were a variety of algae species (59
taxa were reported in 2011 and 95 in 2012) found
in the Garden Pond in the Botanical Garden of the
University of Debrecen. Dominant species was Tra-
chelomonas volvocinopsis.
3 Not reported
2 Authors report, "All experiments were done in
triplicates," which is recommended. Abundance
was reported at about 11x106 in the 2012 experi-
ment, which is outside the range of densities given
in OECD 201 recommendations (e.g. the high-
est recommended cell density is for synechococcus
leopoliensis at 5x104-105).
3 Beakers were plastic and it is unclear whether they
are chemically inert. Measurements of ph, tempera-
ture, 02 cone were taken.
Domain 5: Outcome Assessment
Continued on next page

-------
. continued from previous page
Study Citation: Bacsi, I.,Gonda, S.,B-Beres, V.,Novak, Z.,Nagy, S. A.,Vasas, G.. 2015. Alterations of phytoplankton assemblages treated with chlori-
nated hydrocarbons: effects of dominant species sensitivity and initial diversity. Ecotoxicology 24:823-834
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 3298076
Domain
Metric
Rating1
MWF* Score
Comments' t
Metric 17: Outcome Assessment Methodology
High
Metric 18: Consistency of Outcome Assessment
Low
x 2
x 1
Outcome assessment methodology is described for
changes in growth, and species diversity. Growth
measures are appropriate according OECD guidance
201. Diversity was measured in the following way,
"The phytoplankton samples were processed accord-
ing to the Utermo"hl method (1958) and European
Standard EN 15204:2006. An Olympus CKX31 in-
verted microscope and 4009 magnification was used
for counting and taxa identification."
Details regarding execution of study protocol across
study groups was not reported.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High
Procedures
Metric 20: Outcomes Unrelated to Exposure
Low
X 2 2 Authors measured ph, 02 cone, water temp, and
contols and treatment groups were kept in similar
conditions in the pond in the same basket.
X 1 3 A storm could have affected growth, diversity in this
experiment.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High
Low
High
X 1 1 Significance was determined trough ANOVA and
Tukey test. All experiments were done in triplicate.
X 2 6 Data was reported in figures, but not very well in
text and the exact concentrations at which algae was
exposed is not reported.
X 1 1 Unexpected outcomes were addressed, "The ob-
served decrease of chlorophyll-a content and algal
abundance in the first 24 h probably occurred due to
a summer storm with stormy wind, rain and a tem-
perature drop of 6 C in 2011. Meteorological events
like heavy rainfall, hot weather periods and stormy
hours can have a pronounced impact on hydraulics,
water temperature and nutrient supply. Such short-
term variability is thought to be important in the
changes of algal community structure."
Overall Quality Determination"'"
Unacceptable
4.0
Continued on next page

-------
. continued from previous page
Study Citation: Bacsi, I.,Gonda, S.,B-E>eres, V.,Novak, Z.,Nagy, S. A.,Vasas, G.. 2015. Alterations of phytoplankton assemblages treated with chlori-
nated hydrocarbons: effects of dominant species sensitivity and initial diversity. Ecotoxicology 24:823-834
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	3298076
Domain	Metric	Rating^	MWF* Score	Comments^
Extracted	No
** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
r High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Houde, M.,Douville, M.,Gagnon, P.,Sproull, J.,Cloutier, F.. 2015. Exposure of Daphnia magna to trichloroethylene (TCE) and vinyl
chloride (VC): evaluation of gene transcription, cellular activity, and life-history parameters. Ecotoxicology and Environmental Safety
116
Data Type: Other; Aquatic; Invertebrates
Hero ID: 3502953
Domain Metric Rating"!" MWF* Score Comments"*1"!"
Domain 1: Test Substance
Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity
High x 2
Medium x 1
Low x 1
2 Chemical identified by name and cas
2 Source of TCE was reported but not analytically
verified. Authors report, "Stock standard solutions
of TCE was obtained from Supelco (Bellefonte, PA,
USA) and were diluted in methanol and water."
3 Not reported
Domain 2: Test Design
Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
High x 2
Medium x 1
Low x 1
Methanol control group was included with 6 repli-
cates. methanol is listed as a suitable control sol-
vent for daphnia, and authors report that methanol
concentration never exceeded 0.1 percent which is
recommended in OECD 211 as well.
Control response was reported for some but not all
responses, e.g. no control response was reported for
gene expression.
Not reported
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High x 2
tion
Metric 8: Consistency of Exposure Administration High x 1
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Physical chemical properties were taken into con-
sideration. Authors used standardized methods for
volatile organic compounds (PNLET, 2011). Each
beaker was covered with a watch glass and left at 20
degrees C.
A lot of detail was reported in the paper and supple-
mentary information about exposure administration
timing and actual concentrations. Exposure admin-
istration was described as
Authors measured cone of TCE extensively, report-
ing the measured cone at hour 01234568 24 30
48.
Continued on next page

-------
. . . continued from previous page
Study Citation: Houde, M.,Douville, M.,Gagnon, P.,Sproull, J.,Cloutier, F.. 2015. Exposure of Daphnia magna to trichloroethylene (TCE) and vinyl
chloride (VC): evaluation of gene transcription, cellular activity, and life-history parameters. Ecotoxicology and Environmental Safety
116
Data Type: Other; Aquatic; Invertebrates
Hero ID: 3502953
Domain Metric Rating"!" MWF* Score Comments"i"i"
Metric 10: Exposure Duration and Frequency
High x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High x 1
Considering the volatile properties of the studied
compounds, renewal of the media was conducted ev-
ery 24 h in order to ensure the continuous exposure
of D. magna. New stock solutions were prepared at
every media renewal and water temperature, con-
ductivity, dissolved oxygen, pH, and hardness were-
monitored.
For the chronic toxicity test, only 3 cone were tested,
and OECD 211 recommends 5. Concentrations were
determined by what levels found in the environment
(in river water in Quebec Canada).
Concentrations were far below TCE's solubility.
Domain 4: Test Organism
Metric 13:
Metric 14:
Test Organism Characteristics High
Acclimitization and Pretreatment Conditions High
Metric 15: Number of Organisms and Replicates per High
Group
Metric 16: Adequacy of Test Conditions High
x 2
x 1
x 1
x 1
Daphnia magna were used in this study
Pretreatment conditions were reported and
acceptable. Authors reported, "Geneti-
cally homogenous D. magna were cultured in
growthchamber following Environment Canada" s
method(EnvironmentCanada, 1990). Cul-
tureswerekeptat2071 "C with a photoperiodof
16hlight:8hdark.Organismswerefedgreenalgae
Pseudokirchneriella subcapitata (concentra-
tion: 3.85105 cells/mL) and YCT preparation
(yeast" cerophy 11" trout chow, concentration:
0.0125g/L) everyday. All experiments were per-
formed under the same constant temperature and
diurnal lighting conditions."
OECD recommends 10 animals at each test conc. In
this study 10 animals were used per treatment.
Temperature, light/dark period were within OECD
211 recommended ranges. Food was also appropri-
ate.
Domain 5: Outcome Assessment
Continued on next page

-------
. . . continued from previous page
Study Citation: Houde, M.,Douville, M.,Gagnon, P.,Sproull, J.,Cloutier, F.. 2015. Exposure of Daphnia magna to trichloroethylene (TCE) and vinyl
chloride (VC): evaluation of gene transcription, cellular activity, and life-history parameters. Ecotoxicology and Environmental Safety
116
Data Type: Other; Aquatic; Invertebrates
Hero ID: 3502953
Domain Metric Rating"!" MWF* Score Comments"i"i"
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
Outcome was measured by protein expression and
genes being up or down regulated. No EC50 was es-
tablished, and no effect on overall reproduction was
found.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2
Procedures
Metric 20: Outcomes Unrelated to Exposure High x 1 1
Test setup and procedure seems sufficient
No unexpected health outcomes
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Medium x 1
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
Medium x 2
High x 1
Most of the statistical analysis mentioned were ap-
propriate (recommended in OECD 211, with only
some uncertainty around using the Kruskal-wallis
test for brooding time. Authors report, "Daphnia
brooding times were compared with Kruskal"Wallis
tests. Numbers of neonates were modeled as over
dispersed Poisson variates and animal sizes at the
end of the lOdays exposurewere compared with
ANOVAs. Relative gene transcription between ex-
posed and non-exposed organisms (methanol con-
trols) were compared using Wilcoxon signed ranks
tests (probabilities corrected to control the false
discovery rate). Biomarker responses to contami-
nant concentrations were compared to controls with
ANOVAs and Dunnett"s procedure. Relationships
between biomarkers and genes were assessed using
Spearman" s correlations.The significance threshold
was established at 0.05. All statistical analyses were
conducted using SAS9.4 (SAS Institute Inc.).
Data was reported for each study group for some but
not all end points.
Sufficient discussion of results
Overall Quality Determination"'" High 1.3
Continued on next page

-------
. . . continued from previous page
Study Citation: Houde, M.,Douville, M.,Gagnon, P.,Sproull, J.,Cloutier, F.. 2015. Exposure of Daphnia magna to trichloroethylene (TCE) and vinyl
chloride (VC): evaluation of gene transcription, cellular activity, and life-history parameters. Ecotoxicology and Environmental Safety
116
Data Type:	Other; Aquatic; Invertebrates
Hero ID:	3502953
Domain	Metric	Rating"!" MWF* Score	Comments"i"i"
Extracted	Yes
* MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
^ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
(Metric Score; X MWF;) / ]T\ MWF,
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
tt Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Vidal, M.,Basseres, A.,Narbonne, J.. 2001. Potential biomarkers of trichloroethylene and toluene exposure in Corbicula fluminea.
Environmental Toxicology and Pharmacology 9:87-97
Data Type:	Other; Aquatic; Invertebrates
Hero ID:	3546158
Domain	Metric	Rating^ MWF* Score	Comments^
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Substance was identified by name
Metric 2:
Test Substance Source
Low
X
1
3
Not reported
Metric 3:
Test Substance Purity
Low
X
1
3
Not reported
Domain 2: Test Design






Metric 4:
Negative Controls
High
X
2
2
Controls consisted in two aquariums each containing






10 1 of filtered lake water.Aquariums were covered to






limit water and/or product evaporation.
Metric 5:
Negative Control Response
High
X
1
1
Response of the negative control was reported
Metric 6:
Randomized Allocation
Low
X
1
3
Not reported
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Medium x 2
tion
Metric 8: Consistency of Exposure Administration
Medium x 1
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency
Medium x 1
The loss of TCE during the preparation of the con-
taminated water and during the exposure was very
important in spite of glass covers on aquariumsand
moderate, intermittent aeration to try to reduce
evaporation. Measurements were taken to verify the
actual cone the clams were exposed to. Results of
chemical analysis are presented in Table 1. As TCE
and TOL are volatile products, an important loss
was observed during the preparation of mixtures,due
mainly to homogenisation (loss of 23 up to 44 per-
cent for TCE and 6 up to 45 percent for TOL).
Some loss of test chemical during preparation of mix-
tures but in general the administration was consis-
tent being a one-time static exposure.
Measurements of test substances were taken; nomi-
nal cone were 1.56, 6.25, 25 and 100 mg/1 for TCE
and measured cone were 1.2, 3.6, 14, and 69.4.
Exposure was a one-time static exposure with tox
results assessed at the end of 5 days. The guidelines
for acute tox for bivalves from EPA suggest a 48
hour flow through exposure, but authors in this pa-
per were trying to mimic conditions that may occur
in the field with a one time spill.
Continued on next page

-------
. continued from previous page
Study Citation: Vidal, M.,Basseres, A.,Narbonne, J.. 2001. Potential biomarkers of trichloroethylene and toluene exposure in Corbicula fluminea.
Environmental Toxicology and Pharmacology 9:87-97
Data Type: Other; Aquatic; Invertebrates
Hero ID: 3546158
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
x 1
5 tests concentrations are recommended in EPA test
guidelines for bivalves (850.1055). this test only has
4 test concentration
Test cone were far below TCE solubility level.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Medium x 2
Metric 14: Acclimitization and Pretreatment Conditions High x 1
Metric 15: Number of Organisms and Replicates per Medium x 1
Group
Metric 16: Adequacy of Test Conditions Medium x 1
Corbicula fluminea, a freshwater clam, is not a listed
recommended test species for bivalves in the EPA
850.1055 guidelines but is present in freshwater en-
vironments in Asia, the US, and increasingly in Eu-
rope.
Replicates not reported; number of organisms was
reported.
Temperature, ph, light and dark, lack of crowded
conditions are consistent with what is recommended
for the
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2
Procedures
Metric 20: Outcomes Unrelated to Exposure High x 1 1
There does not seem to be any differences among
study groups in environmental conditions.
No health outcomes unrelated to exposure
Domain 7: Data Presentation and Analysis
Continued on next page

-------
. continued from previous page
Study Citation: Vidal, M.,Basseres, A.,Narbonne, J.. 2001. Potential biomarkers of trichloroethylene and toluene exposure in Corbicula fluminea.
Environmental Toxicology and Pharmacology 9:87-97
Data Type: Other; Aquatic; Invertebrates
Hero ID: 3546158
Domain Metric Rating^ MWF* Score Comments^
Metric 21: Statistical Methods
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
High X 1 1 Authors report, "Statistica software (5.1 release,
Statsoft) was used forall statistical evaluations.
Data distributed normally were submitted to a
Brown" Forsythe test for homogeneity of variances
(across dose groups). Data fulfilling this require-
ment were then submitted to a univariate analy-
sis of variance (ANOVA) with "dose" (of TCE or
TOL) as the independent variable and biochemical
parameters as dependent variables. Whenever a sig-
nificant (P50.05) dose effect was established on a
biochemical parameter response, a Tukey HSD mul-
tiple comparison test was used to check significant
(P50.05) differences between doses. Correlations be-
tween the dose and biochemical parameter responses
were determined with the Pearson correlationcoeffi-
cient (significant for P50.05). As additional stud-
ies, discriminant analysis (DA), often referred to as
canonical variate analysis, were performed to try
todiscriminate doses (of TCE or TOL) from each
other and to determine biochemical parameters al-
lowing the best discrimination between doses. Un-
like univariate analysis, multivariate analysis such
as DA takes intoaccount the whole biochemical pa-
rameter responses and, thus, provides an integrated
approach. DA procedures and its applications in en-
vironmental biomonitoring have been described by
Adams et al. (1994)."
Medium X 2 4 Mechanistic data was reported for each exposure
level, however behavioral patterns mentioned in the
discussion were not reported and would be helpful
in establishing an EC50.
High X 1 1 No unexpected outcomes
Overall Quality Determination"'"
High
1.5
Extracted
Yes

Continued on next page . . .

-------
. continued from previous page
Study Citation: Vidal, M.,Basseres, A.,Narbonne, J.. 2001. Potential biomarkers of trichloroethylene and toluene exposure in Corbicula fluminea.
Environmental Toxicology and Pharmacology 9:87-97
Data Type:	Other; Aquatic; Invertebrates
Hero ID:	3546158
Domain	Metric	Rating^ MWF* Score	Comments^
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Ando, T.,Otsuka, S.,Nishiyama, M.,Senoo, K.,Watanabe, M. M.,Matsumoto, S.. 2003. Toxic Effects of Dichioromethane and
Trichforoethyfene on the Growth of Planktonic Green Algae, Chlorella vulgaris NIES227, Selenastrum capricornutum NIES35, and
Volvulina steinii NIES545. 18:43-46
Data Type: Other; Aquatic; Plants
Hero ID: 3617103
Domain
Metric
Rating"!" MWF* Score
Comments^1
Domain 1: Test Substance
Metric 1:
Metric 2:
Metric 3:
Test Substance Identity
Test Substance Source
Test Substance Purity
High
Low
Low
x 2
x 1
x 1
Test substance was identified by
trichloroethylene, and described as a
organic solvent.
Test substance source was not specified.
The purity not specified.
name,
typical
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
High X 2 2 Controls were included for each species.
High X 1 1 The growth effects were reported for the controls in
the figures.
Low X 1 3 There was no mention of random allocation.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
Metric 8: Consistency of Exposure Administration
High
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency High
x 2
x 1
x 2
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Medium x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
6 According to the authors description, this testing
procedure appeared to bestatic, but details about
the test procedure were lacking. In particular, there-
was uncertainty about whether evaporation of the
solvents occurred.
1 Exposure was administered consistently across study
groups.
6 Test concentrations reported in terms of nominal
concentrations.
1 The test algae were cultured for 10 days and the ab-
sorption of light bychlorophyll a was measured every
2 days as a measure of algal growth.
2 Concentrations were reported as 0, 0.003, 0.03, 0.3
and 3 mg/L. Thiscorresponded to 0, one-tenth, ten-
fold and 100 fold of the Japanese nationaleffluent
standards for TCE. However, there were only 4 test
concentrationswhile OECD recommends at least 5.
1 Test conc. were well below TCE's solubility level.
Continued on next page

-------
. . . continued from previous page
Study Citation: Ando, T.,Otsuka, S.,Nishiyama, M.,Senoo, K.,Watanabe, M. M.,Matsumoto, S.. 2003. Toxic Effects of Dichioromethane and
Trichioroethyiene on the Growth of Planktonic Green Algae, Chlorella vulgaris NIES227, Selenastrum capricornutum NIES35, and
Volvulina steinii NIES545. 18:43-46
Data Type: Other; Aquatic; Plants
Hero ID: 3617103
Domain Metric Rating"!" MWF* Score Comments"*1"!"
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
x 2
Metric 14: Acclimitization and Pretreatment Conditions Medium x 1 2
Metric 15: Number of Organisms and Replicates per High x 1 1
Group
Metric 16: Adequacy of Test Conditions High x 1 1
C. vulgaris, S. capricornutum, and V. Steinii were
used for this study. Strains obtained from Japanese
national collection. Although only one species is an
OECD recommended test organisms, the other or-
ganisms were chosen to cover species that would be
able to grow in a wide range of water qualities.
Algae were incubated prior to addition of test mate-
rial, but details were notprovided, so minor uncer-
tainties remain.
Authors indicated 5 replicates in this study, and
OECD 201 recommends at least 3.
Test vessels were consistent with recommendations
for algal growth potential (AGP) test.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
Absorbance as a measure of growth/biomass was re-
ported for all test concentrations, which can be used
as a surrogate for biomass when measured by a spec-
trophotometer (as it was in the study) according to
OECD TG 201.
Outcomes we assessed consistently across study
groups.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High
Procedures
Metric 20: Outcomes Unrelated to Exposure
x 2
Medium x 1
There were no reported differences among the study
groups in environmental conditions or other factors
that could influence the outcome assessment.
Data on attrition or health outcomes unrelated to
exposure were not reportedfor each study group but
are not likely to have substantial impacts on results.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Low X 1 3 Statistical analysis was performed to determine the
significance of differencesbetween control and test
concentrations, but test methods were not pre-
sented.
Continued on next page

-------
. continued from previous page
Study Citation: Ando, T.,Otsuka, S.,Nishiyama, M.,Senoo, K.,Watanabe, M. M.,Matsumoto, S.. 2003. Toxic Effects of Dichioromethane and
Trichioroethyiene on the Growth of Planktonic Green Algae, Chlorella vulgaris NIES227, Selenastrum capricornutum NIES35, and
Volvulina steinii NIES545. 18:43-46
Data Type:	Other; Aquatic; Plants
Hero ID:	3617103
Domain	Metric	Rating"!" MWF* Score	Comments"*1"!"
Low	X 2	6 Absorbance as a measure of growth/biomass was
reported for all testconcentrations, which can be
used as a surrogate for biomass when measuredby
a spectrophotometer (as it was in the study) ac-
cording to OECD TG 201.And relative absorbance
was reported for each species and each study groupin
the figures. However, no EC50 was derived for the
species that did see aneffect on relative absorbance.
Metric 23: Explanation of Unexpected Outcomes	High	x 1	1 No unexplained outcomes reported.
Metric 22: Reporting of Data
Overall Quality Determination^	High —> Medium 1.7
Extracted	Yes
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Yoshioka, Y.,Ose, Y.,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type:	Other; Aquatic; Invertebrates
Hero ID:	3617749
Domain	Metric	Rating^ MWF* Score	Comments^
Domain 1: Test Substance
Metric 1: Test Substance Identity	High	X 2	2 Chemical identified by name: Trichloroethylene.
Metric 2: Test Substance Source	Low	x 1	3 Source of TCE was not reported, but it was noted
that analytical grade TCE was used.
Metric 3: Test Substance Purity	Medium X 1	2 Purity not reported but it was noted that analytical
grade TCE was used.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
located.
Negative Controls
Negative Control Response
Randomized Allocation
Low
Low
Low
x 2
x 1
x 1
The study refers to a blank but doesn't say what's
in the blank. I assume this is the control for D.
japonica (planarian).
The study reports that most of the planarian in the
blank test regenerated heads normally, but a number
isn't given and TCE isn't discussed specifically.
It's not reported whether animals were randomly al-
Domain 3: Exposure Characterization
Continued on next page

-------
. continued from previous page
Study Citation: Yoshioka, Y.,Ose, Y.,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type: Other; Aquatic; Invertebrates
Hero ID: 3617749
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 7: Experimental System/Test Media Prepara- Low
tion
x 2
Cn
O
Metric 8: Consistency of Exposure Administration
Metric 9:
Low
x 1
x 2
Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency Medium x 1
It's unclear whether the experiement was conducted
in a closed or open system using static or flow
through methods. The study reports, "The breed-
ing liquid for Dugesia japonica was prepared by dis-
solving 3.74 g of NaCl, 0.49 g of KC1, and 8.5 5 g of
CaC12 into distilled water to make 500 ml. This was
diluted 100 times and neutralized by NaHC03 before
use. Dugesiajaponica were collected from a stream
around which there was no source of pollution and
left without food for over 7 days in the breeding
liquid to excrete alimentary canal contents. Those
of about _2 cm long were used. Dugesia japonica
was cut into two parts (head and body part) at the
nearest section to the eyes of the trisected part be-
tween pharynx and eyes. The body part was used
for the head regeneration test. Ten body parts were
put in 100 ml ofa test solution, and this was left
at 20 " 1" C for 7 days. Observation for head re-
generation was carried out with a stereomicroscope
on Days 3, 4, 5, 6, and 7 after head cutting, and the
test solution was replaced at every observatiort. The
degree of regeneration was classified as normal, eye
spot, tetratophthalmic, anophthalmic, aciphthalmic,
and death. The total number of eye spot, tetratoph-
thalmic, anophthalmic, aciphthalmic, and death was
regarded as the abnormal regeneration number. The
ratio of the number to 10 on Day 7 was defined as
the abnormal regeneration rate. The concentration
of the chemical, at which the abnormal regenera-
tion rate reached 50 percent, was defined as EC50"
LC50 of D. japonica was determined at the same
time. LC50 and EC50 values of the test mentioned
above were determined on semilogarithmic paper."
Exposure methods were not reported for each study
group
It was not reported whether nominal or measured
cone were used.
Exposure occurred over 7 days, and observation was
carried out on days 3, 4, 5, 6, and 7 after head cut-
ting, and the test solution was replaced at every ob-
servation.
Continued on next page

-------
. continued from previous page
Study Citation: Yoshioka, Y.,Ose, Y.,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type: Other; Aquatic; Invertebrates
Hero ID: 3617749
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 11: Number of Exposure Groups/Spacing of Ex- Low x 1 3
posure Levels
Metric 12: Testing at or Below Solubility Limit High x 1 1
Not reported for TCE, but for other chemicals it
looks like 4 exposure groups were used plus control.
Substance was tested well below solubility.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Metric 14: Acclimitization and Pretreatment Conditions Low
Metric 15: Number of Organisms and Replicates per Low x 1
Group
Metric 16: Adequacy of Test Conditions
Low X 2 6 Uncertainties about the quality of the test organ-
isms given they were collected from the field and no
acclimation is mentioned. Study reports, "Dugesia
japonica were collected from a stream around which
there was no source of pollution and left without
food for over 7 days in the breeding liquid to excrete
alimentary canal contents. Those of about 2 cm long
were used."
X 1 3 Did not report whether they were acclimatized and
they were collected from the field.
3 The number of animals in each solution was not
clear, possibly ten? The study says "Ten body parts
were put in 100 ml of a test solution, and this was
left at 20 " 1"C for 7 days." Is this 10 body parts
from 10 different individuals? Number of replicates
not reported.
Low X 1 3 Housing not mentioned for planarian.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High x 2
Low x 1
2 Determined an LC50
3 Details of outcome assessment were not reported.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and Medium x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure
Low
x 1
Confounding variables are discussed for planarian.
The study says that confounding may occur due
to the cutting of the head (stress of cutting of the
head).
Data on health and attrition were not reported for
each study group.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Medium x 1
Methods not described clearly
Continued on next page

-------
. continued from previous page
Study Citation: Yoshioka, Y.,Ose, Y.,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type:	Other; Aquatic; Invertebrates
Hero ID:	3617749
Domain Metric
Rating^
MWF*
Score
Comments^
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
Low
Medium
x 2
x 1
6
2
Data for exposure related findings not reported for
each study group
Authors did report unexpected outcomes and ex-
plained some of them, including the planarian num-
bers being very different than the other two species.
Overall Quality Determination"'"
Low

2.5

Extracted
Yes




*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Yoshioka, Y.,Ose, Y. ,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 3617749
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High
Low
Low
X 2 2 Chemical identified by name: Trichloroethylene.
X 1 3 Source of TCE was not reported, but it was noted
that analytical grade TCE was used.
X 1 3 Purity was not reported.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
Unacceptable x 2
N/A
Low
x 1
N/A
3
The study does not mention a control anywhere.
The study refers to a blank for Dugesia japonica
(planarian) but doesn't say what's in the blank, and
doesn't mention a blank for O. latipes (red killifish).
No control reported
It's not reported whether animals were randomly al-
located.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Medium
tion
Metric 8:
Metric 9:
Consistency of Exposure Administration
Low
Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency Low
Metric 11: Number of Exposure Groups/Spacing of Ex- Low
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
X 2 4 Test was completed in a closed container (sealed
with an electrode), but there were some uncertain-
ties about how much air space there was in the flask.
X 1 3 Exposure methods were not reported for each study
group.
X 2 6 It was not reported whether nominal or measured
cone were used.
X 1 3 Exposure occurred over 48 hours, and it sounds like
a static test but it is not clear. OECD recommends
96 hours for fish acute tests.
X 1 3 For TCE, it is unclear how many exposure groups
were used for the LC50 determination. (For the oxy-
gen uptake it looks like 5 exposure groups according
to figure 2 but that was a different test. )
X 1 1 Substance was tested well below solubility.
Domain 4: Test Organism
Continued on next page

-------
. continued from previous page
Study Citation: Yoshioka, Y.,Ose, Y. ,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 3617749
Domain Metric Rating^ MWF* Score Comments^
Metric 13: Test Organism Characteristics
Medium
x 2
Cn
Metric 14: Acclimitization and Pretreatment Conditions Medium
Metric 15: Number of Organisms and Replicates per High
Group
Metric 16: Adequacy of Test Conditions Medium
x 1
x 1
x 1
Minor uncertainties about the quality of the test or-
ganisms given they were collected from the market.
Study reports, "Orizias latipes (ca. 3 cm, 0.3 g) was
obtained from the market and acclimated for at least
1 week in dechlorinated water at 20"C (total hard-
ness was about 80 mg/liter). LC50 was determined
by exposing 10 O. latipes to 2 liters of a chemical
solution at 20 " I "C for 48 hr with the cycle 8 hr
dark and 16 hr light. The oxygen uptake rate was
determined by putting 10 0. latipes in an Erlen-
meyer flask (3-liter) filled with test solution which
was saturated with air, and the flask was sealed with
an electrode. Then it was left without aeration at
20" C for 4 hr. The concentration of dissolved oxy-
gen (DO) was measured by a DO meter (Denkika-
gakukeiki type 3) every 30 min. As the oxygen was
not supplied by aeration during the test, the result
was accepted only when DO concentration was over
3 mg/liter at the end of the test in order to avoid the
influence of the lack of DO. IfDO decreased to under
3 mg/liter, the test was carried out anew with 5 O.
latipes. After the test, the wet weight of O. latipes
was measured in order to calculate the oxygen up-
take rate per wet weight."
Fish were acclimatized for 1 week and OECD rec-
ommends 12 days before they are used for testing.
10 organisms per exposure group. OECD recom-
mends at least 7
10 fish in 2 liters of water which is a little more than
what OECD would recommend. At 0.3 g each and
10 fish per container, it should be a 3 liter flask.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
Low
X 2 2 Derived an LC50
X 1 3 Details of outcome assessment were not reported.
Domain 6: Confounding / Variable Control
Continued on next page . ,

Procedures

a comparison of environmental conditions or other

non-treatment-related factors across study groups,

and the omitted information is likely to have a sub-

stantial impact on study results.
Metric 20:
Outcomes Unrelated to Exposure
Low
X
1
3
Data on health and attrition were not reported for

each study group.
Domain 7: Data Presentation and Analysis

Metric 21:
Statistical Methods
Medium
X
1
2
Methods not described clearly.
Metric 22:
Reporting of Data
Low
X
2
6
Data for exposure related findings not reported for

each study group.
Metric 23:
Explanation of Unexpected Outcomes
High
X
1
1
No unexplained outcomes for the killifish.
Overall Quality Determination"'"
Unacceptable

4.0

Extracted

** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
* MWF = Metric Weighting Factor
r High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
* The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Yoshioka, Y.,Ose, Y. ,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 3617749
Domain Metric Rating^ MWF* Score Comments^
Domain 1: Test Substance
Metric 1:
Metric 2:
Test Substance Identity
Test Substance Source
Metric 3: Test Substance Purity
High
Low
Low
X 2 2 Chemical identified by name: Trichloroethylene.
X 1 3 Source of TCE was not reported, but it was noted
that analytical grade TCE was used.
X 1 3 Purity is not reported.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
Unacceptable x 2
N/A
Low
x 1
N/A
3
The study does not mention a control anywhere.
The study refers to a blank for Dugesia japonica
(planarian) but doesn't say what's in the blank, and
doesn't mention a blank for O. latipes (red killifish).
No control reported
It's not reported whether animals were randomly al-
located.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
Consistency of Exposure Administration Low
Metric 8:
Metric 9:
Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency Low
x 2
x 1
x 2
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Unacceptable x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit
High
x 1
6 It is not reported whether the container was closed
or open, and TCE is a volatile chemical.
3 Exposure methods were not reported for each study
group.
6 It was not reported whether nominal or measured
cone were used.
3 Exposure occurred over 4 hours, and OECD 202 rec-
ommends 48 hours for invertebrate acute tests.
4 For TCE, it is unclear how many exposure groups
were used for the LC50 determination.
1 Substance was tested below solubility.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Low
x 2
Test species is a saltwater invertebrate, and were
used at 5 days old, but the source of the species is
not reported.
Continued on next page

-------
. . . continued from previous page
Study Citation: Yoshioka, Y.,Ose, Y. ,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type: Acute (0-96 hour); Aquatic; Invertebrates
Hero ID: 3617749
Domain Metric Rating^ MWF* Score Comments^
Metric 14: Acclimitization and Pretreatment Conditions Low x 1
Metric 15: Number of Organisms and Replicates per Low x 1
Group
Metric 16: Adequacy of Test Conditions Medium x 1
3 Study did not report acclimating water fleas.
3 Ten organisms per exposure group. For freshwater
invertebrates, OECD 202 recommends at least 20.
2 "Ten M. macrocopa in 100 ml of test solution were
put in a 250-ml vial vessel at 20 " 1"C and the sur-
vivors were counted after 3 hr in order to determine
LC50."
Domain 5: Outcome Assessment

Metric 17: Outcome Assessment Methodology
High
X
2
2
Derived an LC50
Metric 18: Consistency of Outcome Assessment
Low
X
1
3
Details of outcome assessment were not reported.
Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test Design and
Low
X
2
6
The study did not provide enough information to
Procedures

allow a comparison of environmental conditions or

other non treatment related factors across study

groups.
Metric 20: Outcomes Unrelated to Exposure
Low
X
1
3
Data on health and attrition were not reported for

each study group.
Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Medium
X
1
2
Methods not described clearly.
Metric 22: Reporting of Data
Low
X
2
6
Data for exposure related findings not reported for

each study group.
Metric 23: Explanation of Unexpected Outcomes
High
X
1
1
No unexplained outcomes for the water flea.
Overall Quality Determination"'"
Unacceptable

4.0

Extracted
No

Continued on next page

-------
. continued from previous page
Study Citation: Yoshioka, Y.,Ose, Y.,Sato, T.. 1986. Correlation of the Five Test Methods to Assess Chemical Toxicity and Relation to Physical
Properties. 12:15-21
Data Type:	Acute (0-96 hour); Aquatic; Invertebrates
Hero ID:	3617749
Domain	Metric	Rating^	MWF* Score	Comments^
** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, two of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Tsai, K. P.,Chen, C. Y.. 2007. An Algal Toxicity Database of Organic Toxicants Derived by a Closed-System Technique. Environmental
Toxicology and Chemistry 26:1931-1939
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 3617867
Domain
Metric
Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance
Metric 1:
Metric 2:
Metric 3:
Test Substance Identity
Test Substance Source
Test Substance Purity
High x 2 2 Test substance was identified by name. Physical
chemical characteristics such as MW, LogP, HLC,
and solubility were provided for the test material.
Low X 1 3 The source was not provided.
Medium X 1 2 Purity was not provided. Authors described the
chemical purity as "reagent grade."
Domain 2: Test Design
Metric 4: Negative Controls
Medium x 2
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
Low
Low
x 1
x 1
Authors referred to a control when discussing how
they calculated their EC50 value, but additional de-
tails were not reported. The authors indicated that
the details of the test setup can be found at the fol-
lowing source: Lin JH, Kao WC, Tsai KP, Chen CY.
2005. A novel algal toxicity testing technique for
assessing the toxicity of both metallic and organic
toxicants. Water Res 39:1869" 1877.This source in-
dicates that inclusion of a negative control is a part
of the testing procedure.
Negative Control response was not specifically re-
ported in the study, but was incorporated into the
calculation of the percent inhibition.
Researchers did not report how organisms were al-
located to study groups
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High x 2
tion
Metric 8: Consistency of Exposure Administration High x 1
Metric 9: Measurement of Test Substance Concentra- Medium x 2
tion
2 The goal of the study authors was specifically to de-
termine the optimal testing conditions for algae.
1 The goal of the study authors was specifically to de-
termine the optimal testing conditions for algae by
providing a more consistent exposure regime.
4 Test concentrations were reported in terms of nom-
inal concentrations, but analytical confirmation of
the test concentrations was performed at the begin-
ning and end of the test by HPLC. This was intended
to quantify any potential degradation.
Continued on next page

-------
. continued from previous page
Study Citation: Tsai, K. P.,Chen, C. Y.. 2007. An Algal Toxicity Database of Organic Toxicants Derived by a Closed-System Technique. Environmental
Toxicology and Chemistry 26:1931-1939
Data Type: Acute (0-96 hour); Aquatic; Plants
Hero ID: 3617867
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 10: Exposure Duration and Frequency
Medium x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- Low x 1
posure Levels
Metric 12: Testing at or Below Solubility Limit Low x 1
Authors reported, "All tests were conducted in trip-
licate, with a test duration of 48 h. The population
density of the algae was determined using an elec-
tronicparticle counter" 48 hours is acceptable, but
72 hours is recommended in OECD 201.
The study report indicated that both a range finding
and definitive test were conducted but did not report
the test concentrations.
It is unclear what test cone were, but the solubility
of TCE is very high (999-1472 mg/1), and the EC50
determined was relatively low in comparison 26.24
mg/l)
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
Metric 14: Acclimitization and Pretreatment Conditions High
Metric 15: Number of Organisms and Replicates per High
Group
Metric 16: Adequacy of Test Conditions
High X 2 2 Green algae (Pseudokirchneriella subcapitata)
which is a recommended test species in OECD
Guideline 201.
X 1 1 Supplemental methodology indicates that algae are
incubated until a steady-state is reached
X 1 1 3 Replications were used and cell density was and
the initialinoculated cell density was 15,000 cells/
ml. which is in the OECD 201 recommended range
of 5*103 -104 for this species.
High X 1 1 Temperature and light intensity were kept at 24
1C and 65 Em2sl (10 percent),respectively. Algal
growth medium [22] with no ethylenediaminetetra-
acetic acid content was used for toxicity testing. The
initial pH for the growth medium was 7.5, and the
initial inoculated cell density was 15,000 cells/ml.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High x 2
High x 1
Authors proved that closed-system algae testing is
more conservative than batch testing. Were able to
derive an EC50.
Outcomes were assessed consistently across study
groups using the same protocol.
Domain 6: Confounding / Variable Control
Continued on next page

-------
. continued from previous page
Study Citation: Tsai, K. P.,Chen, C. Y.. 2007. An Algal Toxicity Database of Organic Toxicants Derived by a Closed-System Technique. Environmental
Toxicology and Chemistry 26:1931-1939
Data Type:	Acute (0-96 hour); Aquatic; Plants
Hero ID:	3617867
Domain
Metric
Rating^
MWF*
Score
Comments^
Metric 19:
Confounding Variables in Test Design and
Procedures
High
X
2
2
The purpose of the experiment was to determine
whether algae are more sensitive to volatile chem-
icals when tested in closed-systems than in batch
testing. They confirmed that closed system tests
were more sensitive for algae relative to batch tests.
Metric 20:
Outcomes Unrelated to Exposure
Medium
X
1
2
Data on attrition was not reported for each study
group, but is unlikely to have a substantial impact
on results.
Domain 7: Data Presentation and Analysis





Metric 21:
Statistical Methods
High
X
1
1
The EC50 was determined using probit analysis.
Metric 22:
Reporting of Data
Medium
X
2
4
Results did not include effects at each concentration
level; however, this is unlikely to substantially influ-
ence outcomes.
Metric 23:
Explanation of Unexpected Outcomes
High
X
1
1
There were no unexpected outcomes.
Overall Quality Determination"'"
High


1.6

Extracted

Yes





*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / J] . MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Schell, J. D. J.. 1987. Interactions of Halogenated Hydrocarbon Mixtures in the Embryo of the Japanese Medaka (Oryzias latipes).
Data Type: Other; Aquatic; Fish
Hero ID: 3625489
Domain Metric Rating^ MWF* Score Comments"^
Domain 1: Test Substance
Metric 1:
Test Substance Identity
High
X
2
2
Chemical is identified by name
Metric 2:
Test Substance Source
High
X
1
1
Purchased from Fisher scientific
Metric 3:
Test Substance Purity
High
X
1
1
Study reports TCE is certified ACS which I believe
means greater than 99.5 percent.
Domain 2: Test Design
Metric 4:
Metric 5:
Metric 6:
Negative Controls
Negative Control Response
Randomized Allocation
Medium x 2
High
Low
x 1
x 1
Clean rearing solution was used as a control, with
only minor uncertainties about formulation.
Did not report whether allocation to study groups
was random.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- High
tion
Metric 8: Consistency of Exposure Administration High
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency
High
x 2
x 1
x 2
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- High x 1
posure Levels
Volatility of chemical was taken into account in the
study design. "Since the chemicals of interest are
volatile, exchanging solutions on a daily basis in-
sured a more consistent exposure level."
Nominal concentrations were used and were not
measured. TCE is volatile. Rate of loss was deter-
mined for carbon tet and chloroform, but not TCE.
10 day exposure duration (96 hour preferred for
acute, and 28 day preferred for chronic, so this ex-
posure period is sub-chronic) which is recommended
for fish early life stage OECD. Static renewal method
used. Fresh test solutions were exchanged in the
vials each day for 10 consecutive days.
After a range finding test, TCE concentrations were
54, 59, 65, 72, 79, and 87 mg/1 (log-dose intervals).
6 concentrations used plus control; OECD recom-
mends at least 5 for fish early life stage.
Metric 12: Testing at or Below Solubility Limit
High
x 1
Domain 4: Test Organism
Continued on next page

-------
. . . continued from previous page
Study Citation: Schell, J. D. J.. 1987. Interactions of Halogenated Hydrocarbon Mixtures in the Embryo of the Japanese Medaka (Oryzias latipes).
Data Type: Other; Aquatic; Fish
Hero ID: 3625489
Domain Metric Ratingt MWF* Score Commentstt
Metric 13: Test Organism Characteristics High x 2
Metric 14: Acclimitization and Pretreatment Conditions High x 1
Metric 15: Number of Organisms and Replicates per Medium x 1
Group
Metric 16: Adequacy of Test Conditions High x 1
rice fish (Japanese Medaka)
"Adult male and female medaka used for breeding
purposes were obtained from the Carolina Biological
Supply Company (Burlington, N.C.). Upon arrival
in the laboratory, fish were quarantined from exist-
ing laboratory stocks and treated for 24 h with a
mixture of malachite green (0.05 mg/L) and formalin
(20 mg/L) to protect against infection resulting from
the stress associated with handling and transporta-
tion during shipment. Following treatment, the fish
were partitioned into breeding stocks at a ratio of 3
males:2 females with an optimum number of 60 fish
per JO gallon aquarium."
10 embryos per dose group, which is acceptable, but
no mention of how many replicates.
Domain 5: Outcome Assessment
Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome Assessment
High
High
x 2
x 1
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2
Procedures
Metric 20: Outcomes Unrelated to Exposure
Medium x 1
2 No reported differences among the study groups in
environmental conditions or other factors that could
be influencing the outcome assessment.
2 Data on attrition was reported in each exposure
group. Other health outcomes were not reported,
but I consider these only minor uncertainties.
Domain 7: Data Presentation and Analysis
Metric 21: Statistical Methods
Metric 22: Reporting of Data
High x 1
Medium x 2
Metric 23: Explanation of Unexpected Outcomes
High
x 1
Most but not all outcomes were reported, only mi-
nor uncertainties.
Unexplained outcomes were reported and discussed
for the other chemicals in this paper. No unex-
plained outcomes were noted for TCE.
Continued on next page

-------
. continued from previous page
Study Citation:	Schell, J. D. J.. 1987. Interactions of Halogenated Hydrocarbon Mixtures in the Embryo of the Japanese Medaka (Oryzias latipes).
Data Type:	Other; Aquatic; Fish
Hero ID:	3625489
Domain	Metric	Ratingt MWF* Score	Commentstt
Overall Quality Determination"'"	High	1.4
Extracted	Yes
* MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
^ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
(Metric Score; X MWF;) / J] . MWF,
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
tt Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 3665276
Domain
Metric
Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance
Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical identified by name, trichloroethylene, and
by CAS no. 79-01-6.
Medium X 1 2 Unclear where TCE specifically was from. Study
reports, "Almost all test chemicals were purchased
from Aldrich Chemical (Milwaukee, WI, USA), with
only a few supplied by other commercial companies.
Regardless of source, all chemicals were of a high
level of purity (i.e., 95 percent or greater) and were
not repurified before testing."
Medium X 1 2 At least 95 percent purity but unclear what the pu-
rity of TCE is specifically.
Domain 2: Test Design
Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
Medium X 2 4 Controls were used, and assume it was just a lake
water control given the study says "no solvent car-
riers were used and all toxicants were tested at con-
centrations below their reported solubility."
Low X 1 3 No response was reported for control
Medium X 1 2 Concentrations were distributed randomly between
the two diluters, and for the acute test it was not
mentioned whether animals were distributed ran-
domly amongst control groups, but study did report
that procedures followed standard American Society
for Testing and Materials experimental procedure
and as described previously. For the chronic exper-
iment, embryos were added five at a time into 56
different egg cups which then randomly were added
to the treatment and control chambers so that each
tank received one cup.
Domain 3: Exposure Characterization
Continued on next page

-------
. continued from previous page
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 3665276
Domain
Metric
Rating"!" MWF* Score
Comments'^
Metric 7: Experimental System/Test Media Prepara- Medium x 2
tion
Metric 8: Consistency of Exposure Administration High x 1
CJ5
CJ5
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency
High
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- High x 1
posure Levels
Flow through test was conducted, which is better for
volatile chemicals like TCE. It was not specifically
noted whether the containers were covered and how
much headspace was allowed, but the flow through
nature of the test should help with keeping TCE
cone consistent. Also measured concentrations were
taken.
Continuous flow through with a check for any spikes.
See following description "To verify the accuracy of
the method of analyses, known amounts of toxicant
were added to control water so that toxicant con-
centrations could be corrected routinely for spiked
water recoveries. At least one spike sample was pre-
pared for each sample set. A mean percent recov-
ery of 90 to 110 percent usually was observed. All
statistical/model analyses were based on measured
concentrations."
"Methods of chemical analysis included high-
pressure liquid chromatographywith ultraviolet or
diode array/fluorescence detectors, and gasliquid
chromatography with flame ionization or electron
capture detectors. Samples of all test solutions were
taken from the test chambers and toxicant mixing
trays and analyzed according to a monitoring pro-
gram that characterized the toxicant exposures. Du-
plicate measurements were made routinely witheach
analytical series to define the reproducibility (pre-
cision) of the measurements. Percentage duplicate
agreements usually were greater than 90 percent. To
verify the accuracy of the method of analyses, known
amounts of toxicant were added to control water so
that toxicant concentrations could be corrected rou-
tinely for spiked water recoveries. At least one spike
samplewas prepared for each sample set. A mean
percent recovery of 90 to 110 percent usually was
observed. All statistical/model analyses were based
on measured concentrations."
Continuous exposure for 96 hours which is one rec-
ommended option for the fish acute guidelines from
OECD
Authors used 5 exposure groups, and at least 5 is
recommended by OECD 203 test guidelines.
Continued on next page

-------
. . . continued from previous page
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type: Acute (0-96 hour); Aquatic; Fish
Hero ID: 3665276
Domain Metric Rating^ MWF* Score Comments^
Metric 12: Testing at or Below Solubility Limit High X 1 1 TCE concentrations tested were below the solubility
of TCE.
Domain 4: Test Organism
Metric 13: Test Organism Characteristics
High
Metric 14: Acclimitization and Pretreatment Conditions High
Metric 15: Number of Organisms and Replicates per Low
Group
CJ5
Metric 16: Adequacy of Test Conditions
X 2 2 Fathead minnow are an acceptable test species ac-
cording to OECD 203 test guidelines.
X 1 1 Acclimatization took place, and fish were cultured in
the lab with appropriate temp for fathead minnow,
and ph of 7.8 was within the range of OECD 203 test
guidelines. Water hardness was also in the range of
OECD 203 test guidelines.
X 1 3 7 fish are recommended per test conc. It is unclear
how many fish were used in the acute test from the
description in this paper, "The acute toxicity tests
to determine 96-h LC50 values were conducted ac-
cording to a standard American Society for Test-
ing and Materials experimental procedure [28] and
as describedpreviously [27]. By exposing groups of
test organisms to a range of concentrations in con-
tinuous flow-through systems for separate, binary, or
equitoxic multiple chemical mixtures, the percentage
mortality in 96-h was determined for numerous tests.
Toxicity tests were conducted initially withindivid-
ual toxicants and expanded subsequently to numer-
ous binary test solutions and multiple toxicant mix-
tures. All isoboles of acute joint toxic action were
determined for the 96-h LC50 response level. A plot
of 96-h LC50 values and 95 percent confidence limits
was constructed as test data allowed for eachbinary
mixture.
Medium X 1 2 Housing for the most part are acceptable, but test
water was obtained from lake superior and under-
went sand filtration. There could be other contam-
inants in the water, however because controls were
used, and chemicals measurement and analysis took
place, only minor uncertainties regarding this detail.
The appropriate temp for fathead minnow was used,
and the ph of 7.8 within the range of OECD 203 test
guidelines. Water hardness was also in the range of
OECD 203 test guidelines.
Continued on next page

Hero ID: 3665276

Domain Metric Rating^
MWF*
Score
Comments^
Domain 5: Outcome Assessment

Metric 17: Outcome Assessment Methodology High
x 2
2
LD50 was measured for acute toxicity. EC50 and

EC20 were measured and determined for growth for

chronic toxicity.
Metric 18: Consistency of Outcome Assessment High
x 1
1
Outcome assessment was consistent between study

groups.
Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test Design and High
x 2
2
No reported differences in study groups from envi-
Procedures

ronmental conditions of other factors that could in-

fluence the outcome of assessment.
Metric 20: Outcomes Unrelated to Exposure Medium
x 1
2
Health outcomes were not reported for each study

group
Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High
x 1
1
In the 96-h toxicity tests, mortalities were recorded

daily, and an estimate of the LC50 and its 95 percent

confidence limits was determined by the trimmed

Spearman-Karber method [32]. The 96-h LC50s

from the binary mixtures were used to construct

isobole diagrams of joint toxic action for the 50 per-

cent3120 Environ. Toxicol. Chem. 24, 2005 S.J.

Broderius et al. response level. The data on com-

bined effects of binary mixtures were analyzed by

comparison of the observed results with that ex-

pected for the concentration- or response-addition

models.
Metric 22: Reporting of Data Medium
x 2
4
Data were not reported for outcomes with negative

findings, but tis is unlikely to have substantial im-

pact on results.
Metric 23: Explanation of Unexpected Outcomes High
x 1
1
There were no unexplained outcomes.
Overall Quality Determination"'" High

1.5

Extracted Yes
Continued on next page

-------
. continued from previous page
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister> D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type:	Acute (0-96 hour); Aquatic; Fish
Hero ID:	3665276
Domain	Metric	Rating^ MWF* Score	Comments^
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
^ (Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.
^ Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type: Chronic (>21 days); Aquatic; Fish
Hero ID: 3665276
Domain
Metric
Rating"!" MWF* Score
Comments^ f
Domain 1: Test Substance
Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity
High X 2 2 Chemical identified by name, trichloroethylene, and
by CAS no. 79-01-6.
Medium X 1 2 Unclear where TCE specifically was from. Study
reports, "Almost all test chemicals were purchased
from Aldrich Chemical (Milwaukee, WI, USA), with
only a few supplied by other commercial companies.
Regardless of source, all chemicals were of a high
level of purity (i.e., 95 percent or greater) and were
not repurified before testing."
Medium X 1 2 At least 95 percent purity but unclear what the pu-
rity of TCE is specifically.
Domain 2: Test Design
Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation
Medium X 2 4 Controls were used, and assume it was just a lake
water control given the study says "no solvent car-
riers were used and all toxicants were tested at con-
centrations below their reported solubility."
Low X 1 3 No response was reported for control.
High X 1 1 Concentrations were distributed randomly between
the two diluters, and for the acute test it was not
mentioned whether animals were distributed ran-
domly amongst control groups, but study did report
that procedures followed standard American Society
for Testing and Materials experimental procedure
and as described previously. For the chronic test,
embryos were added five at a time into 56 different
egg cups which then randomly were added to the
treatment and control chambers so that each tank
received one cup.
Domain 3: Exposure Characterization
Continued on next page

-------
. continued from previous page
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type: Chronic (>21 days); Aquatic; Fish
Hero ID: 3665276
Domain Metric Rating^ MWF* Score Comments^
Metric 7: Experimental System/Test Media Prepara- Medium x 2
tion
Metric 8: Consistency of Exposure Administration High x 1
Metric 9: Measurement of Test Substance Concentra- High x 2
tion
Metric 10: Exposure Duration and Frequency
High
x 1
Metric 11: Number of Exposure Groups/Spacing of Ex- High x 1
posure Levels
Flow through test was conducted, which is better for
volatile chemicals like TCE. It was not specifically
noted whether the containers were covered and how
much headspace was allowed, but the flow through
nature of the test should help with keeping TCE
cone consistent. Also measured concentrations were
taken.
Continuous flow through with a check for any spikes.
See following description "To verify the accuracy of
the method of analyses, known amounts of toxicant
were added to control water so that toxicant con-
centrations could be corrected routinely for spiked
water recoveries. At least one spike sample was pre-
pared for each sample set. A mean percent recov-
ery of 90 to 110 percent usually was observed. All
statistical/model analyses were based on measured
concentrations."
"Methods of chemical analysis included high-
pressure liquid chromatographywith ultraviolet or
diode array/fluorescence detectors, and gasliquid
chromatography with flame ionization or electron
capture detectors. Samples of all test solutions were
taken from the test chambers and toxicant mixing
trays and analyzed according to a monitoring pro-
gram that characterized the toxicant exposures. Du-
plicate measurements were made routinely witheach
analytical series to define the reproducibility (pre-
cision) of the measurements. Percentage duplicate
agreements usually were greater than 90 percent. To
verify the accuracy of the method of analyses, known
amounts of toxicant were added to control water so
that toxicant concentrations could be corrected rou-
tinely for spiked water recoveries. At least one spike
samplewas prepared for each sample set. A mean
percent recovery of 90 to 110 percent usually was
observed. All statistical/model analyses were based
on measured concentrations."
Continuous exposure for 32 days which is one rec-
ommended option for the fish acute guidelines from
OECD 210 for fathead minnows
5 exposure groups, at least 5 is recommended by
OECD 210 guidelines
Continued on next page . . .

Metric 13:
Test Organism Characteristics
High
X
2
2
Fathead minnow are an acceptable test species ac-
cording to OECD 210 guidelines.
Metric 14:
Acclimitization and Pretreatment Conditions
High
X
1
1
Acclimatization took place, and fish were cultured in
the lab with appropriate temp for fathead minnow,
and ph of 7.8 was within the range of OECD 210
guidelines. Water hardness was also in the range of
OECD 210 guidelines.
Metric 15:
Number of Organisms and Replicates per
High
X
1
1
80 embryos are recommended by OECD 210. This

Group

study did "... 40 embryos were added to each of

the replicated treatment levels and controls." There
were at least two replicates.
Metric 16:
Adequacy of Test Conditions
Medium
X
1
2
Housing for the most part are acceptable, but test
water was obtained from lake superior and under-
went sand filtration. There could be other contam-
inants in the water, however because controls were
used, and chemicals measurement and analysis took
place, only minor uncertainties regarding this detail.
The appropriate temp for fathead minnow was used,
and the ph of 7.8 within the range of OECD 210
guidelines. Water hardness was also in the range of
OECD 210 guidelines.
Domain 5: Outcome Assessment

Metric 17:
Outcome Assessment Methodology
High
X
2
2
EC50 and EC20 were measured and determined for
growth for chronic toxicity.
Metric 18:
Consistency of Outcome Assessment
High
X
1
1
Outcome assessment was consistent between study
groups.
Domain 6: Confounding / Variable Control
Metric 19: Confounding Variables in Test Design and High x 2 2 No reported differences in study groups from envi-
Procedures ronmental conditions of other factors that could in-
fluence the outcome of assessment.
Continued on next page

-------
. . . continued from previous page
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type:	Chronic (>21 days); Aquatic; Fish
Hero ID:	3665276
Domain	Metric	Rating^ MWF* Score	Comments^
Metric 20: Outcomes Unrelated to Exposure	Medium x 1	2 Health outcomes were not reported for each study
group, but this is unlikely to have substantial im-
pacts on results.
Domain 7: Data Presentation and Analysis
Continued on next page

-------
. continued from previous page
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister, D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type: Chronic (>21 days); Aquatic; Fish
Hero ID: 3665276
Domain Metric Rating^ MWF* Score Comments^
Metric 21: Statistical Methods Medium x 1 2 Only minor uncertainties in the method used to de-
rive the EC values. The study says, "Growth (fi-
nal wet wt) was selected as the graded sublethal re-
sponse for the ELS studies because it represents an
integrated activity of the whole organism. For the
ELS tests, growth was defined as the wet weight of
a fathead minnow at the end of a 32-d experiment.
The concentrations that reduce the growth by 50
or 20 percent compared to the controls (i.e., EC50
and EC20 values) were calculated from a toxicity re-
lationship analysis program developed by Erickson
[34]. With this program, the effects of toxicants on
wet weight of fathead minnows from early life-stage
toxicity tests were analyzed by weighted leastsquares
nonlinear regression analysis of the dependant vari-
able (i.e., wet wt) versus the independent variable
x (i.e., toxicant concentration). Three mathemati-
cal forms of the regression curves that were used are
the logistic equation, piecewiselinear, and piecewise-
tailed regression. The regression using the logistic
equation produces a sigmoid-shaped curve with in-
finite tails, a form similar to probit analysis. Piece-
wise linear regression produces an effects curve in
which the effects variable is constant at a control
value below a threshold exposure (ECO) and declines
linearly above this threshold until it reaches a value
of zero at some finite exposure (EC100). The piece-
wise tailed regression provides a curve that is sig-
moid shaped like the logistic equation, but has fi-
nite tails and, thus, a finite ECO and EC100 like
the piecewise linear regression. The wet weight of
fathead minnows that survived from each individual
ELS toxicity test was analyzed by these three regres-
sion models. The model that provided the best vi-
sual and statistical fit to the experimental data was
used to define the test results. Usually that was the
piecewise linear or piecewise tailed regression model.
A logarithm exposure variable transformation was
used in all regression analyses to improve error ho-
mogeneity in regression analyses. The analysis pro-
vides final estimates for the regression parameters,
their standard errors, and 95 percent confidence lim-
its of the best estimate of mean effects. These EC50
estimates and confidence limits then were illustrated
graphically."
Continued on next page . . .

-------
. continued from previous page
Study Citation: Broderius, S. J.,Kahl, M. D.,Elonen, G. E.,Hammermeister> D. E.,Hoglund, M. D.. 2005. A Comparison of the Lethal and Sublethal
Toxicity of Organic Chemical Mixtures to the Fathead Minnow (Pimphales promelas). 24:3117-3127
Data Type:	Chronic (>21 days); Aquatic; Fish
Hero ID:	3665276
Domain Metric
Rating^
MWF*
Score
Comments^
Metric 22: Reporting of Data
Metric 23: Explanation of Unexpected Outcomes
Medium
High
x 2
x 1
4
1
Data were not reported for outcomes with negative
findings, but this is unlikely to have a substantial
impact on results.
No unexpected outcomes reported.
Overall Quality Determination"'"
High

1.4

Extracted
Yes




*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
Overall rating =
(Metric Score; x MWF;) / ^ MWFj
if any metric is Unacceptable
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

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Study Citation: Leblanc, G. A.,Surprenant, D. C.. 1980. The Chronic Toxicity of 8 of the 65 Priority Pollutants to the Water Flea (Daphnia magna).
Data Type: Chronic (>21 days); Aquatic; Invertebrates
Hero ID: 3683110
Domain Metric Rating^ MWF* Score Comments"^
Domain 1: Test Substance
Metric 1:
Metric 2:
Metric 3:
Test Substance Identity
Test Substance Source
Test Substance Purity
High
High
High
X 2 2 Trichloroethylene identified by name
X 1 1 Aldrich chemical co
X 1 1 98 percent purity - lot number 041557
Domain 2: Test Design
Metric 4:
Metric 5:
Negative Controls
Negative Control Response
Medium x 2
Unacceptable x 1
Metric 6: Randomized Allocation
High
x 1
4 a solvent and negative control were used, but it did
not say what the solvent was.
4 Biological response of negative control and solvent
control was reported, but there was only 50 percent
and 57 percent survival of negative and solvent con-
trol. There fore the results of this study are unac-
ceptable.
Domain 3: Exposure Characterization
Metric 7: Experimental System/Test Media Prepara- Low
tion
Metric 8: Consistency of Exposure Administration High
Metric 9: Measurement of Test Substance Concentra- Low
tion
Metric 10: Exposure Duration and Frequency
Low
Metric 11: Number of Exposure Groups/Spacing of Ex- High
posure Levels
x 2
x 1
x 2
x 1
x 1
6 test was conducted in a closed system. Dissolved O,
temperature, and pH were measured throughout the
experiment. However despite efforts to produce a
closed system, volatilization of other volatile chem-
icals did occur. Because TCE is a volatile chemical
other studies have shown rapid volatilization of the
chemical from test media within 24 and 48 hours.
1 No differences were reported.
6 While it was attempted to measure TE concentra-
tions authors were unable to reliably measure the
chemical and therefore relied on nominal concentra-
tions for this chronic test.
3 duration is good at 21 days which is recommended
in OECD 211, however it is unclear how often the
test cone is renewed, which could have substantial
results on the experiment given that this chemical is
highly volatile.
1 Daphnids were exposed to five concentrations of each
compound, and concentrations were determined by
doing an acute range finding test.
Continued on next page

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. . . continued from previous page
Study Citation: Leblanc, G.
A.,Surprenant, D. C.. 1980. The Chronic Toxicity of 8 of the 65 Priority Pollutants to the Water Flea (Daphnia magna).
Data Type: Chronic (>21 days); Aquatic; Invertebrates

Hero ID: 3683110

Domain
Metric
Haling1
MWF*
Score
Commentstt
Metric 12:
Testing at or Below Solubility Limit
High
X 1
1
cone were below solubility level.
Domain 4: Test Organism

Metric 13:
Test Organism Characteristics
High
x 2
2
daphnia magna
Metric 14:
Acclimitization and Pretreatment Conditions
N/A

N/A

Metric 15:
Number of Organisms and Replicates per
High
x 1
1
10 daphnia per container and done in triplicate

Group

Metric 16:
Adequacy of Test Conditions
Unacceptable
x 1
4
it appears the closed nature and no cleaning of the

system during the experiment produced bacteria and

algae growth that affected the health of the daphnia

in both controls between day 15 and 21.
Domain 5: Outcome Assessment

Metric 17:
Outcome Assessment Methodology
High
x 2
2

Metric 18:
Consistency of Outcome Assessment
High
x 1
1

Domain 6: Confounding / Variable Control

Metric 19:
Confounding Variables in Test Design and
High
x 2
2
no reported differences among the study groups in

Procedures

environmental conditions or other factors that could

influence the outcome of the assessment.
Metric 20:
Outcomes Unrelated to Exposure
Unacceptable
x 1
4
controls had very low survival rate of 50 percent and

57 percent
Domain 7: Data Presentation and Analysis

Metric 21:
Statistical Methods
N/A

N/A
not applicable because a chronic toxicity endpoint

was not determined
Metric 22:
Reporting of Data
High
x 2
2
results were reported for each exposure group
Metric 23:
Explanation of Unexpected Outcomes
High
x 1
1
unexpected outcomes were discussed, e.g. the high

rate of mortality in controls and the growth of bac-

teria and algae.
Overall Quality Determination"'"
Unacceptable

4.0

Extracted

Continued on next page

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. continued from previous page
Study Citation:	Leblanc, G. A.,Surprenant, D. C.. 1980. The Chronic Toxicity of 8 of the 65 Priority Pollutants to the Water Flea (Daphnia magna).
Data Type:	Chronic (>21 days); Aquatic; Invertebrates
Hero ID:	3683110
Domain	Metric	Rating^ MWF* Score	Comments'^
** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, three of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the
score is presented solely to increase transparency.
*	MWF = Metric Weighting Factor
t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.
*	The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.
if any metric is Unacceptable
Overall rating =
J]. (Metric Score; x MWF;) / J] . MWFj
(round to the nearest tenth) otherwise
where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft Metrics that are rated 'High' met the criteria for high confidence as expected for this type of study, and may not require additional comments.

-------