Risks of Atrazine Use to
Federally Listed Endangered
Barton Springs Salamanders
(Eurycea sosorum)
Appendix A: Ecological Effects
Characterization
August 22,2006
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Appendix A. Ecological Effects Characterization
A. 1 Toxicity to Birds / Reptiles 2
A. 1.1 Birds: Acute Oral Studies 2
A. 1.2 Birds: Subacute Dietary Studies 3
A. 1.3 Birds: Chronic Studies 5
A. 1.4 Birds/Reptiles: Open Literature 6
A. 1.4a Birds: New Open Literature Data 6
A. 1,4b Reptiles: Open Literature Data from 2003 IRED 7
A. 1.4c Reptiles: New Open Literature Data (2006 Literature Review) 7
A.2 Toxicity to Freshwater Animals 8
A.2.1 Freshwater Fish and Amphibia, Acute 8
A.2.2 Freshwater Fish, Chronic 11
A.2.3 Freshwater Fish/Amphibians, Open Literature Data on Mortality/Survivorship 13
A.2.4 Sublethal Effects, Freshwater Fish and Amphibians (Open Literature) 15
A.2.4a Sublethal Effects: Freshwater Fish (2003 IRED Data): 16
A.2.4b Sublethal Effects: Freshwater Fish (New (2006) Open Literature Data) 19
A.2.4c Sublethal Effects: Amphibians (Summary of the White Paper): 21
A.2.4d Sublethal Effects: Amphibians (New Open Literature Data) 23
A.2.5 Freshwater Invertebrates, Acute 34
A.2.6 Freshwater Invertebrate, Chronic 37
A.2.7 Freshwater Invertebrates, Acute Open Literature Data 38
A.2.8a Freshwater Microcosm/Field Studies (2003 IRED Data) 39
A.2.8b Freshwater Field Studies (New Open Literature Data) 58
A.3 Toxicity to Estuarine and Marine Animals 59
A.3.1 Estuarine and Marine Fish, Acute 59
A.3.2 Estuarine and Marine Fish, Acute (Open Literature 2006 Review) 59
A.3.2 Estuarine and Marine Fish, Chronic 60
A.3.3a Sublethal Effects: Estuarine/Marine Fish (2003 IRED Data) 61
A.3.3b Sublethal Effects: Estuarine/Marine Fish (New Open Literature Data) 62
A.3.4 Estuarine and Marine Invertebrates, Acute 62
A.3.5 Estuarine and Marine Invertebrate, Chronic 65
A.3.6 Sublethal Effects: Estuarine/Marine Invertebrates (New Open Literature Data) 66
A.3.7a Estuarine and Marine Field Studies (2003 IRED Data) 66
A.3.7b Estuarine and Marine Field Studies (New Open Literature Data) 73
A.4 Toxicity to Plants 74
A.4.1 Terrestrial Plants 74
A.4.2 Aquatic Plants 77
A. 5 Effects of Environmental Factors and Life-Stage on Aquatic Atrazine Toxicity 88
A.5.1 Interaction Effects on Atrazine Toxicity to Plants 88
l
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A.5.2 Interaction Effects on Atrazine Toxicity to Aquatic Animals
A. 6 References
90
92
A.l Toxicity to Birds / Reptiles
Given a lack of ecotoxicity data for reptiles, avian acute oral, subacute dietary, and chronic
reproduction data are used as a surrogate for sea turtles. In addition, open literature data are
available for a limited number of reptiles including turtles (red-eared slider [Pseudemys elegans]
and snapping turtles \Chelydra serpentine]) and American alligators (Alligator mississippiensis).
Ecotoxicity data for birds and reptiles are discussed in Sections A. 1.1 through A. 1.4.
A. 1.1 Birds: Acute Oral Studies
An acute oral toxicity study using the technical grade of the active ingredient (TGAI) is required
to establish the toxicity of atrazine to birds. The preferred test species is either mallard duck
{Anasplatyrhynchos; a waterfowl) or bobwhite quail (Colinus virginianus] an upland gamebird).
Results of this test are summarized below in Table A-l.
Table A-l. Avian Acute Oral Toxicity: Technical Grade and Formulations
Surrogate Species
% ai
LD5(i (mg/kg)
Probit Slope
Toxicity Category
MRID No.
Author/Year
Study
Classification1
Northern bobwhite quail
{Colinus virginianus)
14-day old chicks; 8-day test
Tech.
940
slope 3.836
Slightly toxic
000247-21
Fink 1976
Acceptable
Mallard Duck
(Anas platyrhynchos)
6-months old; 14-day test
76%
80 WP
> 2,000
slope none
Practically non-toxic
001600-00
Hudson, Tucker &
Haegle 1984
Supplemental
(only 3 birds)
(formulation)
Ring-necked Pheasant
(Phasianus colchicus)
3-months old; 14-day test
76%
80 WP
> 2,000
slope none
Practically non-toxic
001600-00
Hudson, Tucker &
Haegle 1984
Supplemental
(formulation)
Japanese Quail
(Coturnix c. japonica)
50-60 days old; 14-day test
Tech.
4,237
slope > 6
Practically non-toxic
000247-22
Sachsse and Ullman
1974
Supplemental
(species not
native)
Since the lowest LD50 is in the range of 501 to 2,000 mg/kg, atrazine is categorized as slightly
toxic to avian species on an acute oral exposure basis. According to Hudson et al. (1984), signs
of intoxication in mallards first appeared 1 hour after treatment and persisted up to 11 days. In
pheasants, signs of intoxication disappeared by 5 days after treatment. Signs of intoxication
included weakness, hyper-excitability, ataxia, tremors; weight loss occurred in mallards.
Degradates: Minor atrazine degradates include deethylatrazine (DEA), deisopropylatrazine
(DIA) and diaminochlorotriazine. Acute mammalian LD50 values available for deethylatrazine
and deisopropylatrazine are both more toxic than the parent atrazine. Therefore, a special (70-1)
acute oral toxicity test with the upland gamebird (preferably northern bobwhite) are required to
2
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address the concern for these degradates. Acute avian LD50 data for the atrazine degrdates,
deethylatrazine (DEA) and deisopropylatrazine(DIA), and hydroxyatrazine (HA) are
summarized in Table A-2.
Table A-2. Avian Acute Oral Toxicity: Degradates
Surrogate Species
Degradate
% ai
LD50 (mg/kg-
bw)
Probit Slope
Toxicity Category
MRID No.
Author/Year
Study
Classification1
Northern bobwhite quail
('Colinus virginianus)
18-week old chicks; 14-day test
Northern bobwhite quail
( 2,000
atrazine slope none
(DIA)
96%
Hyrdroxy
atrazine > 2,000
(HA) slope none
97.1%
Desethyl 768
Atrazine Slope = 6.21
(DEA)
(95% CI =
96% 3.19-9.27)
Practically non-toxic
Practically non-toxic
Slightly toxic
465000-07
Stafford, 2005a
465000-08
Stafford, 2005b
465000-09
Stafford, 2005c
Acceptable
Acceptable
Acceptable
The results of the acute avian oral toxicity data with the atrazine degradates shows that DEA is
slightly toxic, while HA and DIA are practically non-toxic, to bobwhite quail. It should be noted
that the LD50 value for DEA (768 mg/kg-bw) is less than the corresponding value for the parent
technical grade of atrazine (940 mg/kg-bw), indicating that the DEA degradate is more toxic to
birds than the parent on an acute oral exposure basis. In the DEA study, 10, 40, 90, and 100%
mortality was observed in quail exposed to DEA at 445, 735, 1212, and 2000 mg/kg-bw by 14
days (MRID # 465000-09). In addition, sublethal treatment-related effects, including reduction
in body weight gain and decreased food consumption, were observed at the lowest treatment
level of 270 mg/kg-bw as well as the higher doses. Although no treatment-related mortality was
observed in the acute oral test using DIA, sublethal effects on reduced body weight gain and
food consumption were observed at concentrations of 445 mg/kg-bw (MRID # 465000-08) and
higher. No mortality and/or sublethal effects were noted in the acute oral test with HA (MRID #
465000-08).
A. 1.2 Birds: Subacute Dietary Studies
Two subacute dietary studies using the TGAI are required to establish the toxicity of atrazine to
birds. The preferred test species are mallard duck and bobwhite quail. Results of these tests are
tabulated below in Table A-3.
3
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Table A-3. Avian Subacute Dietary Toxicity
Surrogate Species
% ai
5-Day LC50
(ppm)1
Toxicity Category
MRID No.
Author/Year
Study
Classification
Northern bobwhite
('Colinus virginianus)
9-days old chicks
99.0
> 5,000
(no mortality)
Practically non-toxic
000229-23
Hill etal. 1975
Acceptable
Northern bobwhite
(1Colinus virginianus)
young adults
Tech.
> 10,000
Practically non-toxic
unknown - Gulf South
Gough & Shellenberger
1972
Supplemental
(Adult birds &
no raw data)
Ring-necked pheasant
(.Phasianus colchicus)
10-days old chicks
99.0
> 5,000
(no mortality)
Practically non-toxic
000229-23
Hill etal. 1975
Acceptable
Japanese Quail
(Coturnix c. japonica)
7-days old chicks
99.0
> 5,000
(7 % mortality
at 5,000 ppm)
Practically non-toxic
000229-23
Hill etal. 1975
Supplemental
(species not
native)
Mallard duck
(Anas platyrhynchos)
10-days old ducklings
99.0
> 5,000
(30 % mortality
at 5,000 ppm)
Practically non-toxic
000229-23
Hill etal. 1975
Acceptable
1 Test organisms observed an additional three days while on untreated feed.
Because the LC50 values are greater than 5,000 ppm, atrazine is categorized as practically non-
toxic to avian species on a subacute dietary exposure basis. In the sub-acute dietary with mallard
ducks, 30% mortality was observed at the highest test concentration of 5,000 ppm (MRID #
000229-23). The time to death was Day 3 for the one Japanese quail and Day 5 for three mallard
ducks (J. Spann at Patuxent Wildlife Center, 1999, personal communication).
Subacute dietary studies using a typical end-use product (TEP) may be required on a case-by-
case basis to establish the toxicity of atrazine formulations to birds. The preferred test species
are mallard duck and bobwhite quail. Results of these tests are summarized below in Table A-4.
Table A-4. Formulation Avian Subacute Dietary Toxicity
% ai
5-Day LC50 (ppm ai)1
Toxicity Category
MRID No.
Study
Surrogate Species
Form
Probit Slope
Author/Year
Classification
Northern bobwhite
76
5,760
Practically non-toxic
000592-14
Supplemental
(
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A. 1.3 Birds: Chronic Studies
Avian reproduction studies using the TGAI are required for atrazine, because the following
conditions are met: (1) birds may be subject to repeated or continuous exposure to the pesticide,
especially preceding or during the breeding season, (2) the pesticide is stable in the environment
to the extent that potentially toxic amounts may persist in animal feed, (3) the pesticide is stored
or accumulated in plant or animal tissues, and/or, (4) information derived from mammalian
reproduction studies indicates reproduction in terrestrial vertebrates may be adversely affected
by the anticipated use of the product. The preferred test species are mallard duck and bobwhite
quail. Results of these tests are provided below in Table A-5.
Table A-5. Avian Reproduction
Surrogate Species/
NOAEC/
Statistically sign. (p<0.05)
MRID No.
Study
Study Duration
% ai
LOAEC Endpoints
Author/Year
Classification
LOAEC (ppm
ai)
Northern bobwhite
97.1
NOAEC 225
29 % red. in egg production
425471-02
Acceptable
(Colinus virginianus)
LOAEC 675
67 % incr. in defective eggs
Pedersen &
20 weeks
27 % red. in embryo viability
DuCharme 1992
6-13 % red. in hatchling body wt.
10-16 % red. in 14-day old body wt.
8.2 % red. in 14-day old body wt.
(after recovery period)
NOAEC < 75
6.7-18 % red. in 14-day old body wt.
LOAEC 75
Mallard duck
97.1
NOAEC 225
49 % red. in egg production
425471-01
Acceptable
(Anas platyrhynchos)
LOAEC 675
61 % red. in egg hatchability
Pedersen &
20 weeks
12-17 % red. in food consumption
DuCharme
1992
NOAEC 75
9-13 % red. in food consumption
LOAEC 225
(During 3 of 11 biweekly periods)
In the bobwhite study, reproductive endpoints were measured after a 3-week recovery period.
During the recovery period, there was a 67% percent increase in the number of defective eggs at
675 ppm as compared to controls; the number of defective eggs during the recovery period was
consistent with the number of defective eggs during the treatment period at 675 ppm (MRID #
425471-02). Bobwhite and mallard tests show similar toxic effects on reduced egg production
and embryo viability/hatchability with LOAEC and NOAEC values of 675 and 225 ppm,
respectively. Although the bobwhite test showed a 7 to 18% reduction in 14-day body weight in
the 75 ppm treatment group, relative to the control group, the reproductive endpoints were
considered to be more biologically significant, given the use of the avian data as a surrogate for
sea turtles in the Chesapeake Bay.
In the 8-day subacute LC50 test with adult Japanese quail, food consumption and body weight
were reduced and egg production stopped after 3 days of exposure to atrazine (Sachsse and
Ullman, 1975; MRID 000247-23).
5
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A. 1.4 Birds/Reptiles: Open Literature
A.1.4a Birds: New Open Literature Data
Two studies were located in the open literature that evaluated the potential for atrazine to affect
endpoints including growth, sexual maturity, liver effects, and endocrine effects in birds
(summarized in Table A-6). Wilhelms et al. (2005; Ecotox Reference # 80632) reported that
dietary exposure to 1000 ppm atrazine resulted in reduced food consumption (15% reduction
compared with controls) and weight gain (31% reduction compared to controls), and elevated
testosterone levels (approximately 3-fold increase relative to controls) in male Japanese quail. It
is possible that the reduced food intake observed in this study represents taste aversion. Atrazine
was not definitively associated with effects on any other endpoint evaluated. Wilhelms et al.
(2006; Ecotox Reference # 82035) observed similar types of effects in female Japanese quail at
comparable dietary concentrations (Table A-6).
This study suggests that atrazine was associated with evidence of toxicity at dietary
concentrations of 1000 ppm in Japanese quail. These open literature studies were less sensitive
than the submitted data summarized in Table A-5.
Table A-6. Avian Reproduction/Growth Effects Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
Endpoint Concentration
in ppb
Citation
(EcoRef. #)
Rationale for Use in Risk
Assessment(1)
Reproduction
dietary studies in
birds / Atrazine
technical 99.9% ai
Male Japanese quail
Seven separate studies were conducted.
Dietary concentrations ranged from 10 to
1000 ppm. Animals were approximately
6-week old males. Endpoints evaluated
included growth, liver effects, sexual
maturation, and anti-estrogenic effects.
Exposure duration was up to 4 weeks.
In addition, studies using SC
administration and silastic implants were
also conducted that evaluated endpoints
including growth, liver effects, testes
weight, and circulating LH levels. Doses
up to 10 mg/kg-bw were tested.
At 1000 ppm, there was a
reduction in growth rate and food
intake and an elevation in
testosterone levels, although the
reduction in testosterone leves
was not consistently observed
across studies. Other statistically
significant observations were
considered spurious and not
related to atrazine treatment.
Wilhelms et
al., 2005
(80632)
Qual: Study did not evaluate a
comprehensive suite of
reproductive effects and was not
the most sensitive study in birds.
Reproduction
maturation in birds
/ Atrazine
technical, 99.9% ai
Female Japanese
quail
Birds were exposed to dietary
concentrations that ranged from 1 ppm to
1000 ppm. The following endpoints were
evaluated: growth, food intake, liver,
ovary, and oviduct weight, and plasma
luteinizing hormone and estradiol levels.
Exposure was up to 4 weeks.
Growth, food intake, liver
weight, and circulating estradiol
levels were significantly
(p<0.05) reduced in birds
exposed to atrazine at 1000 ppm,
3ut not at lower levels.
Wilhelms et
al., 2006
(82035)
Qual: Study did not evaluate a
comprehensive suite of
reproductive effects. No
concentrations between 100 and
1000 ppm were evaluated.
Studies have been submitted that
evaluated concentrations
between 100 and 1000 ppm;
NOAEC/LOAEC values from
those studies are considered to be
more suitalbe.
^ QUAL = The paper is not appropriate for quantitative use but is of good quality, addresses issues of concern to the risk
assessment and is used in the risk characterization discussion.
6
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A.1.4b Reptiles: Open Literature Data from 2003 IRED
Atrazine was tested on eggs of the turtle, red-eared slider (Pseudemys elegans) and the American
alligator (,Alligator mississippiensis) to determine if atrazine produced endocrine effects on the
sex of the young (Gross, 2001). The turtle and alligator eggs were placed in nests constructed of
sphagnum moss treated with 0, 10, 50 100 and 500 |ig/L for 10 days shortly after being laid. The
test temperatures, 27.3 °C for the turtle and 32.8 °C for alligators, normally yield all male young.
No adverse effects were found. Analysis of the embryonic fluids indicated that no atrazine was
present in the eggs at the detection limit (0.5 |ig/L). Under these conditions, atrazine does not
appear to have permeated the leathery shell of reptiles (MRID 455453-03 and 455453-02).
A. 1.4c Reptiles: New Open Literature Data (2006 Literature Review)
Two additional open literature studies on snapping turtle and alligator egg exposures to atrazine
are summarized below (De Solla et al., 2005 and Crain et al., 1999) in Table A-7. The results of
both of these studies suggest that exposure of reptilian eggs to atrazine does not cause significant
alteration in gonadal development and aromatase activity at environmentally relevant
concentrations.
Snapping turtles (Chelydra serpentina) were used to determine if environmentally relevant
exposures to atrazine affected gonadal development (De Solla et al., 2005; Ecotox Reference #:
82032). Eggs were incubated in soil treated with atrazine at a typical field application rate (1.32
lb ai/A), 10-fold this rate (13.2 lb ai/A) and a control rate (no atrazine) for the duration of
embryonic development (-117 days). Measured concentrations of atrazine in the low and high
atrazine treatment groups were 0.64 and 8.1 ppm, respectively. The incubation temperature (25
°C) was selected to produce only males. Although some males with testicular oocytes and
females were produced in the atrazine-treated groups (3.3 - 3.7%), but not in the control group,
no statistical differences were found among the treatment and control groups. In addition, there
was no difference in hatching success and thyroid activity among the different atrazine
treatments and the control. According to the study authors, observations of other turtles suggest
that natural and spontaneous intersexes exist in turtle populations.
Gonadal histology and hepatic steroidogenic activity was measured in American alligator eggs
exposed to atrazine at concentrations of 0, 0.014, 0.14, 1.4, and 14 ppm (Crain et al., 1999;
Ecotox Reference #: 70208). All atrazine treated eggs incubated at female- and male-
determining temperatures produced female and male hatchlings, respectively. No differences in
gonadal and reproductive tract histology or hepatic aromatase activity were observed in any of
the atrazine-treated or control alligators. The results of the study suggest that embryonic
exposure to atrazine does not cause significant alterations in gonadal structure or hepatic
steroidogenic enzyme activity of hatchling American alligators.
7
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Table A-7. Reptilian Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism (Common
and Scientific Name) and
Age and/or Size
Test
Design
Endpoint
Concentration
in ppm
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Chronic lab (117
days) /
Atrazine 480
formulation
(atrazine content =
481 g/L and
unspecified
triazines of 29
g/L)
Snapping turtle {Chelydra
serpentine) eggs
¦ Eggs incubated in soil
treated w/atrazine at 1.32 lb
ai/A (measured cone = 0.64
ppm) and 13.2 lb ai/A
(measured cone = 8.1 ppm)
and control.
- 3 replicates (with 23-24
eggs/replication)/treatment
group.
¦ Incubator temp = 25°
(+1°C) to produce males.
¦ Endpoints: gonadal
development (hatching
success, gonadal
morphology, and thyroid
activity)
NOAEC = 13.2 lb ai/A
(0.81 ppm)
Some males w/testicular
oocytes and females
produced in atrazine-
treated groups (3.3 -
3.7%); however, no
significant differences
between atrazine
treatments and controls
were observed.
Thyroids from each
treatment and control
displayed similar levels
of activity.
De Solla et al.,
2005
(82032)
QUAL:
• no raw data provided
¦ 3 PAHs detected at
non-toxic levels in
control soil, but not
analyzed for in the
atrazine treatment
groups
• low incidence of
intersex or females
precluded ability to
differentiate between a
low incidence caused
?y atrazine exposure
and random sampling
error
Chronic lab
(duration NR) /
Atrazine (99 % ai)
American alligator
[Alligator mississippiensis)
eggs at stage 21 in
embryonic development,
just prior to onset of
gonadal differentiation
¦ Eggs were treated
w/atrazine at 0, 0.014, 0.14,
1.4, and 14 ppm via topical
application to the eggshell
in 50 jul of 95% ethanol.
¦ 5 eggs/treatment were
incubated at temperatures
to produce either 100%
males (33 °C) or 100%
females (30 °C).
¦ Endpoints: gonadal
tiistology and hepatic
steroidogenic activity
NOAEC = 14 ppm
All atrazine treated eggs
incubated at female- and
male-determining temps
produced female and
male hatchlings,
respectively. No
differences in gonadal
tiistology (Mullerian
duct epithelial cell
tieight and medullary
regression) and hepatic
aromatase activity was
noted between atrazine
treated groups and
controls.
Crain et al., 1999
(70208)
QUAL:
• no raw data provided
^ QUAL = The paper is not appropriate for quantitative use but is of good quality, addresses issues of concern to the risk
assessment and is used in the risk characterization discussion.
NR = Not reported.
A.2 Toxicity to Freshwater Animals
A. 2.1 Freshwater Fish and Amphibia, Acute
Two freshwater fish toxicity studies using the TGAI are required to establish the toxicity of
atrazine to fish. The preferred test species are rainbow trout (Oncorhynchus mykiss; a coldwater
fish) and bluegill sunfish (Lepomis macrochirus; a warmwater fish). Results of these tests are
summarized below in Table A-8.
Table A-8. Freshwater Fish Acute Toxicity (TGAI)
Surrogate Species/ 96-hour LC50 (ppb)
Static or (measured/nominal) MRID No. Study
Flow-through test % a.i. Probit Slope Toxicity Category Author/Year Classification
8
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Table A-8. Freshwater Fish Acute Toxicity (TGAI)
Surrogate Species/
Static or
Flow-through test
% a.i.
96-hour LC50 (ppb)
(measured/nominal)
Probit Slope
Toxicity Category
MRID No.
Author/Y ear
Study
Classification
Rainbow trout
(Oncorhynchus mykiss)
Static test
98.8
5,300
(nominal)
slope - 2.723
moderately toxic
000247-16
Beliles & Scott 1965
Acceptable
Brook trout
{Salvelinus tontinalis)
Flow-through test
94
6,300
4,900 (8-day test)
not specified
moderately toxic
000243-77
Macek et al. 1976
Supplemental
(52-gram fish &
no raw data)
Fish from the Nile River
Chrysichthyes auratus
Static-renewal - daily
150 mg/L CaC03; 22EC
96
6,370
(not specified)
moderately toxic
452029-11
Hussein, El-Nasser
& Ahmed 1996
Supplemental
(non-native sp.;
26-gram fish;
no raw data)
Bluegill sunfish
(.Lepomis macrochirus)
Flow-through test
94
> 8,000
6,700 (7-day test)
(not specified)
moderately toxic
000243-77
Macek et al. 1976
Supplemental
(6.5-gram fish &
no raw data)
Tilapia 38 grams
('Oreochromis niloticus)
Static-renewal - daily
150 mg/L CaC03: 22EC
96
9,370
(not specified)
moderately toxic
452029-11
Hussein, El-Nasser &
Ahmed 1996
Supplemental
(non-native sp.;
38-gram fish;
no raw data)
Fathead minnow
(.Pimephales promelas)
24-Hour renewal test
94
15,000
(nominal)
15,000 (5-day test)
slightly toxic
000243-77
Macek et al. 1976
Supplemental
(no raw data)
Carp
('Cyprinus carpio)
Semi-static test
93.7
18,800
(nominal)
slope not reported
slightly toxic
452029-13
Neskovic et al. 1993
Supplemental
(no raw data)
Fathead minnow juvenile
{Pimephales promelas)
Flow-through test;
52 mg/L CaC03
97.1
20,000
(measured)
Slope - 6.889
slightly toxic
425471-03
Dionne 1992
Acceptable
Bluegill sunfish
{Lepomis macrochirus)
Static test
98.8
24,000
(nominal)
no slope
slightly toxic
000247-17
Beliles & Scott 1965
Acceptable
Brown trout
{Salmo trutta) 1.9 gr.
Static-Renewal - daily
pH 6; 10EC;
11 mg/L CaC03
Zebrafish
{Brachydanio rerio)
NR
NR
27,000
(nominal)
37,000
(NR)
slightly toxic
slightly toxic
452029-09
Grande, Anderson &
Berge 1994
MRID # NR
Korte & Greim 1981
Supplemental
(no raw data;
slight aeration &
purity unknown)
Supplemental
(article
unavailable)
Bluegill sunfish
{Lepomis macrochirus)
Static test
100
57,000
(nominal)
slightly toxic
001471-25
Buccafusco 1976
Acceptable
Goldfish
{Carassius auratus)
Static test
98.8
60,000
(nominal)
Slope - 2.695
slightly toxic
000247-18
Beliles & Scott 1965
Supplemental
(not an acceptable
species)
The range of acute freshwater fish LC50 values for technical grade atrazine is 5,300 to 60,000
ppb; therefore atrazine is categorized as slightly (>10,000 to 100,000 ppb) to moderately (>1,000
to 10,000 ppb) toxic to freshwater fish on an acute exposure basis. The freshwater fish acute
9
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nominal LC50 value of 5,300 ppb is based on a static 96-hour toxicity test using rainbow trout
(Oncorhynchus mykiss) (MRID # 000243-77).
Table A-9 presents fish and amphibian toxicity data for formulated products.
Table A-9. Freshwater Fish and Amphibian Acute Toxicity (Formulated Products)
Surrogate Species/
Flow-through or Static
% ai
formul.
96-hour LC50 (ppb)
(measured/nominal)
Toxicity Category
MRID No.
Author/Y ear
Study
Classification
Black Bass - fry
(Micropterus salmoides)
Static test; 20EC
78 mg/L hardness
80
80 W
12,600
(nominal)
slope - 5.86
slightly toxic
452277-17
R. O. Jones 1962
Supplemental
(48-hours;
limited raw data)
Channel Catfish yolk sac
(.Ictalurus punctatus)
Static test; 23.3-25.8EC
78 mg/L hardness
80
80 W
16,000
(nominal)
slope - 3.36
sightly toxic
452277-17
R. O. Jones 1962
Supplemental
(limited raw data)
Bluegill Sunfish fry
(.Lepomis macrochirus)
Static test; 25-27EC
78 mg/L hardness
80
80 W
20,000
(nominal)
no slope
slightly toxic
452277-17
R. O. Jones 1962
Supplemental
(limited raw data)
American Toad - larvae
(Bufo americanus)
Flow-through test
40.8
4L
10,700 late stage
26,500 early stage
(nominal)
slightly toxic
452029-10
Howe et al. 1998
Supplemental
(no raw data)
Northern Leopard Frog
larvae
(.Rana pi pi ens)
Flow-through test
40.8
4L
14,500 late stage
47,600 early stage
(nominal)
slightly toxic
452029-10
Howe et al. 1998
Supplemental
(no raw data)
Coho Salmon
(1Oncorhynchus kisutch)
Renewal daily; 144 hr
40.8*
AAtrex
Liquid
> 18,000
25 % mortality
(measured)
slightly toxic
452051-07
Lorz et al. 1979
Supplemental
(no LC50 value &
12-17 months old)
Rainbow trout
(iOnchorhynchus mykiss)
Flow-through test
40.8
4L
20,500
(nominal)
slightly toxic
452029-10
Howe et al. 1998
Supplemental
(no raw data)
Channel Catfish
(.Ictalurus punctatus)
Flow-through test
40.8
4L
23,800
(nominal)
slightly toxic
452029-10
Howe et al. 1998
Supplemental
(no raw data)
Rainbow trout
(iOncorhynchus mykiss)
Static test
43
Liquid
24,000
(unknown)
slightly toxic
400980-01
Mayer & Ellersieck
1986
Supplemental
(no raw data)
Bluegill sunfish
{Lepomis macrochirus)
Static test
43
Liquid
42,000
(unknown)
slightly toxic
400980-01
Mayer & Ellersieck
1986
Supplemental
(no raw data)
* Percent a.i. assumed based on description as a liquid formulation, AAtrex.
All toxicity values for the atrazine formulations are > 10 and 100 ppm; therefore, the formulated
products are classified as slightly toxic to aquatic invertebrates on an acute exposure basis.
Based on comparison of acute toxicity data for technical grade atrazine and formulated products
of atrazine, it appears that freshwater fish are more sensitive to the TGAI. It should be noted
that available formulated product (40.8% ai for 4L) data for amphibians reports LC50 values
>10,000 ppb.
10
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Degradates Acute fish testing with bluegill and rainbow trout are required to address degradate
concerns. Table A-10 presents freshwater fish toxicity data for hydroxyatrazine.
Table A-10. Freshwater Fish Acute Toxicity (Hydroxyatrazine)
Surrogate Species/ % ai
Flow-through or Static formul.
96-hour LC50 (ppb)
(measured/nominal)
Toxicity Category
MRID No.
Author/Y ear
Study
Classification
Bluegill sunfish
(.Lepomis macrochirus)\
1-15 g
Static test; 20.8- 21.6 °C
125 mg/L hardness
Rainbow trout
('Oncorhynchus mykiss);
0.75 g
Static test; 13.2-14.1 °C
125 mg/L hardness
>3,800
(measured dissolved)
>3,000
(measured dissolved)
moderately toxic*
moderately toxic*
465000-05
Peither, 2005b
465000-04
Peither, 2005a
Acceptable
Acceptable
* Biological results for both studies were based on the mean-measured concentration of dissolved Hydroxyatrazine,
which remained constant at the limit of its water solubility throughout the duration of the tests. Therefore,
hydroxyatrazine is not acutely toxic to bluegill sunfish and rainbow trout at the limit of its water solubility.
Although the freshwater fish LC50 values (>3,000 to >3,800 ppb) for the degradate,
hydroxyatrazine, are within the range classifying it as moderately toxic, the biological results for
both studies were based on dissolved (filtered) mean-measured concentrations of
hydroxyatrazine, which remained constant at the limit of its water solubility (3-4 ppm ai)
throughout the duration of the tests. No mortalities were reported in either study at the
maximum test concentration. Therefore, hydroxyatrazine is technically classified as moderately
toxic to fish on an acute exposure basis; however, given that its solubility limit is close to the
maximum concentration tested, hydroxyatrazine is not likely to be acutely toxic to freshwater
fish at the limit of its water solubility.
A.2.2 Freshwater Fish, Chronic
A freshwater fish early life-stage test using the TGAI is required for atrazine because the end-use
product is expected to be transported to water from the intended use site, and the following
conditions are met: the pesticide is intended for use such that its presence in water is likely to be
continuous and recurrent; an aquatic acute EC50 is less than 1 mg/L (i.e., Chironomus tentans
LC50 0.72 ppm); and the pesticide is persistent in water {i.e., half-life greater than 4 days). The
preferred test species is rainbow trout. Table A-l 1 presents the chronic toxicity data for
freshwater fish.
Table A-ll. Freshwater Fish Early Life Stage Toxicity
Surrogate Species/
Study Duration/ NOAEC/LOAEC
Flow-through or 11 g/L (ppb) Statistically sign. (p=0.05) MRID No. Study
Static Renewal % ai (measured or Endpoints Affected Author/Y ear Classification
nominal)
11
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Table A-ll. Freshwater Fish Early Life Stage Toxicity
Surrogate Species/
Study Duration/
Flow-through or
Static Renewal
% ai
NOAEC/LOAEC
ug/L (ppb)
(measured or
nominal)
Statistically sign. (p=0.05)
Endpoints Affected
MRID No.
Author/Y ear
Study
Classification
Rainbow trout
Tech.
NOAEC 410
sign, delays in hatching @
452083-04
Invalid
(Oncorhynchus mykiss)
LOAEC 1,100
1,100 and 3,800 ng/L
Whale et al.
(DMSO used as
86 days, flow-through
(measured)
sign. red. wet wt. at 30 & 58
1994
solvent, which
50 mg/L CaC03
days @ 1,100 & 3,800 ng/L
aids in transport
sign. red. dry wt. @ 3,800 ju.g/L
of chemicals
58.8 % mortality @ 3,800 ^g/L
across cell
at swim-up
membranes)
Rainbow trout
80
Hardness 50 mg/L:
% normal survival 50/200 mg/L
452029-02
Supplemental
embryo-larvae
WP
LC50 660
19 f^g/L - 94 98
Birge, Black
(short test;
{Oncorhynchus mykiss)
LC01 29
54 - 88 90
& Bruser
no raw data for
27 days; flow-through
Slope 1.2
54** - 68 74
1979
statistical
Hardness 200 mg/L:
5,020 ** - 10 9
analyses)
LC50 810
50,900 ** - 0 0
LC01 77
Slope 1.38
Channel catfish
80
Hardness 50 mg/L:
highly teratogenic in all tests;
452029-02
Supplemental
embryo-larvae
WP
LC50 220
no results for soft water
Birge, Black
(short test;
(Ictalurus punctatus)
Slope 0.977
&
no raw data for
8 days; flow-through
Hardness 200 mg/L:
420 f^g/L - 16%terata
Bruser 1979
statistical
LC50 230
830 f^g/L - 47 % terata
analyses)
Slope 0.84
46,700 jug/L - 86 % terata
Zebrafish
98
NOAEC 300
452029-08
Supplemental
(Brachydanio rerio)
LOAEC 1,300
2 - 3 % sign. incr. in edema
Gorge &
(no raw data)
35 Days; pH 8; 27±1EC
(measured)
45-62 % mortality
Nagel 1990
Flow-through test
35-Day LC50 890
Hardness 24 mg/L
Slope 1.25
In addition to affecting survival of rainbow trout and catfish embryo-larvae, Birge et al. (1979)
also reported that "Atrazine was highly teratogenic in all tests." The frequency of teratogenicity
was reported for channel catfish in hard water and is included in the table above; no data on
frequency was reported for soft water or for rainbow trout. (MRID # 452029-02).
A freshwater fish life-cycle test using the TGAI is required for atrazine because the end-use
product is expected to be transported to water from the intended use site and studies of other
organisms indicate that the reproductive physiology of fish may be affected. The preferred test
species is fathead minnow. Results of four fish life-cycle tests are tabulated below in Table A-
12. Following 44 weeks of exposure to atrazine in a flow-through system, brook trout mean
length and body weight were reduced by 7.2% and 16% at concentrations of 120 ppb, as
compared to the control (MRID 000243-77). The corresponding NOAEC for this study is 65
ppb.
12
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Table A-12. Freshwater Fish Life-Cycle Toxicity
Surrogate Species/
Study Duration/
Flow-through or
Static Renewal
%
ai
NOAEC/LOAEC
Hg/L (ppb)
(measured or
nominal)
Statistically sign. (p<0.05)
End points Affected
MRID No.
Author/Y ear
Study
Classification
Brook trout
(Salvelinus frontinalis)
44 weeks, flow-through
94
NOAEC 65
LOAEC 120
(measured)
7.2 % red. mean length
16 % red. mean body weight
000243-77
Macek et al. 1976
Acceptable
Bluegill sunfish
(Lepomis macrochirus)
6-18 months, flow-through
94
NOAEC 95
LOAEC 500
(measured)
LOAEC based on loss of
equilibrium in a 28-day test
conducted at the same lab.
000243-77
Macek et al. 1976
Supplemental
(Low survival in
the controls)
Fathead minnow
(Pimephales promelas)
39 weeks; flow-through
97.1
NOAEC < 150
LOAEC 150
(measured)
6.7 % red. in Fi length
22 % red. in Fi body wt.
(sign. diff. from neg. control)
425471-03
Dionne 1992
Supplemental
(Failed to identify
a NOAEC)
Fathead minnow
{Pimephales promelas)
43 weeks, static-renewal
94
NOAEC 210
LOAEC 870
(measured)
LOAEC based on 25%
mortality in a 96-hour test
conducted at the same lab.
00024377
Macek et al. 1976
Supplemental
(High mortality in
control adults)
A. 2.3 Freshwater Fish/Amphibians, Open Literature Data on Mortality/Survivorship
Open literature data on the effects of atrazine to mortality/survivorship of amphibians is
summarized in Table A-14. Additional open literature data on amphibian mortality/survivorship
is also included as part of the discussion on sublethal effects for amphibians in Section A.2.4 and
Table A-16. Available acute data for amphibians indicate that they are relatively insensitive to
technical grade atrazine with acute LC50 values > 20,000 ppb. Chronic mortality data for
amphibians confirms that exposure to atrazine does not cause direct mortality to frogs and
salamanders at concentrations ranging from approximately 200 to 2000 ppb; these concentrations
represent the highest tested atrazine treatment levels within each of the studies. Only one study
(Storrs and Kiesecker, 2004; reviewed below) shows counterintuitive patterns of survivorship
(lower survivorship at low atrazine doses as compared to higher doses of atrazine); however,
there are a large number of uncertainties associated with the study, including possible surfactant
effects and variable sampling sizes, which confound the ability to discern a atrazine treatment-
related survivorship effect. Further review of the open literature studies containing chronic
mortality data is included as part of discussion for sublethal effects to amphibians.
Three species of amphibian larvae (tadpoles) were tested with technical grade atrazine (Table A-
14). The leopard frog (Ranapipiens), wood frog (Rana sylvatica), and American toad (Bufo
americanus) tadpoles each have LC50 values of >20,000 ppb atrazine (Allran and Karasov,
Ecotox Reference # 59251). Based on these values, the amphibians evaluated are relatively
insensitive to atrazine on an acute exposure basis. However, sublethal effects were observed at
4.3 mg/L and higher. These effects included elevated ventilation rates (4.3 mg/L and higher) and
reduced feeding (20 mg/L only) in adults and increased incidences of deformities in survivors at
4.3 mg/L and higher (approximately 19% incidence). Deformities included wavy tail (54%),
13
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lateral tail flexure (27%), facial edema (12%), axial shortening (3.5%), dorsal tail flexure (3.3%),
and blistering (0.3%). Similar incidences of deformities were observed for all species tested.
Birge et al. (1983; Ecotox Reference # 19124) tested the effects of atrazine exposure on
developing embryos of bullfrogs and American toads under flow through conditions from
fertilization to 4-days after hatching. Incidences of "gross debilitating" abnormalities were
evaluated. LC50s (mortality + malformation incidences) for atrazine were 410 ug/L and >4800
ug/L in bullfrogs and American toads, respectively. Specific information on the abnormalities
associated with atrazine was not included in the study report, although defects of the head and
vertebral column, dwarfed bodies, partial twining, microcephaly, absent or reduced eyes and
fins, and amphiarthrodic jaws were most commonly reported across the treatments. Also,
insufficient information was included in the study report to allow for an independent evaluation
of data and verification of the statistical analyses. Although an LC50 of 410 ug/L was reported in
bullfrogs, 92% survival was observed at 410 ug/L in the study. Survival did not fall below 50%
until atrazine concentrations exceeded 15,000 ug/L. Therefore, there is considerable uncertainty
in the LC50 reported by Birge et al. (1983) of 0.41 mg/L (410 ug/L). Nonetheless, the data
provide evidence that atrazine exposure to embryo-larvae stages may produce developmental
abnormalities. Developmental abnormalities were generally observed at atrazine levels that also
induced mortality.
Long-term (32 days) static renewal exposure of a commercial formulation of atrazine (Aatrex
Nine-O; 85.5% ai) to four species of tadpole frogs including spring peepers (Pseudacris
crucifer), American toads (Bufo americanus), green frogs (Rana clamitans), and wood frogs
(Rana sylvatica) was studied at early (Gosner stages 25-27) and late (stages 29-36)
developmental stages (Storrs and Kiesecker, 2004; Ecotox Reference # 78290). Nominal
atrazine concentrations were 3, 30, and 100 ppb; measured concentrations at Day 1 were 2.8, 25,
and 64 ppb. With the exception of late stages of the toad and wood frog, there was significantly
lower survival for animals exposed to 2.84 ppb as compared with either the higher treatment
groups. Significant differences in survivorship within the 2.84 treatment group relative to the
control were observed for late stages of the toad and both stages of the green frog. However, no
significant survivorship differences between any of the treatment levels and the control were
observed for late spring peepers, early toads, and late wood frogs. The study author suggests that
greater mortality at lower doses than higher doses is associated with a U-shaped dose-response
pattern characteristic of many endocrine disruptors. However, the reference to the U-shaped
dose-response curve cannot be substantiated with only one statistically significant point. In
addition, there are also many uncertainties associated with the study. Possible impacts related to
the surfactant of the commercial grade of atrazine confound the ability to demonstrate treatment-
related effects. In addition, statistical patterns reported by the study authors may have been
influeced by variable sample sizes, both within treatment levels and between different stages of
tadpole species. In the case of the late stage toad, the sample size was extremely low (< 7 for
each treatment and control). Finally, evidence of survivorship patterns observed in this study has
not been replicated in any other available studies (although different atrazine formulations were
used). Survivorship patterns were presented as survival probability; therefore, it was not
possible to determine or quantify the number of days until death or the overall mortality at the
end of the experiment.
14
-------
Table A-14. Amphibian Mortality/Survivorship Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
Endpoint
Concentration
in ppb
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Acute lab (14
days) / 99% ai
¦ Leopard frog (.Rana
vipiens)
¦ Wood frog {Rana
sylvaticas)
¦ American toad {Bufo
americanus)
¦ Renewal
¦ Hardness (mg/L as CaCOs) =
290
Target Temp: 22 Deg. C
Animals were exposed in the
embryonic stage.
LC50 for all 3 species=
>20,000 (measured).
Effects included
increased incidence of
deformities in embryos
exposed for 4 days after
hatching and elevated
ventilation rate in
exposed adults at 4.3
mg/L and higher.
Allran and
Karasov, 2001
(59251)
QUAL. Study may
provide insight into
effect levels of atrazine
exposed adults and
embryos; however,
reporting limitations
were noted, and study
did not provide
sensitive endpoint.
Chronic (32 d) lab
study / Atrazine
commercial-grade
(Aatrex Nine-O;
85.5% ai)
¦ Spring peeper
(.Pseudacris crucifer)
¦ American toad {Bufo
americanus)
¦ Green frog {Rana
clamitans)
¦ Wood frog {Rana
sylvatica)
¦ All tadpoles at early
(Gosner stages 25-27)
and late (stages 29-
36) developmental
stages
¦ Static renewal (water replaced
every 3 d) at nominal
concentrations of 0, 3, 30, and
100 ppb. Measured cone (after 1
d = ND, 2.84, 25.2, and 64.8
ppb)
¦ Peepers, toads, and early-stage
green frogs kept in 120 ml
polypropylene cups w/100 ml (
treatment in dechlorinated
water); late wood and green
frogs kept in 750 ml poly cups
w/ 500 ml water; #
tadpoles/treatment varied
- Temperature = 22 °C
¦ Photoperiod = 12 h light/dark
¦ Feeding: crushed alfalfa every
3d
¦ Endpoints: Surivorship
Earlv sprin2 peeper:
LOAEL = 64.8;
NOAEL = 25.2
Late sprin2 peeper:
NOAEL = 64.8
Earlv A. toad:
NOAEL = 64.8
Late A. toad:
LOAEL = 2.84
NOAEL = <2.84
Earlv areen froa:
LOAEL = 2.84
NOAEL = <2.84
Late 2reen fro2:
LOAEL = 2.84
NOAEL = <2.84
Late wood fro2:
NOAEL = 64.8
Storrs and
Kiesecker, 2004
(78290)
QUAL:
¦ no raw data provided
¦ time to mortality,
relative to control, was
not discussed
¦ with exception of
green frogs, sample
sizes varied; sample
size for late American
toads was < 7 animals
¦ statistical patterns
likely influenced by
variable sample sizes
¦ possible surfactant
effects
¦ suvivorship patterns
observed have not been
replicated in any other
study
¦ survivorship patterns
expressed as survival
probability; therefore,
parameters such as
number of days until
death and overall
mortality were not
presented
Acute,
developmental
study; Atrazine
technical
unspecified purity
Bullfrog and
American toad
embryos
Eggs were exposed from
fertilization to 4 days post hatch.
Atz Cones: 28 to 4800 ug/L
Exposure: flow through
Endpoints: Presence of gross
debilitating anomalies.
Temp: 12-14 DegC
pH: 7 -7.8
Bullfrog LC50: 410 ug/L
American toad LC50:
>4800 ug/L
Birge et al., 1983.
(19124)
No raw data provided
and reporting
deficiencies were noted.
LC50S were not based on
mortality per se, but on
abnormalities that
would presumably
preclude survival under
natural conditions.
^ QUAL = The paper is not appropriate for quantitative use but is of good quality, addresses issues of concern to the risk
assessment and is used in the risk characterization discussion.
A. 2.4 Sublethal Effects, Freshwater Fish and Amphibians (Open Literature)
15
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A.2.4a Sublethal Effects: Freshwater Fish (2003 IRED Data):
A number of open literature studies were reviewed as part of the 2003 IRED. The results of
these studies, which are summarized below, show sublethal effects to olfaction, behavior, kidney
histology, and tissue growth at atrazine concentrations ranging from 0.1 to 3000 ppb.
Adult largemouth bass (Micropterus salmoides) were exposed to nominal concentrations of
technical grade atrazine (purity 97.1%) at 0, 25, 35, 50, 75, and 100 [j,g/L for 20 days to
determine the potential effects on endocrine-mediated functions (Wieser and Gross, 2002).
Additionally, bass were exposed to commercial grade (purity 42.1%) atrazine at 100 (J,g/L. After
20 days, plasma concentrations of estradiol, 11-ketotestosterone, testosterone, and vitellogenin
(a protein that serves in yolk formation) were measured. Female bass treated with 100 |ig/L
formulated atrazine contained significantly higher plasma estradiol and exhibited plasma
vitellogenin roughly 37 times greater (260 (J,g/ml) than controls (7 (J,g/ml). Male bass treated
with 100 [j,g/L formulated atrazine contained significantly lower plasma 11-ketotestosterone
levels. While not statistically significant, plasma testosterone (286 pg/ml) was lower than
controls (433 pg/ml) and plasma vitellogenin (42 (J,g/ml) was 7 times greater than control (6
(j,g/ml). Although there was considerable variability in plasma vitellogenin levels, atrazine-
treated fish appeared to have elevated plasma vitellogenin relative to controls at 50 and 100 |ig/L
of atrazine. Plasma 11-ketotestosterone was significantly lower in fish exposed to atrazine
concentrations greater than 35 (J,g/L. Treatment of fish with commercial grade atrazine resulted
in a significant increase in plasma estradiol in female fish and a significant decrease in 11-
ketotestosterone in male fish. Although not statistically significant, plasma vitellogenin in both
female and male fish appeared to be increased in fish treated with technical and commercial
grade atrazine.
Although high variability confounds this study's ability to resolve the effects of atrazine on
plasma steroids and vitellogenesis, the study has demonstrated that technical grade atrazine
affects plasma 11-ketotestosterone in males and that the formulated product affects plasma
estradiol in females. The non-guideline study is classified as supplemental and provides useful
information on the potential effects of atrazine (MRID 456223-04).
Effects on behavior were found to be significant (p < 0.0001) in zebrafish (Brachydanio rerio)
following 1-week exposures at 5 to 3125 [j,g/L atrazine (Steinberg etal., 1995). Fish exposed to
atrazine for 1-week showed a pronounced preference (p < 0.0001) for the dark part of the
aquarium compared to the control. Because no significant differences were found between the
effects at the various test concentrations (5 (J,g/L: 79%; 25 (J,g/L: 85%; 125 (J,g/L: 83%; 625 (J,g/L:
81%; 3125 |ig/L: 81%), these changes in swimming behavior appears to be threshold effects.
After 4 weeks at the above exposures, 15 to 24 % more of the treated fish preferred dark habitats
than did the controls. The authors concluded that atrazine may have an affect on the sensory
organs and the nervous system at atrazine concentrations commonly found in surface waters
(MRID #452049-10).
Saglio and Trijase (1998) measured 5 behavioral activities in goldfish following 24-hour
exposures to 0.5, 5 and 50 [j,g/L atrazine. A number of behavioral measurements were
16
-------
statistically significant (p < 0.05) from controls, but in most instances the significance was
inconsistent and failed to show a dose-related effect. The only behavioral effect showing a
consistent, dose-related effect was reduction in grouping (i.e., significant at 5 |ig/L (31%
reduction) and 50 [j,g/L (39% reduction). Other behaviors with statistically significant effects
were surfacing at 5 |ig/L (341% increase), burst swimming at 0.5 and 50 [j,g/L (1.00 and 2.25
units, respectively, the controls showed no effect). Following the introduction of skin extract, 5
[j,g/L of atrazine significantly (p < 0.05) reduced sheltering (81%) and grouping (60%), but these
effects showed no consistency with effects at 0.5 and 50 (J,g/L. This study shows that a 24-hour
exposure at 5 [j,g/L atrazine significantly affected aspects of swimming, positioning in water
column, increased number of mouth openings at the surface, and social behaviors, allow the
results of the study appear to be rather subjective. (MRID # 452029-14).
Fischer-Scherl et al. (1991) reported acute and chronic atrazine-induced alterations in rainbow
trout kidneys affecting renal corpuscles, renal tubules, renal interstitium, and glomerular
filtration. Compared to control fish, chronic 28-day exposures at 5, 10 and 20 [j,g/L reduced
Bowman's space due to a proliferation of podocytes. At higher chronic concentrations (40 and
80 |ig/L) renal corpuscles appeared hypercellular and enlarged (i.e., hypertrophy) due to a
proliferation of podocytes and mesangial cells. Also, the amount of membrane-bound vesicles
with varying electron-dense contents had increased in the urinary space of renal corpuscles.
Fibrillar structures and fibrocytes were found around Bowman's capsule indicating beginning
periglomerular fibrosis. Acute 96-hour exposures at 1.4 and 2.8 mg/L caused a more
pronounced obliteration of Bowman's space due to the proliferation of mesangial cells and more
renal corpuscles were affected. Increasing amounts of cellular debris accumulated in Bowman's
space. Simultaneously, epithelial cells of the parietal layer of Bowman's capsule displayed an
increased number of lysosomes and swollen mitochondria. Also, the number of glomerular
endothelial cells exhibiting vacuolar degeneration increased. Furthermore, light microscopy
shows minor alterations to renal tubules, but electron micrographs revel considerable changes.
First, obvious alterations of tubules appeared at 10 (J,g/L. Basilar labyrinth was dilated and
irregularly arranged. The mitochondria were electron-dense and showed club-shaped ends of
circular structure. At 40 (J,g/L, part of the endoplasmic reticulum appeared foamy and fragments
of endoplasmic reticulum were heavily distended. At 80 [j,g/L in proximal and distal tubular
epithelia lysis of the cytoplasm with formation of vacuoles and vesicles and condension of
mitochondria was prominent. In many tubular epithelia, only remnants of the former parallel-
arranged tubular system were present, mitochondria were swollen, lysosomal structures as well
as a vacuolization of the cytoplasm were detectable. In proximal tubules, lysomes had increased
in number and size. At acute exposures (1,400 and 2,800 (J,g/L), tubular structural lesions similar
to those described at 80 |ig/L were present, but a distinctly higher number of renal tubules was
affected. Extensive cytoplasmic vacuolization was evident and the parallel arrangement of the
basilar labyrinth was completely lost, some mitochondria were dark and condensed. Tubules of
the basilar labyrinth appeared foggy, partly involving mitochondria. Except for an increase in
cells with mitotic figures at concentrations of 5, 10, 20 |ig/L, no conspicuous alterations in basic
interstitial architecture could be detected. Beginning at 40 (J,g/L, a loosening of the hemopoietic
tissue was evident. Cells, preumably macrophages and phagocytizing material, had increased in
number. In addition to these effects, sinusendothelial cells were severely damaged at a
concentration of 80 (J,g/L. They separated from the basement membrane and exhibited numerous
17
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vesicular and lysosomal structures as well as swollen degenerating mitochondria. Alterations in
renal interstitium were considerable at acute exposures with 1,400 and 2,800 |ig/L, Interstitial
tissue was loosened and a state of spongiosus was indicated. Numerous macrophages were
present. Nuclei of interstitial cells were pyknotic or karyorhectic, mitochondria were swollen
and the cytoplasm displayed lytic areas. Cell boundaries in some parts of the interstitium were
lost. Cell organelles were scarce, but lysosomal structures abundant. (MRID # 452029-07)
Davies etal. (1994) exposed three fish species to 0.9, 3.0, 10, 50 and 340 [j,g/L atrazine for a
period of 10 days and measured effects on growth and properties of various tissues, such as
blood, muscle and liver. Statistically significant (p < 0.05) effects occurred at levels as low as
0.9 and 3.0 (J,g/L. The most sensitive, consistent statistically significant effect was with the
species Galaxias maculatus at 10 |ig/L (i.e., 144% increase in muscle RNA/DNA levels), and the
DNA levels were significantly reduced 25%. In Pseudaphritis urvillii consistent significant
effects were found on glutathione (GSH) in the liver at 50 |ig/L (24% reduction) and 340 [j,g/L
(13%) reduction). Consistent, significant effects with rainbow trout were found at 50 and 340
[j,g/L (i.e., reductions of 15% and 14%, respectively, in protein levels in muscle); and at 350 |ig/L
(159%) reduction in growth and a 23% increase in glucose levels) (MRID # 452029-04).
Alazemi etal. (1996) reported gill damage to a freshwater fish; the damage was characterized by
the presence of breaks in the gill epithelium at 500 p,g/L which developed into deep pits at 5,000
Hg/L (MRID 452029-05).
Hussein et al. (1996) exposed two Nile River fish (iOreochromis niloticus and Chrysichthyes
auratus) to 3,000 and 6,000 p,g/L atrazine for up to 28 days. Fish exposed to these
concentrations showed some clinical signs of toxicity, such as rapid respiration and increased
rate of gill cover movements; slower reflexes and swimming movements; reduction in feeding
activities; and loss of equibrium and death. These signs were more pronounced in C. auratus
than O. niloticus. About 25 percent of the treated fish had abdominal swelling (ascites) in the
two species. Exposure to 3,000 and 6,000 p,g/L resulted in significant (p < 0.01) decreases in the
number of red blood cells (RBC), hemoglobin and haematocrit levels compared to controls in
both species. While the data appear to show clear differences from controls, these conclusions
could not be verified from the data given in the article . The authors also reported significant (p
< 0.01) changes in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and
mean corpuscular hemoglobin (MCHC), serum components, and brain and serum AChE levels.
While some of these measurements also appear to show clear differences between 3,000 and
6,000 [j,g/L and the controls, such as brain and serum AChE, whether the effects are significantly
different than the controls could not be confirmed from the data presented in the study. (MRID #
452029-11).
Neskovic etal. (1993) exposed carp to atrazine concentrations of 1,500, 3,000 and 6,000 p,g/L
and found changes in the activity of some enzyme activity levels in serum and some organs.
Serum alkaline phosphatase levels were significantly (p < 0.05) higher at all test levels than in
controls. The greatest drop in alkaline phosphatase activity was found in the liver and ranged
from 26.1% (1,500 (J,g/L) to 50.2% (6,000 (J,g/L). Somewhat weaker effects were found on
glutamic-oxaloacetic (GOT) in the liver and kidney (p < 0.1). No statistically significant (p <
18
-------
0.01) effects were found on glutamic-pyruvic transaminase (GPT). Histopathological effects
include damage to gills ( > 1,500 |^g/L), liver (almost normal at 1,500 [j,g/L and vacuolization of
hepatocytes at > 3,000 (J,g/L), kidney (more or le[j,[j,g/L) and intestine (slightly greater
lymphocyte infiltration and stronger mucous secretion at 6,000 (J,g/L) (MRID # 452029-13).
In addition, effects on olfactory function of Atlantic salmon (Salmo salar) were reported by
Moore and Waring (1998) when mature male Atlantic salmon {Salmo salar L.) parr were
exposed to nominal concentrations of 0.5, 5, 10, and 20 [j,g/L atrazine. Measured exposure
concentrations in the study were 0.04, 3.6, 6.0 and 14.0 [j,g/L and represented 8, 72, 60, and 70
percent of nominal concentrations, respectively. There appears to be uncertainty about actual
exposure concentrations because the water samples were collected only after the test period, and
the authors concluded that atrazine in the water samples suffered rapid degradation as the result
of an unavoidable delay in being analyzed (MRID # 452049-06).
A.2.4b Sublethal Effects: Freshwater Fish (New (2006) Open Literature
Data)
Three open literature studies on the potential of atrazine to induce sublethal effects in fish,
including salmon, rainbow trout, and channel catfish, are summarized in Table A-15. Waring
and Moore (2004; Ecotox Reference # 72625) exposed salmon smolts to atrazine under flow-
through conditions for 7 days. Effects on gill physiology were evaluated. Also, effects on
survival from exposure in freshwater and subsequent transfer to atrazine free saltwater were
evaluated. These data suggest that gill physiology, represented by changes in Na K ATPase
activity and increased sodium and potassium levels, was altered at 1 ug/L and higher. In
addition, transfer of fish exposed to atrazine in freshwater at 1 ug/L and higher into atrazine-free
saltwater resulted in mortality; 43% mortality was observed at 5 ug/L and higher after 24 hours;
15% of fish exposed at 1 ug/L died (all controls survived). However, it is uncertain if the effects
observed in this study are applicable to environmental conditions. For example, salmon were
exposed to atrazine in freshwater then moved directly to full salinity sea water. It is uncertain if
more gradual changes in salinity after freshwater exposures would also produce similar effects.
Also, insufficient information was available for an independent evaluation of data adequacy and
verification of statistical analyses. Taken together, these data provide evidence that atrazine
exposure may affect gill physiology; however, toxicity values from this study are not used to
quantify potential risks due to uncertainties in the correlation between the effects reported from
this study in salmon and survival or reproductive effects in fish (and amphibians) considered in
this assessment. There are additional uncertainties associated with the extrapolation of effects
observed in the laboratory to more variable exposures and conditions in the field. Therefore,
these data will be used to qualitiatvely characterize potential risks.
Moore and Lower (2001; Ecotox Reference # 67727) studied effects of simazine and atrazine
and mixtures of the two triazines on pheromone-mediated endocrine function in the male salmon
parr. This study suggests that short-term exposure of the olfactory epithelium of mature male
Atlantic salmon parr to atrazine (1.0 ug/L) significantly reduced the olfactory response to the
female priming pheromone, prostaglandin (PGF2a)- After parr were exposed to atrazine, the
19
-------
levels of plasma testosterone and 7,20P-dihydroxy-4-pregnen-3-one (17,20BP) were statistically
elevated above the control groups. The study authors suggest that exposure resulted in modified
androgen secretion within the testes. Atrazine exposure decreased the olfactory epithelium
response to the amino acid L-serine. Although the hypothesis was not tested, exposure of smolts
to the pesticides during the freshwater stage may potentially affect olfactory imprinting to the
natal river and subsequent homing of the adults. Overall, the relationship between reduced
olfactory response of males to the female priming hormone in the laboratory and reduction in
salmon reproduction (i.e., the ability of male salmon to detect, respond to, and mate with
ovulating females) in the wild is not established. In addition, EPA (2001) did not use these data
in the development of aquatic life water quality criteria for atrazine because the test material was
not adequately described or translated. Furthermore, the study did not determine whether the
decreased response of olfactory epithelium to specific chemical stimuli would likely impair
similar responses in intact fish. Therefore, the results of the study will be qualitatively discussed
only.
Birge et al. (1983; Ecotox Reference # 19124) suggests that atrazine exposure to developing fish
may induce abnormalities. Specific types of abnormalities associated with atrazine exposure
were not listed by Birge et al. (1983), although the report notes that defects of the head and
vertebral column, dwarfed bodies, partial twining, microcephaly, absent or reduced eyes and
fins, and amphiarthrodic jaws were reportedly most common across the studies and species.
Effect levels (e.g., EC50) for incidences of abnormalities were not presented; however, the LC50
(calculated using mortality + terata incidence) for rainbow trout and channel catfish were 870
ug/L and 220 ug/L, respectively. Comparison to the LC50s in rainbow trout in submitted studies
(no studies in channed catfish have been submitted) suggests that embryo-larval stages may be
more sensitive than more developed life stages of trout, which are typically exposed in acute
toxicity studies. Developmental abnormalities were generally observed at atrazine levels that
also induced mortality. Overall, sufficient information was not included in the study report to
allow for an independent evaluation of data adequacy or verification of statistical analyses.
Therefore, these data are used in a qualitative manner to provide additional characterization of
potential risks to atrazine exposure in the current ecological risk assessment. These data were
not used in the calculation of risk quotients.
20
-------
Table A-15. Freshwater Fish Sublethal Effects Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name)
and Age and/or
Size
Test
Design
Endpoint Concentration
in ppb
Citation
(EcoRef. #)
Rationale for Use in Risk
Assessment(1)
Physiological
changes in gill
and survival
Salmon smolts
Fish were exposed to
atrazine for 7 days at
atrazine concentrations of 1
- 23 ug/L under flow
through conditions.
Emndpoints evaluated
included gill physiology and
survival.
Temp: 10-12.5 deg. C
pH: 7.6
Solvent: Industrial
methylated spirits
Effects on gill physiology
were observed in at least one
experiment at 2 ug/L and
tiigher. Effects included
altered Na K ATPase activity,
increased sodium levels, and
increased potassium levels.
Transfer of fish exposed to
atrazine in freshwater at 1
ug/L and higher into atrazine-
free saltwater resulted in
mortality; 43% mortality was
observed at 5 ug/L and higher
after 24 hours.
Waring and
Moore 2004
(72625)
Qual: These data suggest that
atrazine exposure in
freshwater at sublethal levels
in salmon smolts may
compromise their ability to
survive in saltwater.
However, uncertainties in this
study compared with field
conditions, reporting
deficiencies, and use of
unacceptable solvent preclude
its use in quantifying potential
risks.
Olfactory
detection of
female priming
pheromone,
protogandin F2«
in FW fish
30 min exposure
Simazine,
Atrazine, and
Simazine/
Atrazine
mixtures (% a.i.
NR)
Mature male
Atlantic salmon
(Salmo salar L.)
^arr; length =140
mm; weight = 34.2
g)
>ource:
Environment
Agency, Cynrig
latchery, Wales
Skin and cartilage removed
to expose olfactory rosettes
Olfactory epithelium
perfused with control water
for 30 min, then to atrazine-
treated water at nominal
concentrations of 0.1, 0.5,
and 2.0 ug/1 for 30 min
[results from 0.1 ug/L not
reported presumably due to
lack of atrazine detection at
this concentration].
Significant reduction in the
priming response of male
salmon to PGF2a (increased
levels of expressible milt not
present following exposure to
PGF) were observed at 1.0
ug/L (possible effects were
also observed at 0.5 ug/L).
Moore, A., and
N. Lower, 2001
(67727)
Qual: Endpoint not clearly
directly relevant to assessment
endpoint for freshwater fish.
Developmental
study; Atrazine
technical
unspecified
purity
Rainbow trout
Eggs were exposed for 24
days then hatchlings were
exposed for 4 days at
atrazine concentrations of 28
to 4800 ug/L under flow
through conditions.
Incidence of "gross
debilitating" anomalies was
evaluated.
Temp: 12-14 DegC
pH: 7 -7.8
LC50 (combined mortality +
terata incidences) in rainbow
trout was 870 ug/L.
Birge et al.,
1983. ,
(19124)
Qual: No raw data were
provided and reporting
deficiencies were noted.
LC50S were based on
combination of abnormalities
and mortality.
A.2.4c Sublethal Effects: Amphibians (Summary of the White Paper):
Since the January 2003 IRED, the Agency has conducted an evaluation and review of atrazine
effects data on amphibian gonadal development. This information was presented in the form of a
white paper for external peer review to a FIFRA Scientific Advisory Panel (SAP) in June 2003.
In its white paper (EPA, 2003) dated May 29, 2003, the Agency summarized 17 studies
consisting of both open literature and registrant-submitted studies involving both native and non-
native frog species
21
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(http://www.epa.gov/oscpmont/sap/2003/iune/finaliune2002telconfreport.pdf). Of the 17
studies, seven were laboratory-based, and ten were field studies. All studies were individually
evaluated with regard to the following parameters: experimental design, protocols and data
quality assurance, strength of cause-effect and/or dose-response relationships, mechanistic
plausibility, and ecological relevancy of measured endpoints.
Based on this assessment, the Agency concluded and the SAP concurred that there is sufficient
evidence to formulate a hypothesis that atrazine exposure may impact gonadal development in
amphibians; however, there are currently insufficient data to confirm or refute this hypothesis.
Overall, the weight-of-evidence, based on review of the 17 studies, does not show that atrazine
produces consistent, reproducible effects across the range of exposure concentrations and
amphibians tested. Deficiencies and uncertainties associated with the reviewed studies limit
their usefulness in interpreting potential atrazine effects. Specifically, the demasculinzing (i.e.,
decreased laryngeal dilator muscle area) effects were not replicated in multiple laboratories.
Additionally, the feminizing effects (i.e., intersex, hemaphroditism, and presence of ovotestes) of
atrazine were observed in three laboratory studies whose experimental designs could not be
reconciled and that reported significant effects at different concentrations: one at 25 |ig/L
atrazine and the other two at 0.1 |ig/L. While the feminizing effects observed in these different
studies were consistent qualitatively, there was no consistency across the studies in the reported
dose-response relationships. That inconsistency, together with the limitations in methodology in
each study, does not allow a reliable determination of causality or the nature of any dose-
response relationship. Although the Florida cane toads (Bufo marinus) monitored in the field
exhibited both demasculinizing effects (genetic males with female coloration) and feminizing
effects (oogenesis in male Bidder's organ), there were insufficient data to conclusively link
atrazine exposure to the phenomena. Thus, the available data do not establish a concordance of
information to indicate that atrazine will or will not cause adverse developmental effects in
amphibians.
Because of the inconsistency and lack of reproducibility across studies and an absence of a dose-
response relationship in the data, the Agency determined that the conclusions reached in the
January 2003 IRED regarding uncertainties related to atrazine's effects on amphibians have not
changed. The SAP supported EPA in seeking additional data to reduce uncertainties regarding
potential risk to amphibians (Scientific Advisory Panel, 2003). The data collection for additional
amphibian toxicity data has followed the multi-tiered process outlined in the Agency's white
paper presented to the SAP. In addition to addressing uncertainty regarding the potential of
atrazine to cause these effects, these studies will be helpful in characterizing the nature of any
potential dose-response relationship. A data call-in for the first tier of amphibian studies was
issued in 2005, and the studies are currently underway, although not yet complete. Therefore,
the results of the amphibian toxicity testing, which are expected to become available in 2007, are
not available for inclusion in this endangered species risk assessment.
22
-------
A.2.4d Sublethal Effects: Amphibians (New Open Literature Data)
Open literature data on sublethal effects of atrazine to amphibians, including frogs and
salamanders, are summarized in Tables A-16 and A-17 and discussed in the following
subsections. The following information includes studies identified as part of the 2006 open
literature search that were not reviewed as part the white paper discussed above.
Frogs (Anurans)
A total of eight studies on potential sublethal effects of atrazine to frogs were reviewed as part of
the open literature. Four of the eight studies were classified as acceptable to use in qualitative
sense and the other four were classified as unacceptable. Two of the four qualitative studies are
microcosm/mesocosm tests (one of which includes data for both frogs and salamanders), and two
are chronic lab studies. A review of the qualitative studies is provided below and summarized in
Table A-16. Studies were classified as qualitative because they address issues of concern to the
risk assessment, but are not appropriate for quantitative use due to uncertainties related to a lack
of raw data and limitations in the study design. In summary, the microcosm/mesocosm and
chronic lab data for frogs indicate that sublethal effects to amphibians, such as reduced mass and
length at metamorphosis, may occur at exposure concentrations of approximately 200 ppb and
higher under the conditions tested. Decreased frog weight (and length) at metamorphosis is
hypothesized to result from atrazine's effect on algal populations, which are a primary source of
food for developing anurans. Other factors, such as decreasing DO, pH, and macrophyte
biomass following atrazine exposure may also contribute to observed sublethal effects. In the
lab, plasma testosterone was reduced in male frogs at atrazine concentrations of 259 ppb;
however, an increase in aromatase activity (aromatase increases synthesis of 17P-estradiol
resulting in depletion of testosterone levels) was not observed. Therefore, the mechanism
associated with decreased testorsterone levels in adult males is unclear. The observed effect
level of -200 ppb is greater than the aquatic community-level effect of 10-20 ppb documented
in the 2003 atrazine IRED. In addition, uncertainties and associated limitations in the design of
the reviewed studies are similar to the conclusions of the amphibian white paper.
The effects of technical grade atrazine (% ai unspecified) on survival, mass, and length at
metamorphosis, and days to metamorphosis of larval gray tree frogs (Hyla versicolor) inhabiting
artificial pond microcosms was studied by Diana et al. (2000; Ecotox Reference # 59818). The
interrelationship of these parameters and DO concentrations, water pH, and estimates of
phytoplankton, periphyton, and macrophyte biomass were also evaluated. Gray tree frog larvae
(40 larvae/treatment; 4 replicates/treatment) were exposed to nominal atrazine concentrations of
0, 20, 200, and 2000 ppb atrazine in artifical pond microcosms (16 plastic wading pools; 1.22-m
diameter w/ 90 L pond water) containing phytoplankton, periphyton, and the aquatic
macrophyte, marshpepper knotweed (Polygonum hydropiper). Microcosms were covered with
mesh fiber to exclue predators. Concentrations of atrazine measured in microcosms immediately
following addition were consistent with those intended and showed minimal variation within
treatment groups. By three weeks following addition of atrazine to the microcosms,
concentrations had declined by 21%, 9%, and 16% in the 20-, 200-, and 2000-ppb treatment
groups, respectively. Phytoplankton chlorophyll a concentrations declined slightly during the
23
-------
first week following atrazine addition (in all but the 200 ppb group) and, by Day 14, rebounded
above levels before exposure (in all but the 20 ppb group). Phytoplankton densities in the 200
and 2000 ppb groups increased significantly above the control during the rebound period. Over
the course of study (-40 days), chlorophyll a was lowest in control, highest in 200 ppb, and
intermediate in 20 and 2000 ppb groups. Macrophyte biomass at the end of the study was
decreased, relative to controls, by 30%, 98%, and 99% in the 20, 200, and 2000 ppb groups,
respectively. DO decreased to approximately 20 and 40% of pre-exposure values in the 200 and
2000 ppb treatment groups after 1 d of atrazine treatment. DO in these microcosms returned to
control concentrations by 10 d after treatment, but declined again to approximately 60 to 80% of
control values at 21 d after treatment and remained depressed for the remainder of the study. In
the 200 and 2000 ppb groups, pH decreased similarly within 1 d of atrazine treatment and
returned to control values after 16 d. The DO and pH did not differ significantly between the 0
and 20 ppb groups or the 200 and 2000 ppb groups. Frogs from the two higher treatment groups
were statistically shorter (5% reduction) and had lower body weight at metamorphosis (10%
reduction) than those from the control and low atrazine groups. No difference in length or body
mass at metamorphosis was detectable between the 0 and 20 ppb groups or between the 200 and
2000 ppb groups. Time to metamorphosis was 5% longer in the 2000 ppb groups than in the 200
ppb group, but did not differ statistically from controls in any treatment group. No significant
treatment-related differences were detected for survival rate. Given the lack of decrease in
phytoplankton over time and the subsequent compensatory growth of phytoplankton following
atrazine treatment, it seems unlikely that the effects on amphibian development were due to a
decrease in food. However, the study author's postulate that atrazine-resistant species occurring
in presence of continued atrazine exposure may be less palatable, of lower nutritive value, or
toxigenic. The observed rebound of phytoplankton was likely due to elimination of
macrophytes. Given the modest decline in phytoplankton biomass and the marked effects of
atrazine on DO, it appears likely that the adverse effects on amphibian growth are mediated
primarily by decreased oxygen availability. Other amphibian larval species have shown
increased effort at gill respiration in the presence of low DO at the expense of feeding. Based on
observed decreases in length and mass at metamorphosis, and decreases in pH, DO, and
macrophyte biomass, the study authors suggest that these variables may lead to increased risks of
predation as well as decreased fitness to anurans at > 200 ppb atrazine. The corresponding
NOAEC for this study, based on decreased length and mass, is 20 ppb.
Boone and James (2003; Ecotox Reference # 81455) studied the post-application effects of
atrazine on body mass development, and survival of two anuran species (southern leopard frog,
Rana sphenocephala, and American toad, Bufo americanus) and two caudate species (spotted
salamander, Ambystoma maculatum, and small-mouthed salamander, A. texanum) reared in
outdoor cattle tank mesocosms containing leaf litter and plankton from natural ponds. Screen-
mesh lids covered each pond to exclude predators and other anurans. Animals used in the study
were free-swimming larvae. Natural factors of density and pond hydroperiod were also
considered. Atrazine was added as Aatrex (40.8% ai) at only one concentration of 200 ppb
(mean-measured concentration at Day 1 was 197 ppb). Atrazine (at 197 ppb) reduced
chlorophyll concentration of algal communities and resulted in reduced mass (for toads and
leopard frogs) and lengthened larval periods (for small-mouthed salamanders). While the
presence of atrazine did not cause mortality from reductions in food, it did statistically reduce
24
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metamorph size (i.e, weight). During metamorphosis, salamander larvae lose their gills and
develop lungs that enable it to breathe air. Because size at metamorphosis has been positively
correlated with overwinter survival and future reproduction, atrazine may affect population
dynamics when it reduces metamorph size. Atrazine also interacted with density and decreased
leopard frog survival as compared to the high density (60 tadpoles/1000 L) control group.
According to the study author's, this observation suggests that atrazine reduced the food supply
of leopard frog tadpoles to some extent and increased the likelihood of starvation in high-density
conditions where food was scarcer.
Hecker et al. (2005; Ecotox Reference # 79287) studied the effects of atrazine (97% ai) on
CYP19 gene expression, aromatase activity, plasma sex steroid concentrations including
testosterone (T) and 17P-estradiol (E2), and gonad size (GSI) of adult sexually mature male
African clawed frogs (Xenopus laevis) in the lab for 36 days under static renewal conditions.
Adult male frogs in 40-L aquariums (15 reps/treatment; 20 reps/control) were exposed to
atrazine at nomimal concentrations of 1, 25, or 250 ppb; respective measured concentrations
were 0.8, 24.6, and 259 ppb. There were no effects on any of the parameters measured, except
plasma T concentrations, which were significantly less (54 % reduction) in the 259 ppb group as
compared to untreated frogs. No significant increase in aromatase activity was observed;
therefore, the mechanism associated with decreased testorsterone levels in adults males has not
been demonstrated. The extent to which the suppression of T observed in frogs exposed to 250
ppb atrazine may affect reproductive functions in the wild is unclear. The authors concluded that
aromatase enzyme activity and gene expression were at basal levels in X laevis from all
treatments, and that the tested concentrations of atrazine did not interfere with steroidogenesis
through an aromatase-mediated mechanism of action.
Gucciardo (1999; summarized in Table A-16) exposed three frog species to technical grade
atrazine at concentrations ranging from 30 to 600 ug/L from the first feeding stage through
metamorphosis and evaluated potential effects on growth and development rate. Atrazine
exposure to A. crepitans at 300 ug/L was associated with delayed development (increased time to
metamorphosis) and reduced post metamorphic dry weight. No effects on the other two frog
species tested (R. sylvatica and R. pipiens) were observed. This study did not produce an effect
level more sensitive than than the NOAEC of 65 ug/L observed in submitted chronic fish studies.
25
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Table A-16. Frog Toxicity Tests from O
pen Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
End pomt
Concentration
in ppb
(significant changes as
compared to control)
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Amphibian Microcosm
Study (duration = 6
wks) / TGAI Atrazine
(% ai NR) w/acetone
solvent
¦ Larval gray tree frogs
(Hyla versicolor) 15 d
old and lid posthatch
¦ Aquatic macrophyte
marshpepper knotweed
(Polygonum hydropiper)
¦ Artifical pond microcosms (16
plastic wading pools, 1.22 m
diameter) w/ 90 L pond water
(including phytoplankton &
macrophytes) used. 5
macrophytes were added to each
pool.
¦ Treatment levels (nominal
cone) = 0, 20, 200, and 2000 ppb
(and solvent control)
¦ 40 larvae/treatment; 4
reps/treatment
¦ Microcosms covered to exclude
predators.
¦ Endpoints: Survival, mass, and
length at metamorphosis; days to
metamorphosis; relationshiop of
amphibian endpoints to DO, pH,
and estimates of phytoplankton,
periphyton, and macrophyte
biomass
¦ Survival: no effect;
NOAEC = 2000 ppb
¦ Mass: 10% reduction
(p < 0.001) at 200 ppb
(LOAEC);
NOAEC = 20 ppb
¦ Length: 5% reduction
(p < 0.001) at 200 ppb
(LOAEC);
NOAEC = 20 ppb
¦ Larval period: no
effect; NOAEC = 2000
pbb
Diana et al.,
2000
(59818)2
QUAL:
¦ no raw data provided
¦ pre-metamorphosis
weight and length were
not determined
26
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Table A-16. Frog Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Free-swimming larvae of
2 anuran species and 2
caudate species:
- Southern leopard frog
(.Rana sphenocephala)
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
Endpoint
Concentration
in ppb
(significant changes as
compared to control)
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Amphibian Mesocosm
Study (duration 56-58 d)
I Atrazine formulation
(Aatrex, 40.8%ai)
American toad
[Bufo americanus)
- Spotted salamander
(Ambystoma maculatum)
- Small-mouthed
salamander
(A. texanum)
phytoplankton
- Mesocosm design:
polyethylene cattle tank ponds
(1.85 m in diameter; 1480 L
volume) containing 1000 Ltap
water, 1 kg leaf litter from mixed
deciduous forest, and plankton
from natural pond (500 mL/pond
at 6 times).
Mesh lids covered each pond to
exclude predators and anuran
colonists
- Atrazine added at nomimal
cone of 200 ppb and control
(Day 8 pH = 7.7; temp = 13.3
°C) mean-measured
concentration at Day 1 exposure
197 ppb
- 3 reps/treatment
- Anuran low density = 20
tadpoles/1000 L; high density =
60 tadpoles/1000 L
Hydroperiod manipulated:
constant or drying
- Anuran and caudate species
reared separately and together
- Endpoints: body mass,
developmental stage, SVL (for
salamander larvae only), pond
survival for all species, time to
metamorphosis for toad and
small-mouthed salamander,
chlorophyll a content
Leopard frog
survival and
developmental stage: no
effect; NOAEC = 197
ppb
- survival x high density:
decrease relative to high
density control w/no
atrazine (p = 0.0235);
LOAEC = 197 ppb;
NOAEC = <197 ppb
- mass: decreased at 197
ppb (LOAEC) (p =
0.0052); NOAEC =
<197 ppb
American toad
-survival and time to
met: no effect; NOAEC
= 197 ppb
¦mass: decreased at 197
ppb (LOAEC) (p =
0.0040); NOAEC =
<197 ppb
Spotted salamander
no effect to survival,
mass, SVL, and dev.
stage; NOAEC = 197
ppb
Small-mouthed
salamander
Boone and
James, 2003
(81455)
survival and mass: no
effect; NOAEC = 197
ppb
-mass x hydroperiod:
decreased during drying
periods (p = 0.0202);
LOAEC = 197 ppb;
NOAEC = <197 ppb
- time to met: increasing
w/atrazine exp (p =
0.0084) and combination
of atrazine exp and
hydroperiod (p =
0.0093); LOAEC = 197
ppb; NOAEC = <197
ppb
Chlorophyll a\ reduced
at 12 h at 197 ppb (p =
0.0006)
QUAL:
no raw data provided
only one concentration
of atrazine tested
¦ % difference in effect
of atrazine relative to
control not presented
tap water used in all
ontrol and treatment
test solutions; however,
the chlorine content of
the tap water is not
;pecified
27
-------
Table A-16. Frog Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
Endpoint
Concentration
in ppb
(significant changes as
compared to control)
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Chronic (36 d) lab study
f Atrazine (97.1% ai)
African clawed frog
(Xenopus laevis); adult
sexually mature males
(30-50 g)
- 40-L aquariums (10-L exposure
solution).
Static renewal (50% test
solution renewed every 3 days)
at nominal concentrations of 0, 1,
25, and 250 ppb. Measured cone
(after 36 days = ND, 0.8, 24.6,
and 259 ppb)
15 reps/treatment; 20 reps for
the contol.
- Temp = 19.6 °C ±1.3 °C
- Photoperiod: 12 h light: 12 h
dark
Feeding: Nasco frog brittle
3x/wk ad libitum
Endpoints: Testicular
aromatase activity, CYP19 gene
expression, concentrations of
plasma sex steroids testosterone
(T) and 17p-estradiol (E2), and
gonad size (GSI)
- T concentration: 54%
decrease (p = 0.036) at
259 ppb (LOAEC);
NOAEC = 24.6 ppb
- Testicular aromatase
activity, CYP19 gene
expression, E2
concentration, and GSI:
no effect; NOAEC = 259
ppb
Hecker et al.,
2005
(79287)
QUAL:
no raw data provided
mechanism associated
with suppression of T is
unclear because
aromatase activity was
not increased
extent to which
suppression of T may
affect reproductive
functions in wild is
unclear
Chronic lab study I
atrazine (99% pure)
Cricket frogs (A.
crepitans), wood frogs
(R. sylvatica), Northern
leopard frogs (R.
pipiens)
Tadpoles were exposed to 30,
300, or 600 ug/L atrazine from
the first feeding stage through
metamorphosis. Growth rate,
days to metamorphosis,
metaphorphic success, and
juvenile weight and length were
evaluated.
A statistically significant
(p<0.05) delay in time to
metamorphosis and
decrease in post
metamorphic dry weight
was observed in A
crepitans at 300 ug/L
and above. No effects
on the other two frog
species tested were
observed.
A Frog Embryo
Teratogenesis Assay-
Xenopus resulted in a
96-hr EC50of 13.4
mg/L.
Gucciardo, 1999 QUAL: Study did not
(78286) produce the most
ensitive endpoint and
was not GLP; however,
the study appears to be
well reported and well
onducted. Not all raw
data were included in
the report.
^ QUAL = The paper is not appropriate for quantitative use but is of good quality, addresses issues of concern to the risk
assessment and is used in the risk characterization discussion.
^ Also reviewed as a field study. Phytoplankton density and chlorophyll a concentrations increased over the study duration (~40
days); however, macrophyte biomass was decreased, relative to controls by 30%, 98%, and 99% in the 20, 200, and 2000 ppb
groups. DO decreased to 60% and 80%) of control at 21 days and remained depressed for study duration. pH decreased w/in 1
day of exposure in 200 and 2000 ppb groups, but returned to control values following 16 days.
NR = Not reported.
The following four open literature frog toxicity studies were classified as invalid:
1. Sullivan and Spence, 2003 (Ecotox Reference # 68187; chronic lab study):
Classified as invalid because acetone was added to all atrazine treatment groups;
however, no solvent control was tested.
28
-------
2. Jooste et al., 2005 (Ecotox Reference # 79286; microcosm study): Classified as
invalid due to the presence of testicular oocytes in the reference control (57%)
relative to the atrazine treatment groups (39-59%).
3. Coady et al., 2004 (Ecotox Reference # 78295; chronic lab study): Classified as
invalid because atrazine was detected in the control sample.
4. Coady et al., 2005 (Ecotox Reference # 81457; chronic lab study): Classified as
invalid because atrazine was detected in the control sample.
Salamanders (Caudates)
A total of five studies on potential sublethal effects of atrazine to salamanders were reviewed as
part of the open literature. A discussion of these studies is provided below and summarized in
Table A-17. One of the five studies was classified as invalid. Of the remaining four studies, one
is a mesocosm study (including data for both frogs and salamanders), and the other three are
chronic lab studies. All of the test species in the reviewed open literature studies were
salamanders in the Ambystomatidae family or mole salamanders. Eggs of the Ambystomatidae
family hatch in the water into larvae that metamorphose into terrestrial adults. During
metamorphosis, the feathery external gills of the aquatic larvae are resorbed and lungs develop in
the adult terrestrial form. All reviewed studies were classified as acceptable for qualitative use
because they address issues of concern to the risk assessment, but are not appropriate for
quantitative use due to uncertainties related to a lack of raw data and limitations in the study
design. In summary, the reviewed studies contain variable results with respect to atrazine
exposures and sublethal effects to salamanders. Two chronic studies on the streamside
salamander (A. barbouri) and long-toed salamander (A. macrodactylum) show significant
reduced mass and snout-vent length (SVL) at metamorphosis, in addition to significantly
accelerated metamorphosis, relative to controls, at atrazine concentrations ranging from 184 to
400 ppb. The NOAEC values for these studies range between 18.4 and 40 ppb. In another
study, the time to metamorphosis was increased in small-mouthed salamanders at the only
concentration of atrazine tested (197 ppb); however, no effect in the time to metamorphosis was
observed in spotted salamanders (Ambystoma maculatum) at the same concentration of atrazine.
The interaction of atrazine and one of the iridoviruses (tiger salamander, Ambystoma tigrinum
virus, [ATV]) was studied in long-toed salamanders. ATV is an emerging iridovirus responsible
for epizootics in tiger salamanders through out western North America. Larvae exposed to both
atrazine and ATV had lower levels of mortality and ATV infectivity compared to larvae exposed
to virus alone, suggesting that atrazine may compromise virus efficacy or improve salamander
immune competency. Behaviorial changes in locomotion (i.e., increased activity following
tapping on tanks) were observed in streamside salamanders exposed to 400 ppb; however, this
endpoint is not relevant to the assesssment endpoints chosen for this risk assessment. It is
unclear how increased larval salamander activity due to tank tapping in the lab would translate
into reduced fitness in the wild. Conversely, increased larval activity could result in an increase
in predator avoidance.
The Boone and James (2003) mesocosm study, previously described and summarized in Table
A-16, studied the post-application effects of one concentration of atrazine (197 ppb) on body
29
-------
mass, development, and survival of two larval salamander species including the spotted and
small-mouthed salamanders. There were no effects on survival, mass, SVL, and developmental
stage of the spotted salamander following exposure to atrazine; however, the larval period of the
small-mouthed salamander was statistically lengthened at 197 ppb atrazine as compared to the
controls. According to the study authors, lengthened larval periods for salamanders may be a
result of atrazine increasing energy required for growth and development, although the
mechanism is not clear. Atrazine also interacted significantly with the hydroperiod treatment
(i.e., constant or drying), affecting both time and mass to metamorphosis and resulting in longer
larval periods in constant hydroperiods and smaller mass at metamorphosis in drying
hydroperiods.
Rohr et al. (2003; Ecotox Reference # 71723) exposed streamside salamander (A. barbouri)
embryos and larvae to atrazine (80% ai) for 37 days at nominal concentrations of 4, 40, and 400
ppb in the presence and absence of food. No effect on embryo or larval survival, hatching, or
growth (i.e., mass, SVL, and limb deformities) rates were observed at any of the test
concentrations. Systematically tapping of the tanks using a spring-loaded mousetrap caused
greater activity (observed as movement following the disturbance) in larvae exposed to 400 ppb
atrazine. The study authors attributed this startle response to a nervous system malfunction;
however, the reported malfunction is not statistically documented. In addition, the locomotion
behavioral endpoint is not relevant to the assessment endpoints chosen for this risk assessment.
Hunger stimulated a decrease in refuge use and an increase in activity; however this response
was least pronounced in the larvae exposed to atrazine at 400 ppb.
In 2004, Rohr et al. (Ecotox Reference # 81748) studied the combined effects of food limitation
and drying conditions on the survival, behavior, and metamorphosis of the streamside
salamander from embryo stage through metamorphosis at nominal atrazine concentrations of 4,
40 and 400 ppb. In general, food and atrazine levels did not interact statistically. Exposure to
400 ppb atrazine decreased embryo survival to Day 16 and increased time to hatching. However,
most embryo mortality was associated with a white film covering the embryo, suggesting the
presence of a fungal pathogen. It is unknown whether the fungi caused or simply followed
mortality. Delayed hatching could prolong time in streams and result in mortality from stream
drying or from aquatic predation. Drying conditions and food limitation decreased larval
survival, while 400 ppb atrazine only reduced larval survival in one of the two years tested. The
study author attributes the difference between the years in atrazine-related mortality to possible
condition-dependent mortality. Sublethal effects included elevated activity and reduced shelter
use associated with increasing atrazine cone (400 pbb) and food limitation. Although atrazine-
induced reduction in refuge use and increase in activity did not appear to strongly influence
feeding rates, they may elevate predation risk by increasing conspicuosness and encounters with
predators. Larval period was lengthened by food limitation and shortened by 400 ppb atrazine.
Earlier metamorphosis may provide a benefit to atrazine-exposed animals by reducing exposure;
however, their smaller size at metamorph could result in lower terrestrial survival, lower
reproduction and compromised immune function. Drying conditions accelerated metamorphosis
for larvae exposed to 0 and 4 ppb atrazine, but did not affect metamorphosis timing for the 40 or
400 ppb groups. Therefore, combined effects of stream drying and atrazine exposure may not
pose a greater threat to salamander larvae than either factor alone. Food limitation, drying
30
-------
conditions, and 400 ppb of atrazine reduced size at metamorphosis without affecting body
condition (relationship between mass and length), even though feeding rates did not differ
significantly among atrazine cones at any time during development. The authors suggest that
food limitations, drying conditions and atrazine exposure (at 400 ppb) have the potential to
contribute to decreased amphibian populations in impacted systems because atrazine levels of
400 ppb may result in increased larval energy expenditures, and reduced the feeding duration due
to a shortened larval period. The authors also suggest that because smaller size at
metamorphosis may result in lower terrestrial survival and lifetime reproduction,
Recent studies suggest that agricultural contaminants, such as atrazine, may have suppressive
effects on the amphibian immune system, thereby increasing susceptibility to parasites and
pathogens such as iridoviruses in the genus Ranavirus and the chytrid fungus (Batrachochytrium
dendrobatidis). A study by Forson and Storfer (2006; Ecotox Reference # 82033) tested the
interaction of emerging infectious diseases and atrazine (86.5% ai) in long-toed salamanders (A.
macrodactylum). 6-week old long-toed salamanders were exposed to Ambystoma tigrinum virus
(ATV; 0 or 103 5 plaque-forming units/ml) and sublethal concentrations of atrazine (0, 1.84, 18.4,
and 184 ppb) in a 4x2 factorial design for 30 days. The effects of atrazine and the virus were
tested on weight and snout-vent length (SVL) at metamorphosis and length of larval period as
well as on rates of mortality and viral infectivity. ATV transmission was confirmed, although
infection rates were lower than expected, consistent with the theory predicting lower pathogen
transmission to nonnative hosts. Larvae exposed to both atrazine and ATV had lower levels of
mortality and ATV infectivity (13.3% across all 3 atrazine concentrations) compared to larvae
exposed to virus alone (25%), suggesting atrazine may compromise virus efficacy or improve
salamander immune competency. The highest atrazine level (184 ppb) accelerated
metamorphosis and reduced mass and SVL at metamorphosis relative to controls. The authors
suggest that the mechanism for this effect may be an alteration of the neuroendocrine stress
pathway involving the thyroid hormones and corticoid hormones. Exposure to ATV also
significantly reduced SVL at metamorphosis. Atrazine alone had no significant effect on
mortality. The study suggests moderate concentrations of atrazine may ameliorate ATV effects
on long-toed salamanders, whereas higher concentrations initiate metamorphosis at a smaller
size, with potential negative consequences to fitness. Larger size at metamorphosis is correlated
with higher survival to maturity and reduced time to maturity, thereby increasing fitness relative
to smaller individuals. The study authors suggest that smaller size at metamorphosis may be a
fitness cost resulting from high-level atrazine exposure. Lighter, smaller animals may have
reduced terrestrial locomotor performance and, therefore, reduced ability to avoid predators or
capture prey. Smaller, newly metamorphosed adults also tend to have weakened immune
systems, which could make them more susceptible to disease.
31
-------
Table A-17. Salamander Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
End pomt
Concentration
in ppb
(significant changes as
compared to control)
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Chronic (37 d) lab
study / Atrazine
(80% ai)
Streamside salamander
[Ambystoma barbouri)
smbyos tracked through
larval development
¦ Static renewal (50% test
solution renewed every other
day)
¦ Tested in 3.7 L glass bowls
containing submerged,
translucent, gray semicircular
glass refuge plate
¦ Treatment levels (nominal
cone) = 4, 40, and 400 ppb
including DMSO solvent (and
solvent control containing
DMSO and acetone)
¦ 10 embryos/bowl; 4 reps/
treatment level
- Temperatue = 15 °C
- Photoperiod = 12:12 h
light:dark
¦ Feeding: larvae fed live
blackworms (.Lumbriculus
variegates) ad libitum
¦ Endpoints: Larval behavior in
presence and absence of food,
growth (mass and snout-vent
length [SVL]), and development
(limb deformities); hatching; and
survival
¦ Survival: no effect;
NOAEC = 400 ppb
¦ Growth (mass and
SVL): No effect;
NOAEC = 400 ppb
¦ Hatching: no effect;
NOAEC = 400 ppb
¦ Behavior: Systematic
tapping of tanks caused
greater activity (p <
0.05) in larvae exposed
to 400 ppb (LOAEC);
NOAEC = 40 ppb
Rohr et al.,
2003
(71723)
QUAL:
¦ no raw data provided
¦ solvent control
contained both DMSO
and acetone, whereas
the atrazine treatment
groups contained
DMSO only.
- DMSO is not an
acceptable solvent
because it accelerates
movement of a
chemical across cell
membranes; therefore,
it represents a worst
case scenario
¦ the locomotion
behavior endpoint is not
relevant to the chosen
assessment endpoints
for this risk assessment
32
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Table A-17. Salamander Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
Endpoint
Concentration
in ppb
(significant changes as
compared to control)
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Chronc (—117 d)
lab study /
Atrazine (80% ai)
Streamside salamander
{Ambystoma barbouri)
Embryos through
metamorphosis
¦ Static renewal (50% test
solution renewed every other
day)
¦ Tested in aquaria (37 L)
wrapped in black plastic,
containing refuge plates and a
strip of refuge above the water
line
¦ Treatment levels (nominal
cone) = 4, 40, and 400 ppb
w/DMSO solvent (included
DMSO solvent, but no negative
control)
¦ 31-40 embryos/aquaria; 6
reps/treatment
- Temperatue = 15 °C
- Photoperiod = 12:12 h
light:dark
¦ Feeding: 50% larvae fed live
blackworms ad libitum (high
food); 50% rationed 2.24 g
2x/wk (low food)
¦ Hydroperiods: constant or
lowered water level
¦Endpoints: embryo hatching and
survival to Day 16, larval
survival, larval activity and
refuge use, and metamorphosis
(mass, SVL, and time to met)
¦ Embrvo hatching and
survival: both reduced
at 400 ppb (p < 0.001);
LOAEC = 400 ppb;
NOAEC = 40 ppb
¦ Larval survival: no
effect in 2002; in 2003,
survival was reduced at
400 ppb (p = 0.003);
LOAEC = 400 ppb;
NOAEC = 40 ppb
¦Larval refuse use:
lower at 400 ppb (p <
0.034); LOAEC = 400
ppb; NOAEC = 40 ppb
¦Larval activitv: hisher
at 400 ppb (p = 0.007);
LOAEC = 400 ppb;
NOAEC = 40 ppb
¦Mass at met: reduced at
400 ppb (ppb (p =
0.022); LOAEC = 400
ppb; NOAEC = 40 ppb
¦ Time to met: shortened
at 400 ppb (ppb (p =
0.006); LOAEC = 400
ppb; NOAEC = 40 ppb
¦SVL at met: reduced at
400 ppb (ppb (p =
0.022); LOAEC = 400
ppb; NOAEC = 40 ppb
Rohr et al.,
2004
(81748)
QUAL:
¦There is uncertainty
associated with the
effect on embryos and
tiatched larvae because
of the presence of a
white film covering the
embryo, suggesting a
fungal pathogen, which
may have decreased
survival and increased
time to hatching
¦ Effects on larval
survival were different
for 2002 (no effect) and
2003 (significant effect
for 400 ppb treatment
compared to control)
¦ Metamorphic
parameters for 2003
included outliers and
were not included in the
analyses
¦ Duration of study not
specified
¦ No raw data provided
- DMSO is not an
acceptable solvent
because it accelerates
movement of a
chemical across cell
membranes
¦ No negative control
tested
33
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Table A-17. Salamander Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
End pomt
Concentration
in ppb
(significant changes as
compared to control)
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Chronic (30 day)
lab study /
Atrazine 90DF
(86.5% ai)
Long-toed salamander
{Ambystoma
macrodactylum) 6-weeks
old
¦ Static renewal (water changed
every 3 days)
¦Tested in round, polyethylene
containers (12.7 x 7.62 cm)
containing 500 ml artesian spring
water
¦ Treatment levels (nominal cone
= 0, 2, 20, and 200 ppb);
measured cone = 0, 1.84, 18.4,
and 184 ppb
¦ Also exposed to Ambystoma
tigrinum virus (ATV; 0 or 103 5
plaque-forming units/ml)
¦ Factorial 4x2 design
- Temperatue = 20 + 1 °C
¦ Photoperiod = 15:9 h light:dark
to mimic natural conditions
¦ Feeding: larvae fed live
blackworms 2x/wk ad libitum
¦ Endpoints:mass and SVL at
metamorphosis, larval period,
mortality, and viral infectivity
¦ Larval period
accelerated (p = 0.046);
mass (p = 0.002) and
SVL (p< 0.001) at met.
reduced at 184 ppb;
LOAEC = 184 ppb;
NOAEC = 18.4 ppb
¦ Mortality: no effect;
NOAEC = 184 ppb
- Mortality and ATV
infectivity: lower in
larvae exposed to both
atrazine and ATV
(13.3% across all 3
atrazine cone) as
compared to larvae
exposed to virus alone
(25%)
Forson and
Storfer, 2006
(82033)
QUAL:
¦ No raw data provided
(1) QUAL = The paper is not appropriate for quantitative use but is of good quality, addresses issues of concern to the risk
assessment and is used in the risk characterization discussion.
The salamander open literature toxicity study by Larson et al., 1998 (Ecotox Reference # 60632;
chronic lab study) was classified as invalid because atrazine was detected in the control sample.
A.2.5 Freshwater Invertebrates, Acute
A freshwater aquatic invertebrate toxicity test using the TGAI is required to establish the toxicity
of atrazine to aquatic invertebrates. The preferred test species is Daphnia magna. Results of this
test and others are summarized below in Table A-18.
Table A-18. Freshwater Invertebrate Acute Toxicity
Surrogate Species/ 96-hour LC50/EC50 fig/L
Static or (ppb) Toxicity MRID No.
Flow-through % ai (measured/nominal) Category Author/Year Study Classification
Midge 94
{Chironomus tentans)
Static test
Midge 85.5
(1Chironomus riparius)
Waterflea 85.5
{Daphnia magna)
720
(nominal)
1,000
(unknown)
3,500
(unknown)
highly toxic
highly toxic
moderately
toxic
000243-77
Macek et al. 1976
450874-13
Johnson 1986
450874-13
Johnson 1986
Supplemental
(48-hour LC50 &
raw data are missing)
Supplemental
(raw data are missing)
Supplemental
(raw data are missing)
34
-------
Table A-18. Freshwater Invertebrate Acute Toxicity
Waterflea < 24-hours old
(.Daphnia magna)
26-Hour static test
Waterflea
('Ceriodaphnia dubia)
48-Hour static test
Scud
{Gammarus fasciatus)
Static test
Stonefly (nymph)
(Acroneuria sp.)
Flow-through test
67.4 mg/L CaC03
Waterflea
{Daphnia magna)
Static test
Scud juvenile
(.Hyalella azteca)
Flow-through test
67.4 mg/L Ca C03
Scud juvenile
{Gammarus pulex)
Static-renewal - daily
Leech
{Glossiphonia
complanata)
Static-renewal weekly
Leech
{Helobdella stagnalis)
Static-renewal weekly
Snail
{Ancylus fluviatilis)
Static-renewal weekly
Waterflea <12 hr old
{Ceriodaphnia dubia)
Static 48-hour test
57 mg/L CaC03
Midge
{Chironomus riparius)
Static-renewal - daily
10-Day test
Midge
{Chironomus tentans)
Flow-through
10-Day test; water-spiked
exposure
97
94
3,600
(unknown)
> 4,900
(measured)
Slope - no mortality
5,700
(nominal)
6,700
(measured)
at least
moderately
toxic
unknown
moderately
toxic
moderately
toxic
000028-75
Frear & Boyd 1967
452083-09
Jop 1991
000243-77
Macekefa/. 1976
Brooke 1990
Supplemental
(unknown ai, 26-hour test
& no raw data)
Supplemental
(EC50 value not
determined)
Supplemental
(48-hour LC50&
raw data are missing)
Supplemental
(study not seen;
OW in draft WQC)
94
6,900
(nominal)
moderately 000243-77
toxic Macek etal. 1976
Supplemental
(raw data are missing)
14,700
(measured)
slightly toxic Brooke 1990
Supplemental
(no study; cited by
OW in draft WQC)
99.2
14,900
(measured)
4.4 @ 10 days
> 16,000
(measured)
6,300 fig/L @ 28 days
slightly toxic 452029-17
Taylor, Maund &
Pascoe 1991
slightly toxic 452029-16
Streit & Peter 1978
Supplemental
(raw data are missing)
Supplemental
(raw data are missing)
99.2 > 16,000
(measured)
9,900 |lg/L @ 27 days
99.2 >16,000
(measured)
> 16, 000 ng/L @
40 days
(35 % mortality)
>99 > 30,000
(measured)
Slope - no data
slightly toxic 452029-16
Streit & Peter 1978
slightly toxic 452083-05
Oris, Winner &
Moore 1991
slightly toxic 452029-17
Taylor, Maund &
Pascoe 1991
Supplemental
(raw data are missing)
Supplemental
(raw data are missing)
Supplemental
(raw data are missing)
>33,000
(measured)
18,900 fig/L @ 10 days
Mortality:
LC50 > 24,000
(measured)
(37% mortality)
NOAEC= 16,000
LOAEC = 24,000
Growth (dry weighty
EC50 = 8,300 (measured)
NOAEC <3,200
LOAEC = 3,200
slightly toxic
slightly toxic
000272-04
Drake 1976
459040-01
Putt, 2002
Supplemental
(raw data are missing)
(EC50 115 ppm exceeds
water solubility (33 ppm)
Supplemental
(does not fulfill any
currently-approved U.S.
EPA SEP guideline)
35
-------
Table A-18. Freshwater Invertebrate Acute Toxicity
Midge
{Chironomus temtans)
Static-renewal - to
maintain water quality
10-Day test; sediment-
spiked exposures
slightly toxic
Mortality (measured cone):
SED NOAEC = 130,000
SED LOAEC = 270,000
Pore Water (PW) NOAEC
= 26,000
PW LOAEC = 29,000
(14% mortality)
PW LC50 >30,000
Growth: Dry Weight
(measured concV
SED NOAEC = 24,000
SED LOAEC = 60,000
PW NOAEC = 4,000
PW LOAEC = 21,500
459040-02
Putt, 2003
Supplemental
(does not fulfill any
currently-approved U.S.
EPA SEP guideline)
Formulations
% ai
Product
Waterflea 79.6
(.Daphnia magna) 80 WP
Flow-through test
Waterflea 40.8
{Daphnia pulex) 4 L
Static test; 15EC
282 mg/L hardness
With & without sediment
49,000
(higher cones, than 31,000
ug/L were cloudy)
(measured)
slope 2.433
36,500
(nominal)
46,500
(with sediment)
slightly toxic
slightly toxic
420414-01
Putt 1991
452277-12
Hartman & Martin
1985
Supplemental
for formulation
(EC50 was not identified
due to insolubility)
Supplemental
for formulation
(EC50 exceeds water
solublity and low temp.)
Since the lowest LC50/EC50 is in the range of 0.1 to 1 ppm, atrazine is categorized as highly toxic
to aquatic invertebrates on an acute basis. The freshwater invertebrate LC50 value of 720 ppb is
based on an acute 48-hour static toxicity test for the midge, Chironomus tentans (MRID #
000243-77). The preferred test species, Daphnia magna, was not particularly sensitive to
atrazine; therefore, acute toxicity data from the midge {Chironomus tentans) was chosen as the
most sensitive endoint. The formulated end products were less toxic to aquatic invertebrates
than theTGAI.
Degradates: Acute aquatic invertebrate testing with Daphnia magna (72-2) was completed to
address degradate concerns. Table A-19 presents freshwater invertebrate toxicity data for
hydroxyatrazine.
Table A-19. Freshwater Invertebrate Acute Toxicity (Hydroxyatrazine)
Surrogate Species/
Flow-through or Static
% ai 48-hour EC5o (ppb)
formul. (measured/nominal)
Toxicity Category
MRID No.
Author/Y ear
Study
Classification
Waterflea
{Daphnia magna); 1st
instar (6-24 h old)
Static test
98 >4,100
(measured dissolved)
moderately toxic*
465000-01
Peither, 2005c
Acceptable
* Biological results for the study were based on the mean-measured concentration of dissolved Hydroxyatrazine,
which remained constant at the limit of its water solubility throughout the duration of the test. Therefore,
hydroxyatrazine is not acutely toxic to Daphnia magna at the limit of its water solubility.
36
-------
Although the freshwater invertebrate EC50 value (>4,100 ppb) for the degradate,
hydroxyatrazine, is within the range classifying it as moderately toxic, the biological results for
the study were based on dissolved (filtered) mean-measured concentrations of hydroxyatrazine,
which remained constant at the limit of its water solubility (3-4 ppm ai) throughout the duration
of the test (MRID 465000-01). Therefore, the potential toxicity of hydroxyatrazine appears to be
limited by its solubility.
A.2.6 Freshwater Invertebrate, Chronic
A freshwater aquatic invertebrate life-cycle test using the TGAI is required for atrazine since the
end-use product is expected to be transported to water from the intended use site and the
following conditions are met: the pesticide is intended for use such that its presence in water is
likely to be continuous; an aquatic acute LC50 is less than 1 mg/L; and the pesticide is persistent
in water {i.e., half-life greater than 4 days). The preferred test species is Daphnia magna.
Results of these tests are summarized below in Table A-20.
Table A-20. Freshwater Aquatic Invertebrate Life-Cycle Toxicity
Surrogate Species/
NOAEC/LOAEC
Study Duration/
Hg/L (ppb)
Flow-through or
(measured or
Statistically sign. (p=0.05)
MRID No.
Study
Static Renewal
% ai
nominal)
Endpoints Affected
Author/Y ear
Classification
Scud
94
NOAEC 60
25 % red. in development of Fi to
000243-77
Acceptable
(Gammarus fasciatus)
LOAEC 140
seventh instar.
Macek et al.
30 days / flow-through
(measured)
1976
Midge
94
NOAEC 110
25 % red. in Fo pupation
000243-77
Acceptable
('Chironomus tentans)
LOAEC 230
29 % red. in Fo adult emergence
Macek et al.
38 days / flow-through
(measured)
18 % red. in Fi pupation
1976
28 % red. in Fi adult emergence
Waterflea
94
NOAEC 140
000243-77
Acceptable
{Daphnia magna)
LOAEC 250
54 % red. in F0 young/female
Macek et al.
21 days / flow-through
(measured)
1976
Waterflea
99.2
NOAEC 1,000
452029-15
Supplemental
{Daphnia pulex)
LOAEC 2,000
16 % sign. red. in young/adult
Schober &
(no raw data for
28-Day static-renewal
(nominal)
Lampert 1977
statistical
analyses)
70-Day static-renewal test
31 % red. in young/adult
Waterflea - 6 generations
NR
Cups:
Supplemental
{Daphnia magna)
NOAEC 200
Kaushik,
(methods and
Static-renewal test
LOAEC 2,000
66 % reduction in # of young in
Solomon,
raw data are not
(unknown)
generations 4, 5, & 6.
Stephenson and
reported)
4 L aquarium:
Day 1985
NOAEC ??
LOAEC ??
72% reduction in # of young
(water from
treated corrals)
Leech
99.2
NOAEC <1,000
452029-16
Supplemental
{Helobdella stagnalis)
LOAEC 1,000
65% red. in percent hatch
Streit & Peter
(no raw data for
40 Days
(measured)
1978
statistical
Static-Renewal weekly
analyses)
37
-------
Table A-20. Freshwater Aquatic Invertebrate Life-Cycle Toxicity
Waterflea < 12 hr. old
>99
NOAEC 2,500
452083-05
Supplemental
{Ceriodaphnia dubia)
LOAEC 5,000
sign. red. in mean total number
Oris, Winner and
(no raw data for
Two 7-Day static-renewal
NOAEC 2,500
of young per living female
Moore 1991
analyses)
tests; Renewed M, W, & F
LOAEC 5,000
(3 broods)
57 CaC03; Temp. 25EC
(measured)
Green hydra (normal)
NR
NOAEC <5,000
452029-01
Supplemental
(Chlorohydra viridissima)
LOAEC 5,000
sign. red. in budding rates
Benson & Boush
(no raw data for
21-Day Static test
(nominal)
1983
analyses)
Waterflea 3-day old adult
>99
NOAEC 5,000
452083-05
Supplemental
{Ceriodaphnia dubia)
LOAEC 10,000
sign. red. in mean total number
Oris, Winner and
(no raw data for
Two 4-Day static-renewal
NOAEC 10,000
of young per living female
Moore 1991
analyses)
tests; Renewed M & W
LOAEC 20,000
(3 broods)
57 CaC03; Temp. 25EC
(measured)
Freshwater Snail
99.2
1,000
38-39% red. in egg capsules &
452029-16
Supplemental
(Ancylu sfluviatilis)
4,000
eggs in April/May
Streit & Peter
(no raw data for
40 Days
56-57% red. in eggs in April/May
1978
statistical
Static-Renewal weekly
16,000
15-16% red. in eggs in July/Aug.
analyses)
(measured)
68-73% red. in eggs in April/May
65-71% red. in eggs in July/Aug.
Leech
99.2
1,000
no reduction in egg production
452029-16
Supplemental
(Glossiphonia complanata)
4,000
17 % higher mortality
Streit & Peter
(no raw data for
27-Days
16,000
33 % higher mortality
1978
statistical
Static-Renewal weekly
(measured)
67 % higher mortality
analyses)
Growth stages and/or number of young are reduced by atrazine exposures for insects and
crustaceans. The most sensitive chronic endpoint for freshwater invertebrates is based on a 30-
day flow-through study on the scud (Gammarus fasciatus), which showed a 25% reduction in the
development of Fi to the seventh instar at atrazine concentrations of 140 ppb; the corresponding
NOAEC is 60 ppb (MRID 000243-77).
Daphniapulicaria was tested in a 12-day partial life cycle study to determine whether atrazine
has an effect on the sex ratio (Madsen, 2000). No male Daphnia young were found at measured
test concentrations 0, 0.93, 4.1, 8.7, 44, and 87 [j.g/L (MRID # 452995-04).
A.2.7 Freshwater Invertebrates, Acute Open Literature Data
Johnson et al. (1993) tested juvenile and mature freshwater mussels Anodonta imbecilis under
static conditions in a 48-hour acute study (summarized in Table A-21). These results suggest
that 48-hour exposures at atrazine concentrations up to 60 mg/L do not affect survival of A.
imbecilis.
Table A-21. Acute Aquatic Invertebrate Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
Endpoint Concentration
in ppb
Citation
(EcoRef. #)
Rationale for Use in Risk
Assessment(1)
38
-------
Table A-21. Acute Aquatic Invertebrate Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test Organism
(Common and
Scientific Name) and
Age and/or Size
Test
Design
Endpoint Concentration
in ppb
Citation
(EcoRef. #)
Rationale for Use in Risk
Assessment'1'
Acute toxicity
study in
freshwater snails
! 97% pure
Freshwater snail
A. imbecillis
juvenile and
mature organisms
Anodonta imbecillis (20/group) were
exposed to atrazine for 24-48 hours
under static conditions and evaluated
for survival. LC50 values were
estimated.
LC50 was >60 mg/L in both
juvenile and mature A.
imbecillis.
Johnson et
al. 1993
(50679)
Qual: Study suggests that
A. imbecillis is less
sensitive than other
invertebrates tested;
however, freshwater snails
are underrepresentated
taxa.
(1) QUAL = The paper is not appropriate for quantitative use but is of good quality, addresses issues of concern to the risk
assessment and is used in the risk characterization discussion.
A.2.8a Freshwater Microcosm/Field Studies (2003 IRED Data)
A summary of all the freshwater aquatic microcosm, mesocosm, and field studies that were
summarized as part of the 2003 IRED is included in Tables A-22 through A-24. Freshwater
microcosm data are presented in Table A-22. Summaries of mesocosm and limnocorral studies
for freshwater ponds, lakes, reservoirs are included in Table A-23 and natural and artificial
stream mesocosm data are summarized in Table A-24.
Walker (1964) treated Missouri ponds and plastic-lined limnocorrals with atrazine for aquatic
weed control at levels of 500 to 2,000 [j,g/L and quantitatively examined effects on bottom
organisms. Among the most sensitive organisms were mayflies (Ephemeroptera), caddis flies
(Tricoptera), leeches (Hirudinea) and gastropods (Musculium). The most significant reduction
in bottom fauna was observed during the period immediately following the application of
atrazine. Six to eight weeks after treatment, nine out of fourteen taxonomic groups had not
recovered. The total number of bottom organisms per square foot was 52 percent lower than in
the controls. In addition, three categories of invertebrates (water bugs, mosquitoes, and leeches)
were no longer present. (MRID # 452029-19).
Streit and Peter (1978) reviewed Walker's findings and investigated long-term atrazine effects on
three benthic freshwater invertebrates: Ancylus fluviatilis (Gastropoda - Basommatophora),
Glossiphonia complanata and Helobdella stagnalis (both: Annelida - Hirudinea) in the
laboratory (see Chronic Invertebrate toxicity table). Ingestion rates for G. complanata were
determined over a 27-day period at atrazine concentrations of 1,000, 4,000 and 16,000 ppb. The
total ingestion per individual was measured daily (except between Day 23 and 27). Two
significant results were: (1) Contaminated leeches ate significantly more limpets than the
controls (300, 345 and 405% of control ingestion rates for 1,000, 4,000 and 16,000 [j,g/L atrazine
exposures, respectively). (2) There was a constant feeding intensity from immediately after the
beginning of the exposure period. The same phenomenon was seen for snails, A. fluviatilis, but
the intensity of feeding was much less (i.e., 120, 130 and 140% of control ingestion rates at
1,000, 4,000 and 16,000 (J,g/L, respectively). Other observations included: (1) Leeches were
found sometimes lying on their backs suggesting that they may have difficulty staying firmly
attached to the substrate. (2) With increasing atrazine concentrations, an increasing percentage
-------
of snails could be detected that not wholly eaten. Similar effects were observed with the snails
which suggest that leech and snail behavior might be affected in some way. Compared to
controls, Ancylus egg production was significantly reduced after 40 days exposure to atrazine at
16,000 [j.g/L in March/April, April/May (68% fewer egg capsules and 73% fewer eggs) and
July/August (65%) fewer egg capsules and 71% fewer eggs). Lower Ancylus reproduction was
also found at 4,000 [j,g/L in April/May (56-57 percent) and July/August (15-16 percent). At
1,000 ug/L, fewer capsules and eggs were found only in April/May (38 and 39 percent,
respectively). The average number of eggs per brood in the leech, Glossiphonia complanata was
not affected by 27-days of atrazine exposure. Atrazine treatment did not affect the number of
live-born young of Helobdella stagnalis. At 1,000 and 4,000 (J,g/L only a part of the egg masses
developed. Only about 10 percent of the young in the 16,000 [j,g/L treatment hatched. Atrazine
did not affect the time for normal development (5-6 days). (MRID # 452029-16).
Kettle etal. (1987) monitored effects of atrazine (40.8%) on diet and reproductive success of
bluegill in experimental, Kansas ponds. The 0.045-hectare, 2.1-meter deep ponds were each
stocked with adult fish (50 bluegills, 20 channel catfish and 7 gizzard shad). On July 24, atrazine
was applied to two ponds at 20 (J,g/L, and to another two ponds at 500 |ig/L and two controls.
Atrazine concentrations were measured during the study and 70% of the original concentration
was detected at the end of the 136-day study. Bluegills were the only species to spawn during
the study. Atrazine had no significant effect on mortality of the original stocked fish, but the
number of young bluegills retrieved were significantly (p < 0.01) reduced compared to control
ponds (i.e., 95.7 % fewer in 20 [j,g/L-treated ponds and 96.1 % fewer in 500 |ig/L-treated ponds).
Stomach analyses of adult bluegills indicate that the bluegill controls had significantly (p <
0.001) higher numbers of food items per fish stomach and higher numbers of prey taxa per fish
stomach. The number of food items per stomach were reduced 85 and 78 percent in 20 and 500
[j,g/L -treated ponds, respectively. Reductions in taxa per stomach were 57 and 52 percent in 20
and 500 [j.g/L-treated ponds, respectively. Stomachs of bluegills from treated ponds had fewer
numbers of Ephemeroptera (p < 0.001), Odonata (p < 0.001), Coleoptera (p < 0.01) and Diptera
(not significant, p > 0.05) than the controls. The macrophyte community in treated ponds was
noticeably reduced, relative to controls, throughout the summer. Visual estimates of the
macrophyte communities in the ponds showed roughly a 60 percent decline in the 20 |ig/L ponds
and a 90 percent decline in the 500 [j,g/L ponds two months after atrazine addition. These
estimates were verified by rake hauls which produced these same relative differences. The
following May, 10 months after treatment, when macrophytes are normally well established in
Kansas ponds, the ponds were drained. Relative to control ponds, 20 [^g/L ponds had a 90
percent reduction in macrophyte coverage and the 500 [j,g/L ponds had a >95 percent reduction
in macrophyte coverage. Differences were noted in the macrophyte species present. Control
ponds contained Potamogeton pusillus and P. nodosus, Najas quadalupensis, and small amounts
of Chara globularis, whereas the treated ponds contained mostly C. globularis. (MRID #
452029-12).
40
-------
Tabic A-22. Freshwater Microcosm Tests
Application rate (II) sii/A)
N oin in si 1/ Meii su red ( one.
( 'oncentration ntTecting end point (time to effect)
o percent difference from eontrols
Narrative of Studv Trends
MRU) No.
Author/Yesir
Freshwater microcosm:
Measured close to nominal
throughout the testing
period: concentrations of
0.5, 5, 50, 100, 500, and
5000 ppb
0.5 and 5 ppb o no reduction in net oxygen loss
^ k 0 25-30% reduction in net oxygen loss
40-50% reduction in net oxygen loss
100 ppb 90% reduction in net oxygen loss
5^0(RpJ)pb q 100% reduction to negative net oxygen
production
Spirogyra, Oedogonium, Microcystis, Apthanothece, and
Scenedesmus sp. in mixed culture.
Microcosms inoculated with algae demonstrated effects at
concentrations >50 ppb. Physical appearance of the microcosms
was altered at 5,000 ppb. Observations and reculture demonstrated
that the effects were algistatic.
450874-07
Brockway et al., 1984
Freshwater Microcosm:
(Duration 7 weeks exposure)
Mean measured
concentrations
of 5.08 + 0.03 ng/L;
range: 4.2 - 6.0 jig/L
NOEC: 5 ppb
slight non-sign, shifts in water parameters:
DO decreased from means of 9.4 - 9.9 mg/L
(controls) differing weekly by 0.2 - 0.6 mg/L
0 pH decreased from means of 8.4 - 9.0 (controls)
differing weekly by 0.0 - 0.4 units
conductivity increased from 159.3 - 189.3 p,S/cm
(controls) differing by 0.2 - 10.0 jiS/cm
0 alkalinity increased from means of 1.4 - 2.2 mg/L
(controls) differing by 0.0 - 0.3 mg/L
0 no significant adverse effects on phyto- &
zooplankton, or 15 macro-invertebrate species
Cyclopoida sign, increased in week 3
Laboratory microcosms (4 replicates) were tested with 0 and 5 jug/L
atrazine for 7 weeks. The plankton and macro-invertebrates were
introduced together with 2-cm layer of natural sediments into glass
aquaria with a 50 cm water column with a 14-hour photoperiod.
Water was circulated through the microcosms at a flow rate of 3.5
L/min. during an acclimation period for biota of 3 months.
This test was part of a study of pesticide interaction between atrazine
and chlorpyrifos to determine the adequacy of chronic safety factors.
450874-17
van den Brink et al. 1995
Supplemntal
(raw data unavailable)
Freshwater Microcosm:
Mean measured
concentrations of 3.2, 10,
32, 110, and 337 ppb
NOEC: 10 ppb; LOEC: 32 ppb
dissolved oxygen, magnesium, and calcium;
NOEC: 110 ppb; LOEC: 337 ppb
potassium, chlorophyll-a, protein, and species
equilibrium number
Laboratory microcosms were inoculated with foam blocks taken
from a pond. The effect to protozoans from atrazine exposure was
examined by measuring structure (species number, biomass), and
function (colonization rate, oxygen production, chlorophyll
concentration) of the community as well as ion concentrations of the
biomass after 21 days.
450874-16
Pratt et al. 1988
Supplemental
(raw data unavailable)
Freshwater Microcosm:
(6 weeks)
Meas. peak 20 ppb on day
1, mean measured
concentration of
approximately 10 ppb
10 ppb (6 weeks)
o sign. (0.05) reduced dissolved oxygen (DO), but was
recovering by test termination
Laboratory microcosms were treated with a stock solution of
atrazine and soil to which atrazine was bound. At the end of the
study, no significant effects on plant biomass or daphnid/midge
survival were noted, but DO was affected.
452051-02
Huckins etal. 1986
Supplemental
(raw data unavailable)
41
-------
T.ihk* A-22. l-'ivslm silcr Microcosm Tcsls
Vppliisilioli liilr (II) ili V 1
Noiniiiiil Mciisiiivd ( one.
( oiu-i'iiiiiiiiuii jilTii liiii! ciHlpoinl dime I«»cITtvl i
o pnvciil (lilTcmii'c lioin (oniiols
Virr;ili\c o| s|inl\ 1 ivimK
MUM) No.
V11Ili«»r csii'
Freshwater microcosm:
(30 days):
Macrophytes, algae,
zooplankton and benthic
invertebrates;
Nominal conc. of 10, 100
and 1,000 ppb as a soil
slurry
10 ppb (Day 2)
0 23% red. in gross primary productivity (GPP);
recovery by Day 7 and similar to controls at Day 30
100 ppb (Day 2)
0 32% red. in GPP; recovery by Day 7 and similar
to controls at Day 30
1,000 ppb (Day 2)
91% red. in GPP; no recovery, 70% red.
throughout test
1,000 ppb (Day 30)
o 48% red. (sign. P<0.05 level) macrophyte biomass
o 36% red. (sign., P<0.05) Selenastrum dry weight
1,000 ppb (30-day aged microcosm water)
o 76% red. (sign. P<0.05) Selenastrum dry weight
1,000 ppb (Day 30)
o reduced O2, community respiration, pH
20% increase in conductivity
o 120% increase in alkalinity
0 no effect on soil microbial activity
4-L microcosms were established in the laboratory and treated with a
soil slurry of atrazine. The endpoints examined over the 30-day
experiment included effects to zoo- and phytoplankton as well as
macrophytes (i.e., Lemna sp., Ceratophyllum sp., and Elodea sp.).
Static acute and chronic assays were conducted with Daphnia
magna and Chironomus riparius using treated water that had come
from the microcosm after 30 days or from a vessel that contained the
treated water for 30 days (i.e., aged treated water). The author
concluded that microcosm itself ameliorated the phytotoxic effect at
1,000 ppb. No effect on invertebrates up to 1,000 ppb and effects to
phytoplankton at 10 and 100 ppb were not observed by test
termination (30 days). Conductivity, pH, and alkalinity were also
affected at 1,000 ppb.
450874-13
Johnson, 1986
Supplemental
(raw data unavailable)
Freshwater Microcosm:
Emergent vascular plants;
Nominal water conc. of 10,
50, 100, 500, and 1,500 ppb;
measured water conc. in the
50 and 500 ppb treatments
of 1.3 and 1.6 ppb,
respectively, after 16 weeks
500 ppb (6 weeks)
0 sign. (0.05 level) red. shoot length of Scirpus acutus
1,500 ppb (6 weeks)
0 sign. red. shoot length of Scirpus acutus and Typha
latifolia
Greenhouse microcosms were made by placing rhizome sections in
tubs which were filled with treated water to 1 cm above the soil
surface. The plants were allowed to grow for 16 weeks and shoot
height of hardstem bulrush and broad-leaved cattail was monitored
bi-weekly. Also non-sign, effects of chlorosis and reduced growth
noted at 50 and 100 ppb. A second test demonstrated resiliency of
both plants at 500 ppb.
450874-15
Langan and Hoagland, 1996
Supplemental
(raw data unavailable)
Freshwater Microcosm:
(14 days)
Measured atrazine
concentrations
approximately 75% of
nominal (15 and 153 ppb)
for first application and
150% of nominal (385 and
2,167 ppb) for the second
application
Sign. (0.1 level) reduction in turbidity and chlorophyll (7
days), and increase in phosphorous (day 14) and nitrogen
(days 7 and 14) after the 1st application. Copepod and
rotifer densities were also sign, reduced on days 7 and 14.
Sign, reductions in productivity, chlorophyll, green algal
colonies, rotifers, and Bosmina sp. (zooplankton) after 2nd
application. Phosphorous, nitrogen, and pH were also sig.
affected.
A 3x3 factorial design with three conc. of atrazine (0, 15, and 153
ppb) and three conc. of bifenthrin (0, 0.039, and 0.287 ppb) applied
as soil slurry in May, then again one month later but with atrazine
conc. of 0, 385, and 2,167 ppb and bifenthrin conc. of 0, 0.125, and
3.15 ppb. Atrazine alone caused dose-responsive reductions in
chlorophyll, turbidity, primary production, increases in nitrogen and
phosphorous, and reduced levels of chlorophytes, cladocerans,
copepod nauplii, and rotifers. General recovery after 14 days for
atrazine alone in the first phase, but recovery not complete at
sampling termination after second phase (14 days). No synergistic
or antagonistic effects were noted.
450200-14
Hoagland et al, 1993
Supplemental
(raw data unavailable)
42
-------
T.ihk* A-22. l-'ivslm silcr Microcosm Tcsls
Vppliisilioli liilr (II) ili V 1
Noiniiiiil Mciisiiivd ( one.
( oiu-i'iiiiiiiiuii jilTii liiii! ciHlpoinl dime I«»cITtvl i
o pnvciil (lilTcmii'c lioin (oniiols
Virr;ili\c o| s|inl\ 1 ivimK
MUM) No.
V11Ili«»r csii'
Freshwater microcosm:
(2 months; measured)
Nominal concentrations of
0, 60, 100, 200, 500, 1,000
and 5,000 ppb.
Measurements made three
times during the two month
study.
60 ppb (nominal)
o 14-carbon uptake decreased immediately after
treatment; recovery began after 10 days;
o stimulated production of chlorophyll a;
100 ppb (nominal)
o 14-carbon uptake decreased immediately after
treatment; recovery began after 10 days;
o stimulated production of chlorophyll a;
200 ppb (nominal)
o 14-carbon uptake decreased immediately after
treatment; slight recovery 2 months after treatment;
o stimulated production of chlorophyll a;
o inhibited increases in dissolved oxygen during light
phase and decreases in DO during dark phase
500 ppb (nominal)
o 14-carbon uptake decreased immediately after
treatment; no recovery;
o minimal inhibition of chlorophyll a production;
1,000 and 5,000 ppb (nominal)
o 14-carbon uptake decreased immediately after
treatment; recovery began after 10 days.
EC50s for Days 0-10, 53-60, & Mean (mean measured
conc.)
Time period; 14c uptake; DO (light); DO (dark)
Days 0-10: 103 ppb 126 ppb 106 ppb
Days 53-60: 159 ppb 154 ppb 164 ppb
Days 1-60: 131 ppb 165 ppb 142 ppb
Results of single species assays, microcosm, and pond studies were
compared. 14-Carbon fixation was used as the end-point for all three
study types. Laboratory results with eight algal species ranged from
37 to 308 ppb for carbon uptake inhibition EC50 values. Microcosm
EC50 values ranged from 103 to 159 ppb. The mean pond EC50 was
100 ppb for carbon uptake and 82 ppb for chlorophyll-a inhibition.
The authors stated that multiple laboratory studies or a microcosm
study represent(s) entire ecosystem functional effects.
450200-15
Larsen et ai, 1986
and
450874-19
Stay etal. 1985
Supplemental
(raw data unavailable)
Freshwater microcosm:
(60 days; measured)
Nominal concentrations of
60, 100, 200, 500, 1,000,
and 5,000 ppb.
Concentrations measured on
Days 7, 28, 53, 60.
NOEC < 60 ppb;
60 ppb (1-20 days)
0 sign. (0.05) red. 14-carbon uptake for first 20 days
>100 ppb (2 weeks)
o sign. (0.05 level) red. primary productivity;
o sign. red. in productivity/ dark respiration ratio;
o pH sign, less than control values
> 500 ppb (6 weeks)
o all endpoints declined immediately after treatment
and never recovered during the experiment.
Taub microcosms were 3-L jars inoculated with 10 algal species on
Day 0, Daphnia magna and 4 other animal species on Day 4. On
Day 7, 27 microcosms were treated with atrazine; no other atrazine
treatments um from four different aquatic systems. Community
metabolism was measured for primary productivity and light and
dark respiration. At the high treatment levels (500, 1000 and 5000
ug/L), all process variables declined immediately after atrazine
treatment and did not recover during the experiment. At the low
treatment levels (60, 100 and 200 ug/L), the magnitude of the
responses to atrazine was not constant, but with 3 phases; an
autotrophic phase, daphnid bloom and an equilibrium phase.
450874-19
Stay etal, 1989
Supplemental
(raw data unavailable)
43
-------
T.ihk* A-22. l-'ivslm silcr Microcosm Tcsls
Vppliisilioli liilr (II) ili V 1
Noiniiiiil Mciisiiivd ( one.
( oiu-i'iiiiiiiiuii jilTii liiii! cii100 ppb (2 weeks)
0 sign. (0.05 level) red. primary productivity
o sign. red. in productivity /dark respiration ratio
o pH sign, less than control values
Leffler microcosms were constructed with inoculum from four
different aquatic systems from natural communities and contains
organisms representing several trophic levels. The vessels were
dosed after 6 weeks of seeding and monitoring for 6 more weeks.
The LOEC for 3 of the systems was reported to be 100 ppb, while
the LOEC for the fourth was 200 ppb.
450874-18
Stay etal. 1989
Supplemental
(raw data unavailable)
I'iihlo A-23. l-'ivshwiiloi* Ponds. I.iikos. iiml Rcscnoirs (including Mcsocosms ;md l.imnocornds)
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMV i-Mci l i
o AITrtlril Specie" siimI 1 il'c N;ii»c
Virr;ili\c o| s|inl\ 1 ivimK
MUM) No.
tullior c;ir
Freshwater Lake: Plankton
(Duration 18 days)
Measured = >90% of
nominal over the test period
(18 days): nominal
concentrations of 0.1, 1, 10,
and 100 ppb
NOEC = <0.1 ppb
transient effects on water chemistry
1 ppb (1 week)
o decreased primary production;
0 increased bacterial numbers
decreased in zooplankton numbers
(cladocerans affected greater than copepods)
10 ppb (3 weeks)
o 65% sign, (p < 0.01) red. in daphnid population
growth (combined effect of water & algae)
0 59% sign, (p <0.05) red. in daphnid growth (algae)
100 ppb (3 weeks)
92% sign, (p < 0.01) red. in daphnid growth
(combined)
69% sign, (p < 0.01) red. daphnid growth (algae)
In situ enclosures in a German lake were treated and monitored over
18 days. Dose-responsive reductions in chlorophyll-a and oxygen
and increases in particulate organic carbon were observed at 1, 10,
and 100 ppb. Within 1 week at 1 ppb, primary production decreases
and bacterial number increases were observed. Zooplankton
numbers then decreased, with cladocerans affected more than
copepods. Additional studies at 0.1 ppb also demonstrated transient
effects on water chemistry and biological parameters. Most of the
parameters were recovered or were recovering within 42 days of
application.
450874-14
Lampert et al., 1989
Supplemental
(raw data unavailable)
44
-------
Tiihlc A-23. l-'ivslm silcr Ponds. I.iikcs. iind Resenoirs (including Mosocosms ;ind l.iiiinocornds)
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMV 10 ppb and some
zooplankton populations at 68, 182, and 318 ppb reflect indirect
functional links as a result of altered primary production, gg pp^
up to a 78% reduction in copepod nauplii was found and no increase
in the number of nauplii was found at 182 and 318 ppb. At 182 ppb,
threshold concentrations for direct effects by atrazine were exceeded
in several phytoplankton species. Diatoms appeared to become the
dominant phytoplankton at 182 and 318 ppb. One rotifer species
decreased at 182 ppb and another at 318 ppb and was virtually
absent from Day 18 to the end of the study. Daphnid reproduction
and populations decreased at 318 ppb.
45020022
Juttner et al. 1995
Supplemental
(raw data unavailable)
45
-------
Tiihlc A-23. l-'ivslm silcr Ponds. I.iikcs. iind Resenoirs (including Mosocosms ;ind l.iiiinocornds)
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMVb
sign. (0.05) 9% increase in fluorescence
0 sign. (0.05) 8% decrease in C-14 uptake
20cJ)pb
sign. (0.05) 30% increase in fluorescence
0 sign. (0.05) 12% decrease in C-14 uptake
500 ppb
sign. (0.05) 136% increase in fluorescence
0 sign. (0.05) 88% decrease in C-14 uptake
0
Field pond study results:
20 ppb
sign. (0.05) 51% red. C-14 uptake (4 hr.) (Days 2-7)
o sign. 42% red. phytoplankton biomass (Days 2-7)
0 3% red. growth & 28% red. daphnid reproduction
0 Simocephalus serrulatus correlated with food levels
500 ppb
pH red. 0.3 units lower than controls for a few weeks
o o dissolved 02 generally red. 1-3 mg/L (a few weeks)
sign. 94% red. C-14 uptake (4 hr.) (Days 2-163)
0 usually sign. red. phytoplankton biomass (Days 2-
° 136)
o rapid, nearly complete red. in abundant Peridinium
inconspicuum, a small dinoflagellate and rapid red.
in 7+ other dominate phytoplankton sp. after 7 days
incr. in several flagellate species; mainly Mallomonas
]9seudocoronata, Cryptomonas marssonii & C. erosa
zooplankton dominance shifted to rotifers, mainly
0 Keratella cochlearis after Day 31
o >50% red. in the copepod, Tropocyclops prasinus
mexicanus by Day 14
Single treatment of two 0.045 hectare ponds each with either 20 or
500 ppb atrazine produced dose responsive changes in pH, DO and
daily carbon uptake. Phytoplankton growth was reduced; population
shifts were apparent at 20 and 500 ppb. Effects on phytoplankton
were immediate, within 2 days, for daily carbon-14 uptake and
biomass declines at both treatment levels, which is consistent with
other researchers in laboratory tests. Atrazine concentrations down
to 1 ppb affected photosynthesis in lab tests with phytoplankton
samples from the pond. While atrazine produced direct toxic effects
on just certain members of the aquatic community, their responses
also affected other members of the community. At 500 ppb, one
species of herbivorous zooplankton declined by more than 75%
within 14 days of treatment.
Subsequent laboratory tests demonstrated some atrazine resistance in
phytoplankton and showed zooplankton population effects were due
to loss of food (algae). Further evidence of resistance was indicated
by a dominant phytoplankton species which showed less toxic
responses than the same species in the control pond.
450200-11
DeNoyelles etal. 1982
Supplemental
(raw data unavailable)
46
-------
Tiihlc A-23. l-'ivslm silcr Ponds. I.iikcs. iind Resenoirs (including Mosocosms ;ind l.iiiinocornds)
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMV cITirl i
o AITrtlril ;inil 1 ilf N;ii»c
Virr;ili\c o| s|inl\ 1 ivimK
MUM) No.
Vulli«»r csii'
Artificial freshwater ponds
in
Kansas treated with atrazine
to achieve concentrations of
20 and 500 fig/L
NOAEC < 20 j^g/L
20 jug/L - 29% increase in turbidity.
- initial depressed phytoplankton, followed by
an increase in standing crop and numerical
dominance of resistant species.
_ red. production of Naajas sp. and
Potamogeton spp. in areas excluding carp.
- increase in Chara
- 82% reduction in total insect emergence.
_ 89% red. in non-predator insect emergence.
- 90% red. Labrundinia pilosella emergence.
50% red. in total insect species richness.
_ 57% red. in non-predator insect species
richness.
100 jug/L - 62% increase in turbidity.
_ absence of periphyton on walkway supports,
increase in Chara sp.
83% reduction in total insect emergence.
95% red. in non-predator insect emergence.
_ 96% red. Labrundinia pilosella emergence.
71% red. in total insect species richness.
85% red. in non-predator insect species
richness.
5% red. in insect species evenness.
500 M-g/L - 65% increase in turbidity.
_ absence of periphyton on vascular plants.
_ absence of Chara sp.
70% reduction in total insect emergence.
85% red. in non-predator insect emergence.
_ 90% red. Labrundinia pilosella emergence.
59% red. in total insect species richness.
- 66% red. in non-predator insect species
richness.
- 15% red. in insect species evenness.
Two artificial Kansas ponds each (0.045 ha. and 2.1 m. deep) were
treated with technical atrazine at 20 |ig/L and 100 jug/L and with a
41% ai CO-OP liquid atrazine at 20 jug/L in 1981; two ponds served
as controls. The ponds were treated again on 30 May 1982, but the
41% ai ponds were converted to 500 jug/L with technical atrazine.
The macrophyte community in treated ponds was noticeably
reduced, relative to controls, throughout the summer. For 16
sampling dates between 8 May and 28 September 1982 insect
emergence was monitored in each pond with 4 emergence traps for
48 hour periods. No significant differences between ponds were
found in water level, temperature or oxygen levels. Mean turbidity
varied significantly among treatments (ANOVA), increasing with
increasing atrazine levels up to 100 jig/L.
The phytoplankton community responses to atrazine during the
present study corroborate results from the 1979 study by deNoyelles
etal. (1979). Macrophyte response also paralleled the 1979 study.
The presence of live plants of the primary emergent vegetation,
Typha spp., gradually decreased, as in previous studies, with
increasing atrazine concentration both within and outside carp
exclusion areas (Carney 1983, deNoyelles and Kettle 1983).
452277-06
Dewey 1986
47
-------
Tiihlc A-23. l-'ivslm silcr Ponds. I.iikcs. iind Resenoirs (including Mosocosms ;ind l.iiiinocornds)
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMV cITirl i
o AITrtlril ;inil 1 ilf N;ii»c
Virr;ili\c o| s|inl\ 1 ivimK
MUM) No.
Vulli«»r csii'
Artificial freshwater ponds
in Kansas treated with
atrazine to achieve
concentrations of 20 and
500 ng/L
NOAEC < 20 j^g/L
20 jug/L _ 60% sign, (p < 0.05) reduction in macrophtye
vegetation at summer's end including
elimination of Potamogeton pusillus,
P. nodosus, ScNajas quadalupensis;
95% sign, (p < 0.05) red. macrophyte coverage
in May, 10 months after treatment;
96% sign, (p <0.01) reduction in the number of
young bluegill;
85% sign, (p < 0.001) red. in the number of
food items/ fish stomach;
57% sign, (p < 0.001) red. in the number of
prey taxa/ fish stomach.
500 jug/L - 90% sign, (p < 0.05) reduction in macrophtye
vegetation at summer's end including
elimination of Potamogeton pusillus,
P. nodosus, ScNajas quadalupensis;
- >95% sign, (p < 0.05) red. macrophyte coverage
in May, 10 months after treatment;
96% sign, (p <0.01) reduction in the number of
young bluegill;
78% sign, (p < 0.001) red. in the number of
food items/ fish stomach;
52% sign, (p < 0.001) red. in the number of
prey taxa/ fish stomach.
Two artificial Kansas ponds each (0.045 ha. and 2.1 m. deep) were
treated with 20 jig/L and 500 jig/L on 24 July and two ponds served
as controls. The macrophyte community in treated ponds was
noticeably reduced, relative to controls, throughout the summer.
Visual estimates of the macrophyte communities in the ponds
showed roughly a 60 percent decline in the 20 jug/L ponds and a 90
percent decline in the 500 jug/L ponds two months after atrazine
addition. These estimates were verified by rake hauls which
produced these same relative differences. The following May, 10
months after treatment, when macrophytes are normally well
established in Kansas ponds, the ponds were drained. Relative to
control ponds, 20 jug/L ponds had a 90 percent reduction in
macrophyte coverage and the 500 |J.g/L ponds had a >95 percent
reduction in macrophyte coverage. Differences were noted in the
macrophyte species present. Control ponds contained Potamogeton
pusillus and P. nodosus, Najas quadalupensis, and small amounts of
Chara globularis, whereas the treated ponds contained mostly C.
globularis.
Significant indirect effects were found on bluegill diet and
reproduction.
452029-12
Kettle, de Noyelles, Jr.,
Heacock and Kadoum 1987
Supplemental
(raw data are not available for
analyses)
48
-------
Tiihlc A-23. l-'ivslm silcr Ponds. I.iikcs. iind Resenoirs (including Mosocosms ;ind l.iiiinocornds)
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMV50% red. in the copepod, Tropocyclops prasinus
mexicanus by Day 14
second application 120 - 165 ppb
sign. (0.05) 20% red. dissolved oxygen (Days 37-137)
0 sign. (0.05) 33% increase in Secchi depth
sign. (0.05) 62% increase dissolved inorganic carbon
° sign. (0.05) 103% increase in NO3-NO2-N
0 sign. (0.05) red. periphyton dry weight at depths of
0.5 and 1.5 m on most sampling days
0 sign. (0.05) red. deer, chlorophyll (19 days after
second appl. (Day 54 & on some days thereafter)
0 zooplankton dominance shifted to rotifers, mainly
Keratella cochlearis after Day 31
0 >50% red. in the copepod, Tropocyclops prasinus
mexicanus by Day 14
Elaboration of the 80 ppb treatment from Hamilton et ai, 1987.
After the first application (pulse), blue-green algae were eliminated
and organic matter was significantly reduced. After the second
pulse, organic matter, chlorophyll, biomass, and carbon assimilation
were reduced by between 36 and 67%, along with certain species of
green algae. Diatom numbers were greater in treatment limnocorrals
than in the control limnocorrals for nine weeks after the second
pulse.
450200-12
Herman et al., 1986
Supplemental
(raw data unavailable)
49
-------
Tiihlc A-23. l-'ivslm silcr Ponds. I.iikcs. iind Resenoirs (including Mosocosms ;ind l.iiiinocornds)
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMV100 ppb in the summer after application, but no
effects on microflora or fauna were observed. The year after
treatment (with 10 to 30% of atrazine still in the water column),
Chara sp. replaced Myriophyllum spicatum and Potamogeton natans
at levels >100 ppb. Phytoplankton became dominated with
cyanophytes and then cryptophytes as the concentration of atrazine
increased. Zooplankton numbers at 100 and 300 ppb were also
reduced the following year.
450200-17
Neugebaur et al., 1990
Supplemental
(raw data unavailable)
Measured = nominal (50
ppb) at time zero; declined
to 40% of nominal after 8
weeks
Aquatic plants and fish
Atrazine and esfenvalerate were applied together in mesocosms to
examine possible synergism (reduction of macrophytes leading to
extension of insecticide residues and increased fish mortality).
Combinations of 50 ppb atrazine and esfenvalerate at 0.25 to 1.71
ppb did not result in synergism. However, Chara sp. totally
replaced the co-dominant Naja sp. six weeks after application.
Fairchild et al., 1994
50
-------
Tiihlc A-23. l-'ivslm silcr Ponds. I.iikcs. iind Resenoirs (including Mosocosms ;ind l.iiiinocornds)
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMV
-------
1 iihlc A-24. l-'ivshwiilor \;Kur;il ;ind Arillicul Siiviims
Vppliisilioii liiic (lh iii V i
Nomiiiiil \lc;iMiml ( one.
( oiiii'iiliiilioii iilTci lill!! I'lMlpoinl (lime to dlrt l i
o MTct lt'tl iind IiIV- s|;iot.
N;MT;ili\c | ivmh
MUM) No.
Vulli«»r csii'
Small Canadian first-order
stream adjacent to a tiled-
corn field.
Atrazine of unspecified
purity was applied at 4 liters
per hectare on 6 June 1989.
The Canadian Water Quality
Guidelines (CCREM, 1987)
specify a guideline of 2.0 -
jig/L to protect freshwater
life.
Non-statistical pair-wise comparison of Total Phytoplankton
counts vs sta 9, the control indicates reductions at all
downstream stations with effects generally decreasing with
time and distance.
Downstream station 11 (2.5 km from atrazine source -sta. 5):
0.047 fig/L (range 0.004-0.2|ig/L) atrazine conc.
o all samples with reduced total phytoplankton counts
0 mean reduction of 63 % (range 6-97 %)
o highest red. (97 %) on June 9, first sampling day
o reduced 70 % in final sample on 16 Nov.
Downstream station 10 (50 to 75 m from sta. 5)
0.366 Jig/L (range 0.1 - 1.7 jug/L) atrazine conc.
o 2 out of 11 samples exceed count at sta. 9
0 mean reduction of 45 % (range +55 - 92 %)
o highest red. (92 %) on June 9
o reduced 47 % in final sample on 16 Nov.
Downstream stations 6 & 7 (a few meters from sta. 5)
0.81 (0.17 - 1.89) and 0.05 (0.001-0.224) ^g/L, resp.
o 1 out of 9 samples at sta. 6 exceeds count at sta. 9
o mean reduction sta. 6 of 53 % (range +68 - 99)
0 mean reduction sta. 7 of 66 % (range 3 - 95)
o highest red. (99 and 93 %, resp.) on July 21
o red. 45 & 27 %, resp. in final sample on 16 Nov.
Ditch (station 5) receiving waters from the 4 tile outlets:
2.62 jug/L (range 0.211 - 13.9 jig/L) atrazine conc.
o mean reduction of 79 % (range 46 - 99 %)
o highest red. (92 %) on 3 dates, June 23 - July 21
o reduced 51 % in final sample on 16 Nov.
Atrazine concentrations up to 20.39 jug/L (sta. 4) in field tile water,
13.9 jig/L (sta. 5) in receiving ditch and 1.89 jug/L in a small stream
(sta. 6) were measured in New Brunswick, Canada in a rural
headwater basin of the Petitcodiac River. The fist-order stream
flowed parallel to an 8-hectare sub-surface tile-drained field of
silage corn. The field was divided into 4 plots and each drained
separately into a small canal and into the stream.
Water, phytoplankton and zooplankton were sampled at 15-day
intervals at 11 sampling sites during the growing season.
Total phytoplankton numbers in downstream samples were
consistently much less than those from upstream (control) samples
during the period of low flow and higher atrazine levels (during the
summer). Diatoms dominated the phytoplankton community.
Occurrence of other algal species were erratic between stations and
over time. Zooplankton numbers were too low to discern trends, but
downstream samples were consistently lower in individuals than
control samples.
450200-08
Lakshinarayana, O'Neill,
Johnnavithula, Leger and
Milburn 1992
Supplemental
(Replication of samples and
statistical analyses were not
made)
52
-------
1 iihlo A-24. I'lvshwiilcr N;ilur;il iind Arilliciil Miv.ims
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \li-;is|MV<| ( one.
( oiu-i'iiiiiiiiuii jilTii lini! ciHlpoinl dime lo cITirl i
o MTctlctl specie* jiimI lili' s|;|or
N;MT;ili\c o|'smhI\ | ivmh
MUM) No.
Vulli«»r csii'
Artificial stream test:
(14 day; measured)
Simulated pulsed-
exposures;
5 jug/1 atrazine on Day 1 and
gradually diluted until only
about 1 |ig/L on Day 7
5 |ig/L to about 1 jug/L on Day 7
atrazine concentrations:
o
Day Mean conc.
4.74
1 3.56
10 1.20
14 1.19
Possible atrazine effect:
o 58 to 126 fold increase sign. (p<0.05) in number of
emergent insects on Days 3, 5 and 7;
treatment numbers were equal to or greater
than controls in all samples
No statistical effects found in atrazine treatments on:
periphyton growth measured as chlorophyll a levels;
chlorophyll a levels decreased gradually in all
samples (treatments & controls) over time,
"may have masked an effect of atrazine"
indirect effects on function or taxonomic
composition of benthic community structure
A community of benthic, stream invertebrates from the Patrick
Brook in Hinesburg, Vermont, located in the LaPlatte River
watershed. Microbial community growth was incubated for 2 weeks
this substrate was placed in lOx 10x7 cm polyethylene boxes and
placed in the stream for invertebrate colonization for 3 weeks in July
1993. During the same 3-week period glass slides were placed in
the stream for algal settling and growth.
Four benthic invertebrate boxes and 9 periphyton slides were
randomly placed in each of six replicate tanks. The flow rate was
calculated as 20.8 L/min. throughout the test. After a 24-hour
equilibration period, treatment at 5 j-ig/L atrazine was introduced to 3
replicates and 3 controls. On Day 3, about 15 percent of the water
was replaced; on Days 6 and 7 water replacements were 50 percent
each day; about 15 % was replaced on Day 11 during the 14-day
test.
"Dewey (1986) also observed herbivorous insects emerging earlier
from artificial ponds treated with 20 jug/L atrazine compared to
controls. Dewey suggested that the changes she saw were the
indirect effect of atrazine exposure, which had reduced the amount
of food available to herbivorous insects."
450874-11
Gruessner and Watzin 1996
Supplemental
(raw data unavailable for
statistical analyses)
Artificial stream tests:
(14 day; measured)
One dose and recirculation;
two atrazine levels (40.8%
ai):
15.2 + 1.4 and 155.4+1.4
jug/1 atrazine on Day 1;
17.5+ 1.2 and 135.0+ 4.5
jig/L on Day 28
Interaction test with alachlor
discussed under the section
on pesticide interactions.
15.2 Jig/L (initial atrazine concentration):
45% red. in benthic algal biovolume after 1 week
° sign, (p <0.05);
35% red. in benthic algal biovolume after 2 weeks
non. sign, (p < 0.05);
45% red. in benthic algal biovolume after 4 weeks
sign, (p <0.05).
155.6 Jig/L (initial atrazine concentration):
45% red. in benthic algal biovolume after 1 week
sign, (p <0.05)
0 50% red. in benthic algal biovolume after 2 weeks
sign, (p <0.05);
57% red. in benthic algal biovolume after 4 weeks
° sign, (p <0.05).
Time-dependent analyses showed sign, (p = 0.0083)
reduction in algal biovolume treated with both 15.2 and
155.6 jug/L atrazine throughout the test, but no sign, (p =
0.3629) difference between 15.2 and 155.6 fig/L levels.
A benthic mud community of epipelic algae were collected from
various locations of Wahoo Creek and acclimated for 6 weeks prior
to atrazine treatments. Stream water came from Wahoo Creek on
March 25, 1993. Wahoo Creek is a third-order, sediment-dominated
Nebraska stream draining primarily agricultural land and subject to
major runoff events.
Each model stream was constructed from a 114-L oval-shaped
plastic tub and lined with two-layers of 4-mil clear plastic. Stream
velocities ranged from 0.05 to 0.1 m/sec. in the sending segment and
0.01 to 0.05 m/sec. in the returning segment. Lighting was 12
hour/12 hour light/dark cycle. To replace evaporated water, stream
water from the transport tank was mixed for 24 hours prior addition
to each stream. Epipelic algae were sampled immediately before
herbicide atrazine addition, 24 hours after addition, and after 1, 2
and 4 weeks. Algal samples were analyzed for cell density, cell
biovolume and the relative abundance of 6 dominant taxa.
450200-02
Carder & Hoagland 1998
53
-------
Table A-24. I"reslm;i(er \;Kur;il ;iihI Arilliciil Mre.ims
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \lr;iMMV 0.05)
sign. (p<0.001) increase in night drift in number
of hydroptylid larvae on days 1, 2, 4, and 9
sign. (p<0.001) increase in night drift in number
0 of hydropsychid larvae on days 2, 4, and 9
The effects of invertebrate drift at site 2 were associated with
increased spray drift, during the 12 hours immediately
following application. Poor habitat and limited taxa at site 2
precluded drift analyses on specific taxa.
o no sign, affect on mean densities of benthic
invertebrates, number of taxa or taxa proportions
71% sign. (p<0.01) increase in trout population at
0 site 2 sustained over four months
no sign, effect on fish mortality or physiology
Tasmanian stream, Big Creek, with a catchment area of 36 km2 was
studied for atrazine aerially sprayed on two forest areas of 20 and 66
hectares, at rates of 3 and 6 kg ai/ha on 13 and 14 October 1987,
respectively. Three sampling sites were picked: Site 1 above the 2
plantations, sites 2 and 3 were just below each plantation. Each site
consisted of an upstream riffle for invertebrate samples and an area
100 m downstream for sampling brown trout (Salmo trutta).
Atrazine levels in 174 water samples from 44 sites from 24 streams
averaged 2.85 jLtg/L (range< 0.01-53 mg/L). Only 9.6% of samples
were below detection limit (O.ljig/L) and only 24 % were below 1.0
jig/L. In forestry areas, the mean stream conc. was 2.00 jLtg/L (range
<0.01-8.9 fig/L) with 35% below the detection limit of 1.0 fig/L.
The initial measured concentration in Big creek was 22 |ig/L, 2
weeks later atrazine averaged 2.5 (range 1.2-4.6) jLtg/L, and over the
following 2 months ranged from 0.01 to 0.09 |ig/L. Atrazine levels
in a small seepage draining the 2 plantations range 0.8- 68 jug/L over
the next 2 months. Site 2 sediments ranged from 1.6 to 22 jug/kg wet
weight two weeks after spraying.
No fish mortality or behavioral changes were recorded during
applications. However, brown trout movement within the
application area was significantly different (increased) than the
upstream control movement. No changes in trout physiology were
observed.
450200-03
Davies et al., 1994
(Species are not native to
North America;
Raw data unavailable for
statistical analyses)
TT
54
-------
1 iihlo A-24. I'lvshwiilcr N;ilur;il iind Arilliciil Miv.ims
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \li-;is|MV<| ( one.
( oiu-i'iiiiiiiiuii jilTii liiii! ciHlpoinl dime I«»cITtvl i
o MTctlctl specie* jiimI lilt' s|;|or
N;MT;ili\c o|'smhI\ | ivmh
MUM) No.
V11Ili«»r csii'
Artificial stream in
laboratory
Technical Atrazine: 98.2%
Experiment 1:
Constant 12-day exposures
at 0, 24 & 134 j^g/L atrazine
Experiment 2 involved
pulsed exposures of 4
herbicides mixed together at
nominal concentrations of:
Atrazine at 135 jug/L;
Alachlor at 90 f^g/L;
Metolachlor at 200 fig/L;
Metribuzin at 20 jug/L.
Full concentrations on Days
8 & 9, halved on Days 10 &
11, and discontinued on Day
12.
Constant 12-day exposure tests (Days 8-17) 10 and 25EC:
0 24 ng/L:
- 24% red. sign. (p<.001) in ash-free dry wt. at 25EC
- 30% red. sign. (p<.01) in chlorophyll a at 25EC
o 134 ug/L:
- 47% red. sign. (p<.001) in ash-free dry wt. at 10EC
- 31% red. sign. (p<.001) in ash-free dry wt. at 25EC
- 44% red. s ign. (P<.001) in chlorophyll a at 25EC
- 30% red. s ign. (P<.01) in chlorophyll a at 10EC
Nutrient uptake was affected more by the 15EC difference,
than the atrazine concentrations. Raw data were absent
and statistically analyses could not be assessed. As cited:
- 35% red. N uptake at 134 f^g/L at 10EC; not sign.
- 25% red. N uptake at 134 jig/L at 25EC; not sign.
- 31% red. silica uptake at 134 |ig/L at 10EC; not sign.
- 58% red. silica uptake at 134 jug/L at 25EC; not sign.
- 14% red. P uptake at 134 jig/L at 10EC; not sign.
- 8 % red. P uptake at 134 jig/L at 25EC; not sign.
Six artificial streams consisting of a 7.5 cm OD x 123 cm long Pyrex
glass tube were tested concurrently for pesticide effects on aufwuchs
productivity and nutrient uptake (NO2, NO3, phosphorus PO4 and
silica were tested after an 7-day colonization period with natural
waters from a third order stream in the Sandusky Basin, Ohio. Two
experimental designs (continuous and pulsed exposures) were tested
under constant lighting, flow rates of 7.8 mL/min. natural creek
water and 1.0 mL/min. nutrient water for 20-day periods.
Experiment 1. Two "streams" were exposed to continuous nominal
atrazine concentrations of 0, 50 and 200 jug/L at 25EC and then
repeated at 10EC on Days 8-17.
Experiment 2. Three streams were treated to pulsed exposures of a
mixture of four herbicides. These results are not relevant to the risk
assessment for atrazine.
450200-07
Krieger, Baker and Kramer
1988
Supplemental
(The solvent methanol
0.00057% v/v was not added
to controls;
raw data unavailable for
statistical analyses)
55
-------
1 iihlo A-24. I'lvshwiilcr N;ilur;il iind Arilliciil Miv.ims
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \li-;is|MV<| ( one.
( oiu-i'iiiiiiiiuii jilTii liiii! ciHlpoinl dime I«»i-Mci l i
o MTci li-il spi i ii s jiimI 1 ili- shim*
Vu niliw'o| siinl\ 1 ivimK
MUM) No.
V11Ili«»r csii'
Two artificial model streams
in laboratory continuously
exposed for 30 days with
60-day recovery period and
repeated 4 times in one year.
Nominal concentration of 25
jig/L technical grade
atrazine dissolved in
DMSO; atrazine
concentrations in streams
were not measured.
25 jig/L Atrazine:
After one year of 4 treatment and recovery cycles, it was
reported that the treatment did not have any significant or
lasting effect on macroinvertebrate population structure,
periphyton standing biomass or rates of primary production
and community respiration.
Two out of 200 statistical tests showed significant effects for
atrazine treatment: equitability (p < 0.029) during Winter,
month 3, and taxa/sample (P < 0.001) during the Spring,
month 3.
Macroinvertebrate drift in streams increased abruptly upon
injection in both controls and treatments which was
attributed to the solvent rather than to atrazine.
Initial drift samples were collected only in the autumn and
summer. Drift in the summer samples were "substantially
higher" in the atrazine-treated streams than in the DMSO-
treated control. Pulses in the number of drifting organisms
following toxicant/solvent injection were primarily due to
Baetis mayflies.
Continuous-flow stream treatment for 30 days at 25 ppb, followed
by 60 days of no treatment, and repeated 4 times for one year in
artificial, 3.96 m.-long concrete-lined streams inside a laboratory.
Invertebrate populations were introduced by colonization from
incoming drift with water flowing from a natural creek over a one
year period before treatment. Atrazine was injected into the flowing
water for periods as described above.
Benthic invertebrate populations as follows: two samples (10.2-cm
diameter cores) during pretreatment were collected at 45-day
intervals for 1 year. Three post-treatment samples were made every
30 days.
24-Hour invertebrate drift samples were collected were collected on
days 1, 5, 10, 20, and 29 during treatment and on days 14, 42 and 60
during recovery periods.
Dry and ash weights of periphyton standing crop on four 25 x 75
mm glass slides were sampled at 4-day intervals for 28 days before
and after each treatment.
24-Hour gross primary production and community respiration rates
(O2 levels) were measured during the autumn on days 2, 4, 8, 15, 24
and 29 after treatment and on days 20, 42, 54 and 60 during the
recovery period.
450200-09
Lynch et al, 1985
Supplemental
DMSO is not an acceptable
solvent, because it accelerates
the movement of chemicals
across cell membranes. As
such it represents a worst case
exposure.
Raw data were not available
for statistical analyses. Three
or four samples are considered
inadequate for field samples to
show anything short of severe
effects.
Artificial model streams in
laboratory:
(7 days; nominal)
Single applications to spring
water; Brazos, Texas.
Nominal test concentrations:
0, 100, 1000 and 10,000
Hg/L
o statistically significant reductions (*) in net stream
community productivity compared to controls:
Day 1 Day 3 Day 7
100 |ig/L 736%* 117%* 34%
1000 ^g/L 1367%* 227%* 119%*
10,000 jig/L 264%* n,
1716 %*
o sign. (p<0.02) increase in Nitzschia cell numbers
o no significant effect on other dominant algal groups
o no significant effect on community respiration rates
o no significant effect on conductivity or alkalinity
Four replicate recirculating artificial streams per treatment. Each
stream (2.43 m long, 12.5 cm wide and 6 cm deep) was lined with
polyethylene plastic and a single layer of gravel. Water from Minter
Spring is a nearly anoxic and has a constant temperature (21EC).
The flow rate was about 5 cm/sec. The principal algae genera were
Anabaena, Nitzschia, Rhopalodia and Navicula. Five weeks for
colonization of benthic algae on glass slides. Each stream received a
single treatment which was recirculated. Nominal conc. were 0, 0.1,
1.0 and 10 jig/L. Endpoints were net community productivity,
respiration rate, cell numbers of dominant species, conductivity and
alkalinity.
450200-10
Moorhead and Kosinski 1986
Supplemental
(raw data unavailable)
Not assayed, nominal conc.
of 5, 25, and 125 ppb
Snail
(.Lymnaea palustris)
Snails exposed to one time dosing in mesocosm of either 5, 25, or
125 ppb and monitored for 12 weeks, no affect on growth, fecundity,
or saccharide metabolism.
450200-13
Baturo et ai1995
56
-------
1 iihlo A-24. I'lvshwiilcr N;ilur;il iind Arilliciil Miv.ims
Vppliisilioli liilr (II) ili V 1
Noiniiiiil \li-;is|MV<| ( one.
( oiu-i'iiiiiiiiuii jilTii liiii! ciHlpoinl dime I«»cITtvl i
o MTctlctl specie* jiimI lili' s|;|or
\;UT;ili\r o| s|inl\ 1 ivimK
MUM) No.
V11Ili«»r csii'
Mean concentrations over
two months of 5, 10, 22, 68,
182, and 318 ppb
Phyto- and
zooplankton
Mesocosms in Bavaria were treated with atrazine 3 times over 3
summer months. Dose responsive reductions in dissolved oxygen
and pH were noted at concentrations greater than 5 ppb. Substantial
biological effects were generally noted at concentrations >182 ppb.
Some effects on copepod nauplii were noted at 68 ppb. Diatoms
appeared to become the dominant phytoplankton.
450200-22
Juttner et al., 1995
Supplemental
(raw data unavailable)
Nominal concentrations of
20, 100, 200, and 500 ppb.
Measurements bi-weekly or
monthly but results based on
nominal concentration
Phytoplankton
Results of single species assays, microcosm, and pond studies were
compared. Carbon fixation was used as the end-point for all three
study types. Laboratory results with eight algal species ranged from
37 to 308 ppb for carbon uptake inhibition EC50 values. Microcosm
EC50 values ranged from 103 to 159 ppb. The mean pond EC50 was
100 ppb for carbon uptake and 82 ppb for chlorophyll-a inhibition.
Authors stated that multiple laboratory studies or a microcosm study
represent(s) entire ecosystem functional effects.
450200-15
Larsen et al., 1986
Supplemental
(raw data unavailable)
57
-------
A.2.8b Freshwater Field Studies (New Open Literature Data)
Based on the results of the 2003 IRED for atrazine, potential adverse effects on sensitive aquatic
plants and non-target aquatic organisms including their populations and communities, are likely
to be greatest when atrazine concentrations in water equal or exceed approximately 10 to 20 [j.g/L
on a recurrent basis or over a prolonged period of time. Given the large amount of
microcosm/mesocosm and field data for atrazine, only effects data that are less than or more
conservative than the 10 [j,g/L aquatic-community effect level were considered. In addition, data
for taxa that are directly relavent to the endangered species evaluated as part of this assessment
were also considered. Field study data for amphibians, including frogs and salamanders are
included in Section D.2.3. Based on the selection criteria for review of new open literature, all
of the available studies show effects levels to freshwater fish and invertebrates at concentrations
greater than 10 (J,g/L.
One open literature artifical stream mesocosm study was reviewed because it provides data on
freshwater snails, which may be used as surrogate for the endangered dwarf mussel. The results
of this study, which are summarized as part of Table A-25, show potential indirect effects to
grazing behavior (i.e., increased searching velocity and movement patterns) at 15 [j,g/L atrazine,
due to a decrease in periphyton biomass (Roses et al., 1999; Ecotox Reference # 60860). No
significant effects were observed in rates of snail mortality and biomass. An increase in snail
activity may represent a change in resource quantity, resulting in increased searching speed when
the biomass of periphyton decreases.
Table A-25. Freshwater Mesocosm Study from Open Literature (2006 Review)
Study type/
Test material
Test Organism (Common
and Scientific Name) and
Age and/or Size
Test
Design
Endpoint
Concentration
in ppm
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Artifical stream
18 day exposure
Atrazine (% ai
NR)
Freshwater snails (Physa
acuta mdAncylus
fluviatilis)
¦ U-shaped artifical streams
(170 cm L x 20 cm W x 20
cm deep); water velocity =
1 cm/sec; depth = 1.9 - 2.2
cm; photoperiod: 8:16 h
light/dark; channel bottoms
contained surfaces for
algae attachment.
¦ Atrazine injected
continuously at 15 ppb in 3
ponds, 3 ponds = control
¦ Endpoints: snail
mortality, biomass, and
activity; chlorophyll a
concentration
LOAEC = 15 ppb
Sign, changes in grazer
behavior, increased
searching velocity, and
different movement
patterns at 15 ppb. No
sign, effects on snail
mortality or biomass
Roses, et al., 1999
(60860)
QUAL:
• no raw data provided
• only one atrazine
concentration tested
• relevance of increased
searching velocity in
mails to survival,
growth and
reproductive success is
uncertain
I~1) QUAL = The paper is not appropriate for quantitative use but is of good quality, addresses issues of concern to the risk
assessment and is used in the risk characterization discussion.
NR = Not reported.
58
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A.3 Toxicity to Estuarine and Marine Animals
A. 3.1 Estuarine and Marine Fish, Acute
Acute toxicity testing with estuarine/marine fish using the TGAI is required for atrazine because
the end-use product is expected to reach this environment due to its use in coastal counties. The
preferred test species is sheepshead minnow. Results of these tests are summarized in Table A-
26
Table A-26. Estuarine/Marine Fish Acute Toxicity
Surrogate Species/
Static or Flow-through/
Salinity & Temperature
% ai
96-hour LC50 (ppb)
(measured/nominal)
Probit Slope
Toxicity Category
MRID No.
Author/Y ear
Study
Classification
Sheepshead Minnow
larvae < 24-hours old
{Cyprinodon variegatus)
Static test, T - 20EC
Salinity 25, 15, 5 g/L;
97.1
Sal. 25 g/L 2,000
Sal. 15 g/L 2,300
Sal. 5 g/L 16,200
(measured)
Slope - no data
moderately toxic
452083-03 &
452277-11
Hall, Jr., Ziegenfuss,
Anderson, Spittler &
Leichtweis 1994
Supplemental
(no raw data
on mortalities)
Spot
(.Leiostomus xanthurus)
Static test
Salinity - 12 g/L; T - 22±1EC
97.4
8,500
(nominal)
Slope - no data
moderately toxic
452029-20
Ward &
Ballantine 1985
Supplemental
(no raw data)
Sheepshead minnow
{Cyprinodon variegatus)
Flow-through test
Salinity - 31 g/L; T - 22-23EC
97.1
13,400
(measured)
Slope 4.377
slightly toxic
433449-OI
Machado 1994
Acceptable
Spot (juvenile)
{Leiostomus xanthurus)
Flow-through test
Salinity - 29 g/L; T - 28EC
99.7
> 1,000
(nominal)
Slope - none
unknown
402284-01
Mayer 1986
Supplement
(48-hour test)
Sheepshead minnow
{Cyprinodon variegatus)
Flow-through test
97.4
> 16,000
(30 % mortlity)
(measured)
Slope - none
unknown
452029-20
Ward &
Ballantine 1985
Supplemental
(no raw data)
Since the LC50 values are in the range of 1,000 - 10,000 ppb, atrazine is categorized as
moderately toxic to estuarine/marine fish on an acute exposure basis. Toxicity data on
sheepshead minnow, Cyprinodon variegatus, indicates that atrazine toxicity increases with
increasing salinity levels. The acute effects endpoint for estuarine/marine fish is based on the
LC50 value of 2,000 ppb for sheepshead minnow at a salinity of 25 °/00 (MRID 452083-03 and
452277-11).
A.3.2 Estuarine and Marine Fish, Acute (Open Literature 2006 Review)
A.3.3. Acute Marine/Estuarine Toxicity Data - Degradates
59
-------
A special acute estuarine fish test (72-3) is required to address concerns for the toxicity of
atrazine degradates to estuarine fish (preferably sheepshead minnow). Table A-27 presents
estuarine/marine fish toxicity data for hydroxyatrazine.
Table A-27. Marine/Estuarine Invertebrate Acute Toxicity (Hydroxyatrazine)
Surrogate Species/
Flow-through or Static
% ai
formul.
96-hour LEC50 (ppb)
(measured/nominal)
Toxicity Category
MRID No.
Author/Year
Study
Classification
Sheepshead minnow
('Cyprinodon variegatus)
Static test; T = 21-24 °C
Salinity = 32%o
97.1
>1,900 (no mortality)
(measured)
moderately toxic*
465000-06
Sayers, 2005a
Acceptable
* Biological results for the study were based on the mean-measured concentrations of Hydroxyatrazine, which
remained constant at the limit of its water solubility throughout the duration of the tests. Therefore, hydroxyatrazine
is not acutely toxic to estuarine/marine fish at the limit of its water solubility.
Although the estuarine/marine fish LC50 value (>1,900 ppb) for the degradate, hydroxyatrazine,
is within the range classifying it as moderately toxic, the biological results for the study were
based on mean-measured concentrations of hydroxyatrazine, which remained constant (>90%
recovery of nominal concentrations) at the limit of its water solubility (~1 ppm ai) throughout the
duration of the test (MRID 465000-06). Therefore, the solubility of hydroxyatrazine may limit
its toxicity to marine and estuarine invertebrates.
A. 3.2 Estuarine and Marine Fish, Chronic
An estuarine/marine fish early life-stage toxicity test using the TGAI is required for atrazine
because the end-use product may be applied directly to the estuarine/marine environment or is
expected to be transported to this environment from the intended use site, and the following
conditions are met: the pesticide is intended for use such that its presence in water is likely to be
continuous; an aquatic acute LC50 or EC50 is less than 1 mg/L; and the pesticide is persistent in
water (i.e., half-life greater than 4 days). The preferred test species is sheepshead minnow.
Results of this test are summarized below in Table A-28.
60
-------
Table A-28. Estuarine/Marine Fish Early Life-Stage Toxicity Under Flow-through Conditions
Surrogate Species/
Study Duration/
Flow-through or Static
Salinity & Temperature
% ai
NOAEC/LOAEC
jig/L (ppb)
(measured or nominal)
Statistically sign. (p=0.05)
Endpoints Affected
MRID No.
Author/Y ear
Study
Classification
Sheepshead Minnow 97.4 NOAEC 1,900
('Cyprinodon variegatus) LOAEC 3,400
Study duration - unknown (measured)
Flow-through test
Salinity-13g/L; T30+1EC
Sheepshead Minnow 97.1 NOAEC = 1,100
{Cyprinodon variegatus)
Study duration - 28 days PH LOAEC = 2,200
Flow-through test , ,
Salinity = 29-31 700 (measured
T = 24 - 27 °C
J % red. in juvenile
survival
17% reduction in mean
length; 46% reduction in
mean wet weight
452029-20
Ward &
Ballantine 1985
466482-03
Cafarella, 2005a
Supplemental
(no raw data for
statistical
analyses)
Supplemental
(only 2 replicate
aquaria were
tested [ 4 reps
are required],
time to hatch
endpoint was not
assessed, and
study was
terminated at 28
day PH [32 days
are required])
In the 2003 atrazine IRED, chronic estuarine/marine fish data from Ward and Ballentine (1985;
MRID # 452029-20) were used to evaluate chronic risks to estuarine/marine fish, based on 89%
reduction in juvenile survival of sheepshead minnow (Cyprinodon variegates). However, the
results of more recent chronic estuarine/marine fish data from Caferalla, 2005a (MRID #
466482-03) show that juvenile growth may be a more sensitive endpoint than survival. Although
no effect on pre- or post-hatch survival was observed at atrazine concentrations ranging from
1,500 to 2,200 ppb, juvenile length and wet weights were significantly decreased at the 2,200
ppb treatment level, relative to the control. The NOAEC and LOAEC values, based on growth
(i.e., larval length and wet weight) are 1,100 and 2,200 ppb, respectively. Because juvenile
growth appears to be the more sensitive endpoint, chronic risks associated with estuarine/marine
fish exposure to atrazine are based on respective NOAEC and LOAEC values of 1,100 and 2,200
ppb (MRID # 466482-03).
A.3.3a Sublethal Effects: Estuarine/Marine Fish (2003 IRED Data)
Biagianti-Risbourg and Bastide (1995) exposed juvenile gray mullets (Liza ramada) to 170 |ig/L
atrazine for 9, 20, and 29 days in static tests and for 11 days followed by 18 days of
decontamination; and then measured the sublethal effects on the liver. At 170 |ig/L, 10, 25 and
60 percent mortality occurred following 9-, 20- and 29-day exposures, respectively; control
mortality was a constant 10 percent throughout the test. Treated mullets showed normal
behavior until Day 20 after which they stopped feeding and rapidly died; which is in contrast to
the 90 percent survival of the treated fish that were transferred to clean water after 11 days of
exposure. After 3-days exposure, a number of abnormalities were found in the liver (i.e., hepatic
parenchyma with a few cytologically detectable perturbations and hepatocytes had particularly
large lipofuscin granules (MRID # 452049-02).
61
-------
A.3.3b Sublethal Effects: Estuarine/Marine Fish (New Open Literature Data)
Studies identified in the open literature on potential effects to salmon from exposure to atrazine
were presented in the freshwater fish discussion. In summary, these studies demonstrated an
associated between atrazine exposure and effects on gill physiology (Waring and Moore, 2004)
and olfactory function (Moore and Lower, 2001). These effects occurred at or below 1 ug/L.
Together, these data suggest that atrazine exposure may cause treatment related effects in
salmon; however, the relevancy of these measurement endpoints to assessment endpoints is
unclear. The olfaction-related effects are not clearly associated with decreased survival or
reproduction in the species considered in this assessment and their relevancy under field
conditions is questionable.
In addition, Alvarez (2005; ECOTOX No. 81672) reported results from a chronic study in red
drum (Sciaenops ocellatus) larvae. However, the study was classified as invalid because a
negative control was not used.
A. 3.4 Estuarine and Marine Invertebrates, Acute
Acute toxicity testing with estuarine/marine invertebrates using the TGAI is required for atrazine
because the end-use product is expected to reach this environment due to its use in coastal
counties. The preferred test species are mysid shrimp (Americamysis bahia) and eastern oyster
(Crassostrea virginica). Results of these tests for the TGAI and formulations of atrazine are
provided below in Tables A-30 and A-31.
Table A-30. Estuarine/Marine Invertebrate Acute Toxicity
Surrogate Species/
Static or Flow-through/
Salinity & Temperature
% ai.
96-hour LC50/EC50
|ig/L (ppb)
(measured/nominal)
Probit Slope
T oxicity
Category
MRID No.
Author/Y ear
Study
Classification
Copepod (Acartia tonsa)
Static-renewal - daily
Salinity-31 g/L; T22°C
70
Tech.
88
(measured)
Slope 0.947
very highly toxic
452029-18
Thursby et al.
1990 memo
Supplemental
(12% control
mortality)
Copepod {Acartia tonsa)
Static test
Salinity - 20 g/L; T 20+1 °C
97.4
94
(nominal)
Slope - none
very highly toxic
452029-20
Ward &
Ballantine 1985
Supplemental
(no raw data)
Copepod {Acartia tonsa)
Static-renewal - daily
Salinity-31-32 g/L; T 22 °C
70
Tech.
139
(measured)
Slope 0.543
highly toxic
452029-18
Thursby et al.
1990 memo
Supplemental
(20% control
mortality)
Copepod
nauplii < 24 hours old
{Eurytemora afflnis)
Static test; T - 20 °C
Salinity - 5, 15 & 25g/L
97.1
Sal. 5 g/L 500
Sal. 15 g/L 2,600
Sal. 25 g/L 13,300
(measured)
Slope - no data
highly toxic
to
slightly toxic
452083-03 &
452277-11
Hall, Ziegenfuss,
Anderson, Spittler
& Leichtweis 1994
Supplemental
(no raw data on
mortality)
Mysid Shrimp
{Americamysis bahia)
Flow-through test
97.4
1,000
(Measured)
Slope - none
highly toxic
452029-20
Ward & Ballantine
1985
Supplemental
(no raw data)
Salinity 26 g/L; T 22+1 °C
62
-------
Table A-30. Estuarine/Marine Invertebrate Acute Toxicity
Surrogate Species/
Static or Flow-through/
Salinity & Temperature
% ai.
96-hour LC50/EC50
jig/L (ppb)
(measured/nominal)
Probit Slope
T oxicity
Category
MRID No.
Author/Y ear
Study
Classification
Brown Shrimp (juvenile)
(.Penaeus aztecus)
Flow-through test
Salinity - 30 g/L; T 27 °C
Copepod - 17 days old
(Acartia tonsa)
Flow-through test
Salinity-31-33/L,T-20 °C
Mysid Shrimp
(Americamysis bahia)
Flow-through test
Salinity -32 g/L; T 25-26 °C
Pink Shrimp
{Penaeus duorarum)
Static test
Salinity 26 g/L; T 22±1 °C
Copepod {Acartia clausii)
Static-renewal - daily
Salinity-31 g/L; T 6-6.2 °C
Grass Shrimp
{Palaemonetes pugio)
Static test
Salinity - 26 g/L; T 22±1 °C
Eastern oyster (juvenile)
{Crassostrea virginica)
(Shell deposition)
Flow-through test
Salinity - 28 g/L; T - 28 °C
Eastern oyster (juvenile)
{Crassostrea virginica)
(Shell deposition)
Flow-through test
Salinity 31-32 g/L; T =20-21
°C
Mud Crab
{Neopanope texana)
Static test
Salinity & T - unknown
99.7
97.1
97.1
97.4
70
Tech.
97.4
99.7
97.1
Tech.
1,000
(nominal)
Slope - none
4,300
(measured)
Slope - 2.467
5,400
(measured)
Slope 4.513
6,900
(nominal)
Slope - none
7,900
(nominal)
Slope 0.958
9,000
(nominal)
Slope - none
> 1,000
no effect
(nominal)
Slope - none
> 1,7 00
no effect
(measured)
Slope - none
> 1,000
(nominal)
Slope - none
at least highly
toxic
moderately toxic
moderately toxic
moderately toxic
moderately toxic
moderately toxic
unknown
unknown
slightly toxic
402284-01
Mayer 1986
452083-08
McNamara 1991
433449-02
Machado 1994
452029-20
Ward &
Ballantine 1985
452029-18
Thursbyefa/. 1990
memo
452029-20
Ward &
Ballantine 1985
40228-01
Mayer 1986
466482-01
Caferalla, 2005b
000247-19
Bentley & Macek
1973
Supplemental
(48-hr LC50
& no raw data)
Supplemental
(cloudy with no
0.45 f^m filter
of undissolved
test material)
Acceptable
Supplemental
(no raw data)
Acceptable
Supplemental
(no raw data)
Supplemental
(EC50 has not
been identified
& no raw data)
Acceptable
Supplemental
(LC50 exceeds
water
solubility)
Since the lowest acute LC50/EC50 value is 94 ppb (i.e., <0.1 ppm), atrazine is categorized as very
highly toxic to estuarine/marine invertebrates on an acute exposure basis. The estuarine/marine
LC50 value of 94 ppb is based on an acute static toxicity test for the copepod, Acartia tonsa
(MRID # 452029-20).
Toxicity data for a different copepod, Eurytemora affinis, indicates that atrazine toxicity
decreases with increasing salinity levels. The pattern of decreasing toxicity for estuarine/marine
invertebrates is opposite to the atrazine toxicity data pattern for estuarine/marine fish,
63
-------
sheepshead minnows (C. variegates) where toxicity increased with increasing salinity. The acute
toxicity shows that estuarine/marine mollusks, including the Eastern oyster (Crassostrea
virginica) are less sensitive to atrazine with shell deposition EC50 values >1,700 ppb (MRID #
466482-01).
Table A-31. Estuarine/Marine Invertebrate Acute Toxicity - Formulations
Surrogate Species/
Static or
Flow-through
% ai.
Product
96-hour LC50/EC50
jig/L (ppb)
(measured/nominal)
Probit Slope
Toxicity
Category
MRID No.
Author/Y ear
Study
Classification
Eastern Oyster
('Crassostrea virginica)
(Shell deposition)
Flow-through test
Salinity -11.8 mg/L; T 21EC
79.6
80 WP
>800
no effect
(nominal)
Slope - none
unknown
000247-20
Wright & Beliles
1966
Supplemental
(EC50 has not
been identified)
Pacific Oyster
('Crassostrea gigas)
24-Hour Static-Renewal
??
> 100
(nominal)
0.1 - 50% dead at 22 days
0.2 - 50% dead at 18 days
unknown
452277-22
Moraga &
Tanguy 2000
Supplemental
(no 96-hour
LC50 value)
European Brown Shrimp
(Crangon crangon)
Static test; 15EC
??
WP
10,000-33,000
(nominal)
no slope
slightly toxic
452277-28
Portmann 1972
Supplemental
(only 48 hours &
no raw data)
European Cockle
('Cardium edule)
Static test; 15EC
??
WP
> 100,000
(nominal)
no slope
practically
non-toxic
452277-28
Portmann 1972
Supplemental
(only 48 hours;
LC50 exceeds
water solubility
& no raw data)
Fiddler Crab
(Uca pugilator)
Static test
Salinity - 30 g/L; T 19EC
79.6
80 WP
198,000
(nominal)
Slope - none
unknown
000243-95
Union Carbide
Corp. 1975
Supplemental
(LC50 exceeds
water solubility)
Fiddler Crab
{Uca pugilator)
Static test
Salinity - 30 g/L; T 19EC
Unknown
4-1-3-1
WDL
239,000
(nominal)
Slope - none
unknown
000243-95
Union Carbide
Corp. 1975
Supplemental
(LC50 exceeds
water solubility)
The toxicity of formulated atrazine products to marine/estuarine invertebrates are uncertain,
because the EC/LC50 values are not definitive.
Degradates: Estuarine invertebrate acute tests (72-3) are required to address concerns for the
toxicity of atrazine degradates to estuarine invertebrates (preferably Americamysis bahia). Table
A-32 presents estuarine/marine invertebrate toxicity data for hydroxy atrazine.
Table A-32. Estuarine/Marine Invertebrate Acute Toxicity (Hydroxyatrazine)
Surrogate Species/ % ai 96-hour LEC50 (ppb) MRID No. Study
Flow-through or Static formul. (measured/nominal) Toxicity Category Author/Year Classification
64
-------
Table A-32. Estuarine/Marine Invertebrate Acute Toxicity (Hydroxyatrazine)
Surrogate Species/
Flow-through or Static
% ai
formul.
96-hour LEC50 (ppb)
(measured/nominal)
Toxicity Category
MRID No.
Author/Year
Study
Classification
Mysid Shrimp
(Americamysis bahia)
Static test
Salinity +22-25 g/L; T 25-
26 °C
97.1
>2,000 (5% mortality
(measured)
moderately toxic*
465000-03
Sayers, 2005b
Acceptable
* The highest concentration tested in this study approximated the functional water solubility of
hydroxyatrazine in natural seawater; therefore, hydroxyatrazine is not toxic to mysids on an acute basis at the
limit of its water solubility.
Although the estuarine/marine invertebrate LC50 value (>2,000 ppb) for the degradate,
hydroxyatrazine, is within the range classifying it as moderately toxic, the highest concentration
tested in this study approximated the functional water solubility of hydroxyatrazine in natural
seawater; therefore, hydroxyatrazine is not likely to be acutely toxic to estuarine/marine
invertebrates at the limit of its water solubility. During the 96-hour test, mortality was 5% in the
control and mean-measured 500 and 2000 ppb a.i. treatment groups and 0% in the mean-
measured 62, 130, 250, and 1000 ppb a.i. treatment groups (MRID # 465000-03). No sub-lethal
effects were observed during the exposure period.
A. 3.5 Estuarine and Marine Invertebrate, Chronic
An estuarine/marine invertebrate life-cycle toxicity test using the TGAI is required for atrazine
because the end-use product may be applied directly to the estuarine/marine environment or is
expected to be transported to this environment from the intended use site, and the following
conditions are met: the pesticide is intended for use such that its presence in water is likely to be
continuous and recurrent; an aquatic acute EC50 is less than 1 mg/L; and the pesticide is
persistent in water (e.g., half-life greater than 4 days). The preferred test species is mysid
shrimp. Results of this test are summarized below in Table A-33.
Table A-33. Estuarine/Marine Invertebrate Life-Cycle Toxicity
Species/
NOAEC/LOAEC
Duration/
jig/L (ppb)
Statistically sign. (P=0.05)
MRID No.
Study
Flow-through/ Static-renewal
% ai
(measured/noml)
Endpoints Affected
Author/Y ear
Classification
Mysid
97.4
NOAEC 80
37 % red. in adult survival
452029-20
Supplemental
(Americamysis bahia)
LOAEC 190
Ward &
(no raw data
Duration of test - unknown
(measured)
Ballantine 1985
for statistical
Flow-though test
analyses)
Salinity 20 g/L; T 25+1 °C
Mysid
97.1
NOAEC 260
9.8% red. in male length
466482-02
Supplemental
(Americamysis bahia)
LOAEC 500
Study Duration = 28 days
11% red. in male dry weight
Cafarella, 2005c
(no raw data
Flow-though test
(measured)
8.5% red. in female dry
for statistical
analyses)
Salinity 19-21 g/L; T 26±2 °C weight
65
-------
The chronic endpoint for estuarine/marine invertebrates is based on a 37% reduction in adult
mysid survival at a concentration of 190 ppb, with a corresponding NOAEC of 80 ppb (MRID
452029-20).
A. 3.6 Sublethal Effects: Estuarine/Marine Invertebrates (New Open Literature Data)
Two studies in the marine invertebrate copepod were located; one study was considered invalid
because a negative control was not used. The remaining study is summarized in Table A-34.
Forget-Leray et al. (2004) reported results from a 96-hour, a 10-day, and a 30-day exposure
study. An acute 96-hour LC50 of 125 ug/L in the copepod E. affinis nauplii. In a 10-day study
reported in the same study report, a NOAEC of 25 ug/L (LOAEC of 49 ug/L) was reported for
mortality. Delayed maturity was also observed at 25 ug/L in a 30-day exposure study. These
studies, however, were limited because DMSO was used as a solvent. DMSO is not an
acceptable solvent, because it accelerates the movement of chemicals across cell membranes. As
such it represents a worst case exposure. For this reason, this study was not used to quantify
potential risks to marine/estuarine invertebrates, but was used to qualitatively characterize such
risks. In addition, the relationship between delayed maturity and survival and reproductive
success is uncertain.
Table A-34. Estuarine/Marine Invertebrates Sublethal Effects Toxicity Tests from Open Literature (2006 Review)
Study type/
Test material
Test
Design
Test Organism
(Common and
Scientific Name)
and Age and/or
Size
Endpoint Concentration
in ppb
Citation
(EcoRef. #)
Rationale for Use in Risk
Assessment'1'
Acute and
chronic studies /
Atrazine
unspecified
purity
Study duration: 4-30
days
Atz Cones: not reported
(acute); 25 ug/L (10-day
study)
Exposure: Static
(acute); semi-static (10-
day study)
Endpoints: Survival,
development
Temp: 18DegC.
Solvent: DMSO
Copepods
(.Eurytemora
affinis) from the
Seine river estuary
(France).
An acute 96-hour LC50 was
estimated for the copepod E.
affinis nauplii of 125 ug/L for
atrazine. A 10-day study was
conducted using E. affinis
(nauplius stage) that produced a
NOAEC for survival of 25 ug/L
and a LOAEC of 49 ug/L.
Delayed maturity was also
observed at 25 ug/L in the 30-day
exposure study.
Forget-Leray et
al., 2004
(80951)
Qualitative. No chronic value was
previously available in copepods.
However, reporting limitations
and use of DMSO as a solvent
preclude its use to calculate RQs.
Reporting limitations included
number and identification of test
concentrations, % mortality at the
LOAEC, and control responses.
Bejarano and Chandler (2003; ECOTOX No. 73333) reported results from a 2.5 generation
reproduction study in copepods. This study was considered invalid because a negative control
group was not used.
A.3.7a Estuarine and Marine Field Studies (2003 IRED Data)
66
-------
A summary of all the estuarine/marine aquatic microcosm and mesocosm field studies that were
summarized as part of the 2003 IRED is included in Tables A-35 and A-36, respectively.
67
-------
I'iihlo A-35. Miii'iiio/l''.sliiiii'ino Microcosm Tcsls
Vpplii-iili«»n I'silc dh iii V i
Nuiiiiiisil \lc;i">uml ( "in.
( oiu-i'iiiiiiiioii ii lli'i'l in** mil point dime In rlTcil i
o MTci li-il spi i ii s siimI lili- slsij»c
N;MTiili\c olMu
-------
I'iihlo A-35. Miii'iiio/l''.sliiiii'ino Microcosm Tcsls
Vpplii-iili«»n I'silc dh iii V i
Nuiiiiiisil \lc;i">uml ( "in.
( oiu-i'iiiiiiiioii ii lli'i'l in** mil point dime l<» rlTcil i
o MTctlctl specie* sumI lilt' s|;i«c
N;MTiili\c o| s(inl\ | mi
-------
I'iihlo A-35. Miii'iiio/l''.sliiiii'ino Microcosm Tcsls
Vpplii-iili«»n I'silc dh iii V i
Nuiiiiiisil \lc;i">uml ( "in.
( oiu-i'iiiiiiiioii ii lli'i'l in** mil point dime l<» rlTcil i
o MTci li-il spi i ii s siimI lili- slsij»c
N;MTiili\c o| s(inl\ | mi
-------
I'iihlo A-35. Miii'iiio/l''.sliiiii'ino Microcosm Tcsls
Vpplii-iili«»n I'silc dh iii V i
Nuiiiiiisil \lc;i">uml ( "in.
( oiu-i'iiiiiiiioii ii lli'i'l in** mil point dime l<» rlTcil i
o MTci li-il spi i ii s siimI lili- slsij»c
Virniliw- o| sunl\ I rnnh
MUM) No.
tullior ciir
Estuarine microcosm:
4 weeks
Mean-measured concentrations in water were 130
ppb for the "low" treatment and 1200 ppb for the
"high" treatment over a 4 week period
130 ppb (Week 1):
no photosynthesis
130 ppb (Weeks 2-4)
0 sign, reduction in plant total biomass, no
change in biomass for 3 weeks
130 ppb (Weeks 1-4)
o sign.; averaged 50% reduction in
photosysnthesis of Potamogeton perfoliatus during
the test; steady recovery after first week, but not
fully recovered
1200 ppb (Weeks 1-4)
o sign. 100% red. in photosynthesis
throughout the test
1200 ppb (Weeks 2-4)
o sign, plant biomass steadily reduced
1200 ppb (Weeks 3-4)
o sign. 80% reduction in shoot density
Aquatic plants were planted and atrazine-treated
sediments were added to 700-L microcosms. On
Day 1.5, 93.4% of the total atrazine was dissolved
in water. In addition to photosynthesis, it was
demonstrated that shoot growth was relatively
unaffected at 130 ppb, but total biomass was sign,
reduced after 2-4 weeks. Plant biomass reductions
followed a 1 week lage after photosynthesis
reduction. At 1200 ppb, plant biomass had been
virtually eliminated by the end of the test. Mean
shoot length in the controls declined after week 1
and after week 3 for 1200 ppb.
450874-03
Cunningham et al, 1984
Supplemental
(raw data unavailable)
Estuarine microcosm:
22-23 days
Single dose
Day 0: 30000 ppb - nominal; measured only Day
22-23: 16400-17000 ppb
30,000 ppb (Day 5-22):
sign, (p < 0.05) red. average ratio of # or
ramets (branches): initial # or ramets
30,000 ppb (Day 22 or 23):
sign, (p < 0.05) 46-58% reduction in total
above-ground biomass
sign, (p < 0.05) 18% reduction in average
dry weight per ramet
Experiments were conducted with seagrass
Halodule wrightii, examining the effect of atrazine
and any interactions of salinity (15, 25, 35 ppt),
light intensity (115, 140, 173 uEm'V1), and
cropping (either cut at 4-cm or 6-cm). None of
these environmental factors affected the response of
the grass to atrazine.
452051-01
Mitchell, 1987
Supplemental
(raw data unavailable)
71
-------
Tabic A-36. Marinc/Estuarinc IVlesocosm Tests
Application rate (lb ai/A)
Nominal/Measured Cone.
Concentration affecting endpoint (time to effect)
o Affected Species and Life Stage
Narrative of Study Trends
MRID No.
Author/Year
Marine Mesocosm:
Open Ocean: Phytoplankton:
(15 days; measured conc.)
Measured = nominal at time
zero, concentrations of 0.12,
0.56, and 5.8 ppb
0.12 ppb (differences compared to controls)
sign, lower pH levels (Days 5-14); indicative of
reduced photosynthesis
higher dissolved organic nitrogen (DON) (Days 6-11)
°o up to 50% red. primary production (Days 3-11)
up to 60% red. particulate carbohydrates (Days 5-15)
up to 70% red. chlorophyll (Days 4-15)
0.5§ ppb
sign, lower pH levels (Days 5-13)
0 incr. total dis. organic phosphate (DOP) (Days 3-14)
° higher DON (Days 5-15)
up to 50 % red. primary production (Day 3-13)
up to 85% red. particulate carbohydrate (Days 5-15)
up to 80% red. chlorophyll (Days 4-15)
5.8°ppb
sign, lower pH levels (Days 5-11)
0 up to 200% increase in total DOP (Days 3-14)
° up to 200 % increase in total DON (Days 2-15)
up to 50% red. in primary productivity (Days 3-7)
up to 60% red. in partic. carbohydrates (Days 5-15)
up to 30% red. in chlorophyll conc. (Days 4-15)
Mesocosms (2 m2) inoculated with the diatoms Thalassiosira punctigera,
T. rotula, Nitzschia pungens and Skeletonema costatum and a
prymnesiophtye, Phaeocystis globosa. evidenced a dose-responsive
elevation in dissolved nitrogen and phosphorous and reduction in primary
production at 0.12, 0.56, and 5.8 ppb. The NOEL was reported to be <0.12
ppb. Atrazine at concentrations at 0.12, 0.56 and 5.8 ppb, adversely effects
primary production of unicellar algal species at certain growth phases and
causes increases in "excretions" of dissolved organic nitrogen and
phosphorus. "Excretions" may be caused by atrazine stress on cells or
lysis of cells.
450200-21
Bester etal, 1995
Supplemental
(raw data unavailable)
Nominal applications of 0.4,
4.5, or 45 lb ai/A
Salt marsh edaphic alage
Elaboration of Plumley et ai, concerning the carbon uptake for algae in the
top 0.5 cm of enclosure sediment. Carbon fixation was significantly
reduced at the 0.45 and 4.5 lb ai/A treatment levels for 16 days and at the
45 lb ai/A treatment level for 42 days.
450874-06
Plumley and Davis, 1980
72
-------
A.3.7b Estuarine and Marine Field Studies (New Open Literature Data)
As previously discussed, the 2003 IRED identified 10-20 [j,g/L as the range of atrazine
concentrations in freshwater that are likely to have adverse effects on sensitive aquatic plants and
non-target aquatic organisms including their populations and communities. As such,
estuarine/marine field data from the open literature were considered only when the relevant
endpoints were less than or more sensitive than the 10 [j,g/L aquatic-community effect level. In
addition, data for taxa that are directly relavent to the endangered species evaluated as part of this
assessment were also considered. Based on the selection criteria for review of new open literature,
all of the available studies show effects levels to estuarine/marine fish, invertebrates, and plants at
concentrations greater than 10 (J,g/L.
One estuarine/marine field study on saltwater eelgrass (Zostera capricorni) was reviewed as part
of the open literature because it provides data on seagrass, a potential food item and source of
habitat for sea turtles (Macinnis-Ng, 2003; Ecotox Reference # 72996). The results of this study,
which are summarized as part of Table A-37, show that atrazine is unlikely to affect the
chlorophyll a concentration of estuarine/marine sea grasses at exposure concentrations ranging
from 10 to 100 ppb.
Table A-37. Estuarine/Marine Field Study from Open Literature (2006 Review)
Study type/
Test material
Test Organism (Common
and Scientific Name) and
Age and/or Size
Test
Design
Endpoint
Concentration
in ppm
Citation
(EcoRef. #)
Rationale for Use in
Risk Assessment(1)
Field study
10 h exposure
Atrazine (% ai
NR)
Seagrass (Zostera
capricorni)
¦ open-bottom cylindrical
containers enclosed grasses
within a seagrass meadow;
salinity = 35 ppt; temp = 25
11 °c
¦ Atrazine doses = 0, 10,
and 100 ppb at one
application
¦ Endpoints: total
chlorophyll a
concentration, effective
quantum yield via
fluorescence measurements
NOAEC = 100 ppb
No difference in total
chlorophyll a
concentration between
treatments and control.
Reduction in effective
quantum yield (via
fluorescence
measurements) at both
treatments relative to
the control, but recovery
to control values by end
of 10 hour exposure
period.
Macinnis-Ng and
Ralph, 2003
(72996)
QUAL:
• no raw data provided
• low number of
replicates (2)
• relevance of
fluorescence endpoints
is of limited use in risk
assessment.
I~1) QUAL = The paper is not appropriate for quantitative use but is of good quality, addresses issues of concern to the risk
assessment and is used in the risk characterization discussion.
NR = Not reported.
73
-------
A.4 Toxicity to Plants
A. 4.1 Terrestrial Plants
Terrestrial plant testing (seedling emergence and vegetative vigor) is required for herbicides that
have terrestrial non-residential outdoor use patterns and that may move off the application site
through volatilization (vapor pressure >1.0 x 10"5mm Hg at 25°C) or drift (aerial or irrigation)
and/or that may have endangered or threatened plant species associated with the application site.
For seedling emergence and vegetative vigor testing the following plant species and groups
should be tested: (1) six species of at least four dicotyledonous families, one species of which is
soybean (Glycine max) and the second is a root crop, and (2) four species of at least two
monocotyledonous families, one of which is corn (Zea mays).
Terrestrial Tier II studies are required for all herbicides and any pesticide showing a negative
response equal to or greater than 25% in Tier I tests. Tier II tests measure the response of plants,
relative to a control, and five or more test concentrations at a test level that is equal to the highest
use rate (expressed as lbs ai/A). Results of Tier II seedling emergence and vegetative vigor
toxicity testing on the technical material are summarized below in Tables A-38 and A-39.
Based on the results of the tests, it appears that emerged seedlings are more sensitive to atrazine
via soil/root uptake exposure than emerged plants via foliar routes of exposure. However, all
tested plants, with the exception of corn in the seedling emergence and vegetative vigor tests and
ryegrass in the vegetative vigor test, exhibited adverse effects following exposure to atrazine.
For Tier II seedling emergence, the most sensitive dicot is the carrot and the most sensitive
monocots are oats. EC25 values for oats and carrots, which are based on a reduction in dry
weight, are 0.003 and 0.004 lb ai/A, respectively; NOAEC values for both species are 0.0025 lb
ai/A.
For Tier II vegetative vigor studies, the most sensitive dicot is cucumber and the most sensitive
monocot is onion. In general, dicots appear to be more sensitive than monocots via foliar routes
of exposure with all tested monocot species showing a significant reduction in dry weight at EC25
values ranging from 0.008 to 0.72 lb ai/A. In contrast, two of the four tested monocots showed no
effect to atrazine (corn and ryegrass), while EC25 values for oats and onion were 0.61 and 2.4 lb
ai/A, respectively.
Table A-38. Nontarget Terrestrial Plant Seedling Emergence Toxicity (Tier II)
Surrogate Species
EC25 / NOAEC (lbs ai/A)
% al Problt Slope
lull point Affected
MRID No.
Author/Y ear
Classification
Study
Monocot - Corn
(Zea mays)
97.7 >4.0/>4.0
No effect
420414-03 Acceptable
Chetram 1989
Monocot - Oat
(Avena sativa)
97.7
0.004 / 0.0025
red. in dry weight
420414-03 Acceptable
Chetram 1989
74
-------
Table A-38. Nontarget Terrestrial Plant Seedling Emergence
Toxicity (Tier II)
Surrogate Species
% ai
EC25 / NOAEC (lbs ai/A)
Probit Slope
Endpoint Affected
MRID No.
Author/Y ear
Study
Classification
Monocot - Onion
(Allium cepa)
97.7
0.009/0.005
red. in dry weight
420414-03
Chetram 1989
Acceptable
Monocot - Ryegrass
(.Lolium perenne)
97.7
0.004/0.005
red. in dry weight
420414-03
Chetram 1989
Acceptable
Dicot - Root Crop - Carrot
(.Daucus carota)
97.7
0.003 I 0.0025
red. in dry weight
420414-03
Chetram 1989
Acceptable
Dicot - Soybean
(4.0/>4.0
No effect
420414-03
Chetram 1989
Acceptable
Monocot - Oat
(Avena sativa)
97.7
2.4 / 2.0
red. in dry weight
420414-03
Chetram 1989
Acceptable
Monocot - Onion
(Allium cepa)
97.7
0.61 / 0.5
red. in dry weight
420414-03
Chetram 1989
Acceptable
Monocot - Ryegrass
(.Lolium perenne)
97.7
>4.0/>4.0
No effect
420414-03
Chetram 1989
Acceptable
Dicot - Root Crop - Carrot
(Daucus carota)
97.7
1.7 / 2.0
red. in plant height
420414-03
Chetram 1989
Acceptable
Dicot - Soybean
(Glycine max)
97.7
0.026 / 0.02
red. in dry weight
420414-03
Chetram 1989
Acceptable
Dicot - Lettuce
(Lactuca sativa)
97.7
0.33 / 0.25
red. in dry weight
420414-03
Chetram 1989
Acceptable
Dicot - Cabbage
(Brassica oleracea alba)
97.7
0.014/0.005
red. in dry weight
420414-03
Chetram 1989
Acceptable
Dicot - Tomato
(Lycopersicon esculentum)
97.7
0.72 / 0.5
red. in plant height
420414-03
Chetram 1989
Acceptable
Dicot - Cucumber
(Cucumis sativus)
97.7
0.008/ 0.005
red. in dry weight
420414-03
Chetram 1989
Acceptable
A summary of safety studies evaluating phytoxicity of atrazine to woody plants (target species)
was submitted to the Agency in 2006 (Wall, 2006). A total of 35 species were tested in 13
75
-------
separate trials at application rates of 1.5 to 4.0 lbs a.i./Acre. Signs of phytotoxicity were
summarized and reported. These data are summarized in Table A-39b below.
Table A-39b. Summary of woody plant safety study (Hall, 2006).
Species
Application Rate (lbs a.i./Acre)
Phytotoxicity
(%)
Abies balsamea
2
0
4
3% General
Azalea
2
5% General
2
5% General
Barberry
2
50% General11
Black pine
2
Chlorosis (IS-l)a
Boxwood
2
3.8% General
Chitalpa
2
0%
Common Lilac
2
22% chlorosis
25% necrosis
Conifer shrubs and trees
2
0%
Crabapples
2
0%
Crape-Myrtle
2
Chlorosis (IS-1)a
Creeping juniper
2
7.5% General
Cupressocyparis leylandii
2
0-1% General
Cypruss leylandii
2
Chlorosis (IS-1)a
Ginko
2
0%
Gleditsia triacanthos
2
7.5% General
Hydrangea
2
4 - 16% General
Juniperus
1.5
0%
Ligustrum
2
0%
Locust
2
0%
Macadamia nuts
2
0%
Maple
2
0%
Oak
2
0%
Pears
2
0%
Pinus palustris
1.5
70% General'
Pinus strobus
2
5% General
Pinus virginiana
1.5
90% General0
Pseudotsuga menziesii
4.3
0%
Purpleleaf plum
2
Chlorosis (IS-1)a
Raywood ash
2
Chlorosis (IS-1)a
Redbud, Eastern
2
2.5% chlorosis
0.3% necrosis
10% general
Rhododendron, catawba
2
5
Shrubby althaea
2
100% chlorosis
40% necrosis
Spiraea
2
16"
76
-------
Table A-39b. Summary of woody plant safety study (Hall, 2006).
Species
Application Rate (lbs a.i./Acre)
Phytotoxicity
(%)
Spruce
2
0
Tilia
2
0
a IS rating grades chlorosis severity (normal to excessive color) and ranges from 1 to 5
b Phytotoxicity in controls was up to 45%; other pesticides were included in trial, and sprayer may not have been adequately cleaned,
c Effect was noted as being atypical for conifers, and the effect may not be related to atrazine treatment
A.4.2 Aquatic Plants
Aquatic plant testing is required for any herbicide that has outdoor non-residential terrestrial uses
that may move off-site by runoff (solubility >10 ppm in water), by drift (aerial or irrigation), or
that is applied directly to aquatic use sites (except residential). Aquatic Tier II studies are
required for all herbicides and any pesticide showing a negative response equal to or greater than
50% in Tier I tests. The following species should be tested at Tier II: Kirchneria subcapitata,
Lemna gibba, Skeletonema costatum, Anabaena flos-aquae, and a freshwater diatom. Aquatic
plant testing is required for atrazine because it is applied on crops outdoors and appears to be
mobile with a water solubility value of 33 ppm.
Results of Tier II toxicity testing on technical grade and typical end-use products (TEP) are
tabulated below. The data are presented in four toxicity tables separating the freshwater data from
the marine data and the short, 7-day or less tests from the longer tests. Tables A-40 and A-41
summarize freshwater plant toxicity for short-term (i.e., < 7 days exposure) and longer-term tests.
Tables A-42 and A-43 summarize short-term (< 10 days exposure) estuarine/marine plant toxicity
for technical grade and formulations of atrazine, respectively. Toxicity data for longer-term
exposure of atrazine to estuarine/marine plants are provided in Table A-44.
Field studies involving atrazine toxicity to freshwater and estuarine/marine aquatic plants are
summarized as part of Sections A.2.8 and A.3.7, respectively.
Table A-40. Nontarget Freshwater Plant Toxicity: short-term (< 7 days) (Tier II)
Surrogate Species/
Duration/Measured/nominal
Cone, (ppb)
% ai Probit Slope % Response
MRID No.
Author/Year Study Classification
Vascular Plants:
Duckweed
{Lemna gibba)
5-Day test; Static-Renewal
Duckweed
{Lemna gibba)
7-Day test; Static-Renewal
97 170 50% red. in growth
(nominal)
Slope 3.93
97 170 50% red. in growth
(measured)
Slope 2.2
410652-03d Supplemental
Hughes 1986 (5 days, not 14 days)
420414-04 Supplemental
Hoberg 1991 (7 days, not 14 days)
Non-Vascular Plants:
77
-------
Table A-40. Nontarget Freshwater Plant Toxicity: short-term (< 7 days) (Tier II)
Surrogate Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit Slope
% Response
MRID No.
Author/Y ear
Study Classification
Cyanophyceae
Oscillatoria lutea
(lweek; nominal)
76
80 W
< 1
1,000
93% red. chlorophyll
production
100% red. chlorophyll
prod.
Torres and
O'Flaherty
1976
Supplemental
(raw data unavailable)
Chlorophyceae
Stigeoclonium tenue
(1 week; nominal)
76
80 W
< 1
1,000
67% red. chlorophyll
production
90% red. chlorophyll
production
Torres and
O'Flaherty
1976
Supplemental
(raw data unavailable)
Green Algae - Chlorophyceae
Chlorella vulgaris
(1 week; nominal)
76
80 W
1
1,000
50% red. chlorophyll
production
80-87% red. chlorophyll
production
Torres and
O'Flaherty
1976
Supplemental
(raw data unavailable)
Xanthophyceae
Tribonema sp.
(1 week; nominal)
76
80 W
1
1,000
42% red. chlorophyll
production
75% red. chlorophyll
production
Torres and
O'Flaherty
1976
Supplemental
(raw data unavailable)
Xanthophyceae
Vaucheria geminata
(1 week; nominal)
76
80 W
1
1,000
41% red. chlorophyll
production
100% red. chlorophyll
production
Torres and
O'Flaherty
1976
Supplemental
(raw data unavailable)
Chlorophyceae
Chlamydomonas reinhardi
(24 hour; nominal)
Unk.
19
44
48
50% red. carbon uptake;
media: Taub & Dollar
(TD)
450200-15
Larsen et al.
1986
Supplemental
(raw data unavailable)
Chlorophyceae
Kirchneria subcapitata
=Selenastrum capricornutum
(96 hours; nominal)
Tech.
26
26
50% red. cell growth
50% red. floresence
Caux,
Menard, and
Kent 1996
Supplemental
(NOAEC and raw data
unavailable)
Chlorophyceae
Kirchneria subcapitata
= Selenastrum capricornutum
(24 hours; nominal)
Unk.
34
42
53
50% red. 14-carbon
uptake; media: Taub &
Dollar (TD); algal assay &
TD, respect.
450200-15
Larsen et al.
1986
Supplemental
(raw data unavailable)
Cyanophyceae
Anabaena cylindrica
(?? hours; nominal)
97
37
50% red. in photosynthesis
Stratton &
Corke 1981
Supplemental
(no raw data)
Chlorophyceae
Scenedesmus obliquus
(24 hour; nominal)
Unk.
38
49
57
50% red. 14-carbon
uptake; media: Taub &
Dollar (TD)
450200-15
Larsen et al.
1986
Supplemental
(raw data unavailable)
Chlorophyceae
Kirchneria subcapitata
=Selenastrum capricornutum
(120 hours; measured)
97.1
49
NOAEC 16
Slope 4.002
50% red. cell growth
430748-02
Hoberg 1993
Acceptable
Cyanophyceae
Anabaena inaequalis
(?? hours; nominal)
97
50
50% red. in photosynthesis
Stratton &
Corke 1981
Supplemental
(no raw data)
Chlorophyceae
Kirchneria subcapitata
= Selenastrum capricornutum
(120 hours; nominal)
97.4
53
NOAEC <32
LOAEC 32
Slope 4.127
50% red. growth
17% red. growth
410652-04
Parrish 1978
Supplemental
(NOAEC, method &
raw data unavailable)
78
-------
Table A-40. Nontarget Freshwater Plant Toxicity: short-term (< 7 days) (Tier II)
Surrogate Species/ Cone, (ppb) MRID No.
Duration/Measured/nominal % ai Probit Slope % Response Author/Year Study Classification
Bacillariophyceae
Navicula pelliculosa
(120 hours; nominal)
97.1
60
NOAEC <10
LOAEC 10
Slope 2.31
50% red. growth
410652-03a
Hughes 1986
Acceptable
(EC50 extrapolated;
and NOAEC was not
determined)
Chlorophyceae
Ankistrodesmus sp.
(24 hours; nominal)
Unk.
61
72
219
50% red. 14-carbon
uptake; media: Taub &
Dollar (TD), TD & algal
assay, respect.
450200-15
Larsen et al.
1986
Supplemental
(raw data unavailable)
Ulothrix subconstricta
Tentative species identification
(24 hours; nominal)
Unk.
88
50% red. 14-carbon
uptake;
medium: Taub & Dollar
(TD)
450200-15
Larsen et al.
1986
Supplemental
(raw data unavailable)
Cyanophyceae
Anabaena variabilis
(?? hours; Nominal)
97
100
50% red. in photosnythesis
Stratton &
Corke 1981
Supplemental
(no raw data)
Stigeoclonium tenue
Tentative species Identification
(24 hours; nominal)
Unk.
127
224
50% red. 14-carbon
uptake; media: Taub &
Dollar (TD)
450200-15
Larsen et al.
1986
Supplemental
(raw data unavailable)
Chlorophyceae
Kirchneria subcapitata
=Selenastrum capricornutum
(96 hours; measured)
97
130
NOAEC 76
Slope 6.628
50% red. cell growth
420607-01
Hoberg 1991
Supplemental
(higher light intensity
than recommended)
Cyanophyceae
Anabaena cylindrica
(24 hour; nominal)
Unk.
178
182
253
50% red. 14-carbon
uptake; media: Taub &
Dollar (TD), algal assay, &
TD, respect.
450200-15
Larsen et al.
1986
Supplemental
(raw data unavailable)
Cyanophyceae
Anabaena flos-aquae
(120 hours; nominal)
97
230
NOAEC <100
LOAEC 100
Slope 1.95
50% red. growth
22% red. growth
410652-03a
Hughes 1986
Acceptable
(NOAEC was not
determined)
Chlorophyceae
Chlorella pyrenoidosa
(120 hours; nominal)
97.4
282
NOAEC 130
Slope 4.216
50% red. growth
7% red. growth
410652-04
Parrish 1978
Supplemental
(NOAEC, method &
raw data unavailable)
Chlorophyceae
Chlorella vulgaris
(24 hours; nominal)
Unk.
293
305
325
50% red. 14-carbon
uptake; media: Algal
assay, Taub & Dollar
(TD), & TD, respect.
450200-15
Larsen et al.
1986
Supplemental
(raw data unavailable)
Table A-41. Longer Term, Nontarget Freshwater Plant Toxicity
Surrogate Species/
Duration/ % ai
Measured/nominal
Cone, (ppb)
Probit Slope
% Response
MRID No.
Author/Y ear
Study Classification
Vascular Plants:
Broad Waterweed ????
Elodea canadensis
(20 days; measured)
NOAEC 2
LOAEC 10
200% incr. dark
respiration
33% incr. net
photosynthesis
452277-14
Hofmann and
Winkler 1990
Supplemental
(raw data unavailable)
79
-------
Table A-41. Longer Term, Nontarget Freshwater Plant Toxicity
Surrogate Species/
Duration/
Measured/nominal
% ai
Conc. (ppb)
Probit Slope
% Response
MRID No.
Author/Y ear
Study Classification
Pondweed
Potamogeton perfoliatus
(4 weeks; initial conc.
nominal,
terminal conc. measured)
???
30
Week 3: LOAEC 5
NOAEC < 5
4 Weeks: LOAEC 50
NOAEC 5
50% red. 02 product,
sign. red. 02 product.
sign. red. O2 product.
Kemp et al.
1985
Supplemental
(raw data unavailable)
Duckweed
Lemna gibba
(14 days; measured)
97.1
37
LOAEC 3.4
NOAEC <3.4
Slope 1.716
50% red. growth
19% red. growth
(frond production)
430748-04
Hoberg 1993
Supplemental
(NOAEC was not
determined)
Duckweed - Lemna gibba
(14 days; measured)
97.4
43
NOAEC 10
Slope 1.995
50% red. growth
(frond production)
430748-03
Hoberg 1993
Acceptable
Duckweed
Lemna gibba
(14 days; measured)
Includes recovery phase
98.5
64
67
NOAEC = 18
Slope 3.96+ 0.316
50% red biomass
50% red frond count
461509-01
Desjardins et
al., 2003
Acceptable
Broad Waterweed
Elodea canadensis
(3 weeks; nominal)
???
80
50% red. shoot length
450874-10
Forney and
Davis 1981
Supplemental
(raw data unavailable)
Eurasian Water-Milfoil
Myriophyllum spicatum
(4 weeks; initial conc.
nominal,
terminal conc. measured)
????
91
NOAEC 5
LOAEC 50
50% red, 02 product.
Sign. red. O2 product.
Kemp et al.
1985
Supplemental
(raw data unavailable)
Non-Vascular Plants:
36 freshwater algal strains
(2 weeks; nominal)
99.0
10
1,000
growth 95
30
100
300
50% red. cell count
50% red. growth rate
50% red.
photosynthesis
450874-01
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Chlorophyceae
Ankistrodesmus braunii
(11 days; nominal)
99.9
60
50% red. cell growth
452277-03
Burrell et al.
1985
Supplemental
(raw data unavailable)
Chlorophyceae
Scenedesmus quadricauda
(12-14 days1; nominal)
>95
100
200
300
50% red. cell count
50% red. growth rate
50% red.
photosynthesis
450874-01
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Chlorophyceae
Chlorella pyrenoidosa
(12-14 days1; nominal)
>95
300
1,000
500
50% red. cell count
50% red. growth rate
50% red.
photosynthesis
80
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
-------
Table A-41. Longer Term, Nontarget Freshwater Plant Toxicity
Surrogate Species/ Cone, (ppb) MRID No.
Duration/ % ai Probit Slope % Response Author/Year Study Classification
Measured/nominal
Cyanophyceae
Anabaena cylindrica
(12-14 days; nominal)
>95
1,200
3,600
500
50% red. cell count
50% red. growth rate
50% red.
photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Cyanophyceae
Anabaena variabilis
(12-14 days; nominal)
>95
4,000
5,000
100
50% red. cell count
50% red. growth rate
50% red.
photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Table A-42. Nontarget Marine/Estuarine Plant Toxicity (Tier II)
Surrogate Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit Slope
% Response
MRID No.
Author/Year
Study Classification
Vascular Plants:
Fontinalis sp.
(24 hours; measured)
NR
NR
NR
NR
NOAEC 2
LOAEC 10
red. net O2 production
Supplemental
(raw data unavailable)
Pondweed (Estuarine)
Potamogeton perfoliatus
(2 hours; nominal)
77
50% red. O2 evolution
452277-18
Jones and
Winchell 1984
Supplemental
(Insufficient duration;
raw data unavailable)
Pondweed
Potamogeton perfoliatus
(2 hours; nominal)
80
650
50% red. 02 product.
87% red. 02 product..
452277-18
Jones et al.
1986
Supplemental
(Insufficient duration;
raw data unavailable)
Zannichellia palustris
(2 hours; nominal)
91
50% red. O2 evolution
452277-19
Jones and
Winchell 1984
Supplemental
(Insufficient duration;
raw data unavailable)
Pondweed (Estuarine)
Potamogeton perfoliatus
(2 hours; nominal)
NR
100
52 to 69% red. in
photosynthesis
450874-04
Jones & Estes
1984
Supplemental
(raw data unavailable)
Widgeon-Grass (Estuarine)
Ruppia maritima
(2 hours; nominal
NR
102
50% red. 02 evolution
452277-19
Jones and
Winchell 1984
Supplemental
(Insufficient duration;
raw data unavailable)
Non-Vascular Plants:
Blue-green - Cyanophyceae
Oscillatoria lutea
(lweek; nominal)
76
80 W
1
1,000
93% red. chlorophyll
production
100% red.
chlorophyll prod.
000235-44
Torres and
O'Flaherty
1976
Supplemental
(raw data unavailable)
Green Algae - Chlorophyceae
Stigeoclonium tenue
(1 week; nominal)
76
80 W
1
1,000
67% red. chlorophyll
production
90% red. chlorophyll
production
000235-44
Torres and
O'Flaherty
1976
Supplemental
(raw data unavailable)
Green Algae - Chlorophyceae
Chlorella vulgaris
(1 week; nominal)
76
80 W
1
1,000
50% red. chlorophyll
production
80-87% red.
chlorophyll prod.
000235-44
Torres and
O'Flaherty
1976
Supplemental
(raw data unavailable)
81
-------
Table A-42. Nontarget Marine/Estuarine Plant Toxicity (Tier II)
Surrogate Species/ Cone, (ppb) MRID No.
Duration/Measured/nominal % ai Probit Slope % Response Author/Year Study Classification
Xanthophyceae
Tribonema sp.
(1 week; nominal)
Xanthophyceae
Vaucheria geminata
(1 week; nominal)
Chrysophyceae
Isochrysis galbana
(120 hours; nominal)
Marine Diatom
Skeletonema costatum
(120 hours; nominal)
Marine Diatom
Skeletonema costatum
(120 hours; measured)
Marine Green -
Chlorophyceae
Chlamydomonas sp.
(72 hours; nominal); Salinity
30 g/L
Marine Yellow -
Chrysophyceae
Monochrysis lutheri
( 72 hours; nominal); Salinity
30 g/L
Marine Red - Rhodophyceae
Porphyridium cruentum
(72 hours; nominal); Salinity
30 g/L
Marine Green -
Chlorophyceae
Neochloris sp.
(72 hours; nominal); Salinity
30 g/L
Marine Bacillariophyceae
Cyclotella nana
(72 hours; nominal); Salinity
30 g/L
Marine Bacillariophyceae
Achnanthes brevipes
(72 hours; nominal); Salinity
30 g/L
Marine Yellow -
Chrysophyceae
Isochrysis galbana
(240 hours; nominal); Salinity
30 g/L
76
80 W
76
80 W
97.4
97.4
97.1
99.7
99.7
99.7
99.7
99.7
99.7
99.7
1
1,000
1
1,000
22
NOAEC < 13
LOAEC 13
Slope 3.065
24
NOAEC < 13
LOAEC 13
Slope 3.343
53
NOAEC 14
Slope 2.798
60
77
79
82
84
93
100
42% red. chlorophyll
production
75% red. chlorophyll
production
41% red. chlorophyll
production
100% red.
chlorophyll prod.
50% red. growth
30% red. growth
50% red. growth
14% red. growth
50% red. cell growth
50% red. 02 production
000235-44
Torres and
O'Flaherty
1976
000235-44
Torres and
O'Flaherty
1976
410652-04
Parrish 1978
410652-04
Parrish 1978
430748-01
Hoberg 1993
402284-01
Mayer 1986
50% red. in 02
production
50% red. in 02
production
50% red. in 02
production
50% red. in 02
production
50% red. cell growth
402284-01
Mayer 1986
402284-01
Mayer 1986
402284-01
Mayer 1986
402284-01
Mayer 1986
Supplemental
(raw data unavailable)
Supplemental
(raw data unavailable)
Supplemental
(NOAEC, method &
raw data unavailable)
Supplemental
(NOAEC, method &
raw data unavailable)
Core
Supplemental
(72 hrs & endpoint)
50% red. 02 production 402284-01 Supplemental
Mayer 1986 (72 hrs & endpoint)
Supplemental
(72 hrs & endpoint)
Supplemental
(72 hrs & endpoint)
402284-01 Supplemental
Mayer 1986 (72 hrs & endpoint)
Supplemental
(72 hrs & endpoint)
Supplemental
(NOAEC unavailable)
82
-------
Table A-42. Nontarget Marine/Estuarine Plant Toxicity (Tier II)
Surrogate Species/
Duration/Measured/nominal % ai
Cone, (ppb)
Probit Slope % Response
MRID No.
Author/Year
Study Classification
Marine Green - 99.7
Chlorophyceae
Chlorococcum sp.
(240 hours; nominal); Salinity
30 g/L
Marine Green - 99.7
Chlorophyceae
Platymonas sp.
(72 hours; nominal); Salinity
30 g/L
Marine Bacillariophyceae 99.7
Thalassiosira fluviatilis
(72 hours; nominal); Salinity
30 g/L
Marine Bacillariophyceae 99.7
Stauroneis amphoroides
(72 hours; nominal); Salinity
30 g/L
Marine Algae 97.4
Microcystis aeruginosa
(120 hours - nominal)
Marine Green - 99.7
Chlorophyceae
Chlorella sp.
(72 hours; nominal); Salinity
30 g/L
Blue-green - Cyanophyceae Unk.
Anabaena cylindrica
(24 hour; nominal)
Marine green - 97
Chlorophyceae
Dunaliella tertiolecta
(120 hours; nominal)
Marine Yellow - 99.7
Chrysophyceae
Phaeodactylum tricornutum
(240 hours; nominal); Salinity
30 g/L
Marine Bacillariophyceae 99.7
Nitzschia closterium
(72 hours; nominal); Salinity
30 g/L
Marine Bacillariophyceae 99.7
Amphora exigua
(72 hours; nominal); Salinity
30 g/L
Marine Green - 99.7
Chlorophyceae
Dunaliella tertiolecta
(240 hours; nominal); Salinity
30 g/L
100
110
110
129
NOAEC 65
Slope 3.162
140
178
182
253
180
NOAEC < 100
LOAEC 100
Slope 1.95
200
290
300
300
50% red. cell growth
402284-01
Mayer 1986
50% red. 02 production 402284-01
Mayer 1986
50% red. growth
7% red. growth
50% red. 02 production
50% red. 14-carbon
uptake; media: Taub &
Dollar (TD), algal assay,
& TD, respect.
50% red. growth
34% red. growth
50% red. cell growth
410652-04
Parrish 1978
402284-01
Mayer 1986
450200-15
Larsen et al.
1986
410652-03
Hughes 1986
402284-01
Mayer 1986
50% red. 02 production
50% red. cell growth
402284-01
Mayer 1986
402284-01
Mayer 1986
Supplemental
(NOAEC unavailable)
100 50% red. 02 production 402284-01 Supplemental
Mayer 1986 (72 hrs & endpoint)
50% red. 02 production 402284-01 Supplemental
Mayer 1986 (72 hrs & endpoint)
Supplemental
(72 hrs & endpoint)
Supplemental
(NOAEC, method &
raw data unavailable)
Supplemental
(NOAEC unavailable
Supplemental
(raw data unavailable)
Supplemental
(NOAEC unavailable)
Supplemental
(NOAEC unavailable)
50% red. 02 production 402284-01 Supplemental
Mayer 1986 (72 hrs & endpoint)
Supplemental
(72 hrs & endpoint)
Supplemental
(NOAEC unavailable)
83
-------
Table A-42. Nontarget Marine/Estuarine Plant Toxicity (Tier II)
Surrogate Species/
Duration/Measured/nominal % ai
Cone, (ppb)
Probit Slope
% Response
MRID No.
Author/Year
Study Classification
Marine Red - Rhodophyceae 97.4
Porphyridium cruentum
(120 hours)
Marine Bacillariophyceae 99.7
Nitzschia (Ind. 684)
(72 hours; nominal); Salinity
30 g/L
Marine Green -Chlorophyceae 97.4
Kirchneria subcapitata
(120 hours; nominal)
Marine Bacillariophyceae 99.7
Navicula inserta
(72 hours; nominal); Salinity
30 g/L
308
NOAEC <130
LOAEC 130
Slope 2.449
430
431
NOAEC 200
Slope 4.217
460
50% red. growth
16% red. growth
50% red. O2 production
5% red. in growth
4% red. in growth
50% red. in 02
production
410652-04
Parrish 1978
402284-01
Mayer 1986
410652-04
Parrish 1978
402284-01
Mayer 1986
Supplemental
(NOAEC, method &
raw data unavailable)
Supplemental
(72 hrs & endpoint)
Supplemental
(NOAEC, method &
raw data unavailable)
Supplemental
(72 hrs & endpoint)
Table A-43. Formulation Nontarget Marine/Estuarine Algal Toxicity (Tier II)
Species/
Cone, (ppb)
MRID No.
Duration/Measured/nominal
% ai
Probit slope
% Response
Author/Y ear
Study Classification
Mar. Yellow - Chrysophyceae
76
100 (240 hrs)
50% red. cell growth
402284-01
Supplemental
Isochrysis galbana
80 WP
Mayer 1986
(NOAEC unavailable)
(nominal); Salinity 30 g/L
200 (2 hrs)
50% red. 02 production
Mar. Yellow Chlorophyceae
76
100 (240 hrs)
50% red. cell growth
402284-01
Supplemental
Chlorococcum sp.
80 WP
Mayer 1986
(NOAEC unavailable)
(nominal); Salinity 30 g/L
400 (2 hrs)
50% red. O2 production
Mar. Yellow - Chrysophyceae
76
200 (240 hrs)
50% red. cell growth
402284-01
Supplemental
Phaeodactylum tricornutum
80 WP
Mayer 1986
(NOAEC unavailable)
(nominal); Salinity 30 g/L
200 (2 hrs)
50% red. O2 production
Mar. Green - Chlorophyceae
76
400 (240 hrs)
50% red. cell growth
402284-01
Supplemental
Dunaliella tertiolecta
80 WP
Mayer 1986
(NOAEC unavailable)
(nominal); Salinity 30 g/L
600 (2 hrs)
50% red. O2 production
Table A-44. Longer-term (> 10 days exposure) Nontarget Marine/Estuarine Plant Toxicity
Surrogate Species/ Cone, (ppb) MRID No.
Duration/Measured/nominal % ai Probit Slope % Response Author/Year Study Classification
Vascular Plants:
Sago Pondweed (Estuarine)
NR
Salinity 12 ppt:
450882—31
Supplemental
Potamogeton pectinatus
NOAEC
7.5
Chesapeake
(raw data unavailable)
(28 days; measured/nominal)
LOAEC
14.3
sign. red. dry weight
Bay Program
Salinity 1 &
: 6 ppt:
1998
NOAEC
14.3
LOAEC
30
sign. red. dry weight
84
-------
Table A-44. Longer-term (> 10 days exposure) Nontarget Marine/Estuarine Plant Toxicity
Surrogate Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit Slope
% Response
MRID No.
Author/Y ear
Study Classification
Estuarine rush 97.1
Juncus roemerianus
(5 weeks - 1 year; measured
Pondweed
Potamogeton perfoliatus
(4 weeks; initial conc.
nominal,
terminal conc. measured)
NR
NR
Pondweed (Estuarine)
Potamogeton perfoliatus
(3 weeks; nominal)
Eelgrass (Estuarine)
Zostera marina
(10 days; measured)
Estuarine Eelgrass
Zostera marina
(21 days; nominal)
Wild Celery (Estuarine)
Vallisneria americana
(6 weeks; nominal)
Seagrass (Estuarine) Atrazi
Halodule wrightii ne
(22 - 23 days; measured) 4L
NR
NR
NR
LOAEC 30
NOAEC 30
NOAEC < 30
250 ppb
3, 800 ppb
30
Week 3: LOAEC
5
NOAEC < 5
4 weeks: LOAEC
50
NOAEC 5
53
est. 69
50
100
NOAEC 10
163
30,000
sign. red. chlorophyll a in
5 weeks
(1 year)
partial recovery (1 yr)
practically no survival
50% red. 02 product,
sign. red. 02 product.
sign. red. O2 product.
50% red. ????
50% red. leaf growth
25% red. leaf growth
62% red. leaf growth
21-day LC50 red.
production
50% red. shoot length
no difference at 0, 3, or
6 parts/thousand
46-58% red. total above-
ground biomass
450874-05
Lytle & Lytle
1998
452277-20
Kemp et al.
1985
450874-10
Forney and
Davis 1981
452277-29
Schwarzschild
et al. 1994
452277-05
Delistraty and
Hershner 1984
450874-10
Forney and
Davis 1981
452051-01
Mitchell 1987
Supplemental
(raw data unavailable)
Supplemental
(raw data unavailable)
Supplemental
(raw data unavailable)
Supplemental
(raw data unavailable)
Supplemental
(raw data unavailable)
Supplemental
(raw data unavailable)
Supplemental
(raw data unavailable)
Non-Vascular Plants:
Marine Brown macroalgae
Laminaria hyperborea
(18 days; nominal)
NR
NOAEC < 10
LOAEC 10
50 & 100
sign. red. growth rate
delayed sporophyte
formation
???? Supplemental
Hopkin &Kain (raw data unavailable)
1978
Marine Yellow - 99.7
Chrysophyceae
Isochrysis galbana
(240 hours; nominal); Salinity
30 g/L
Marine Green - 99.7
Chlorophyceae
Chlorococcum sp.
(240 hours; nominal); Salinity
30 g/L
Marine Yellow - 99.7
Chrysophyceae
Phaeodactylum tricornutum
(240 hours; nominal); Salinity
30 g/L
100
100
200
50% red. cell growth
50% red. cell growth
50% red. cell growth
402284-01
Mayer 1986
402284-01
Mayer 1986
402284-01
Mayer 1986
Supplemental
(NOAEC unavailable)
Supplemental
(NOAEC unavailable)
Supplemental
(NOAEC unavailable)
85
-------
Table A-44. Longer-term (> 10 days exposure) Nontarget Marine/Estuarine Plant Toxicity
Surrogate Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit Slope
% Response
MRID No.
Author/Y ear
Study Classification
Marine Green -
Chlorophyceae
Dunaliella tertiolecta
(240 hours; nominal); Salinity
30 g/L
99.7
300
50% red. cell growth
402284-01
Mayer 1986
Supplemental
(NOAEC unavailable)
The Tier II results for freshwater aquatic plants indicate that atrazine causes a 41 to 98%
reduction in chlorophyll production of freshwater algae; the corresponding EC50 value for four
different species of freshwater algae is 1 ppb, based on data from a 7-day acute study (MRID #
000235-44). Non-vascular plants are less sensitive to atrazine than their freshwater vascular
counterparts with an EC50 value of 37 ppb, based on reduction in duckweed growth (MRID #
430748-04).
The Tier II results indicate that the marine algae Isochrysis galbana is the most sensitive
nonvascular aquatic plant (EC50 = 22 ppb; MRID # 410652-04), and the most sensitive vascular
aquatic plant is Sago pondweed (7.5 ppb; MRID # 450882-31).
Comparison of atrazine toxicity levels for three different endpoints suggests that the endpoints in
decreasing order of sensitivity are cell count, growth rate and oxygen production (Stratton 1984).
Walsh (1983) exposed Skeletonema costatum to atrazine and concluded that atrazine is only
slightly algicidal at relatively high concentrations (i.e., 500 & 1,000 ppb). Caux etal. (1996)
compared the cell count IC50 and fluorescence LC50 and concluded that atrazine is algicidal at
concentrations which effect cell counts. Abou-Waly etal. (1991) measured growth rates on days
3, 5, and 7 for two algal species. The pattern of atrazine effects on growth rates differ sharply
between the two species. Atrazine had a strong early effect on Anabaena flos-aquae followed by
rapid recovery in clean water (i.e., EC50 values for days 3, 5, and 7 are 58, 469, and 766 ppb,
respectively). The EC50 values for Selenastrum capricornutum continued to decline from Day 3
through 7 (i.e., 283, 218, and 214 ppb, respectively. Based on theses results, it appears that the
timing of peak effects for atrazine may differ depending on the test species.
Degradates: Special tests are required for algal and vascular plant species (123-2) to address
concerns for the toxicity of atrazine degradates to aquatic plants. A summary of the degradate
aquatic plant toxicity data for deethylatrazine, deisopropylatrazine, diamino-atraine, and
hydroxyatrazine is provided in Tables A-45 through A-48, respectively.
Table A-45. Degradate Deethylatrazine Nontarget Aquatic Plant Toxicity (Tier II)
Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit slope
% Response
MRID No.
Author/Y ear
Study Classification
Fresh. Blue-Green - Cyanophyceae
>95
1,000
50% red. cell count
Stratton 1984
Supplemental
Anabaena inaequalis
4,000
50% red. growth rate
(NOAEC and raw data
(12-14 days1; nominal)
2,500
50% red. photosynthesis
unavailable)
86
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Table A-45. Degradate Deethylatrazine Nontarget Aquatic Plant Toxicity (Tier II)
Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit slope
% Response
MRID No.
Author/Y ear
Study Classification
Freshwater Green - Chlorophyceae
Scenedesmus quadricauda
(12-14 days; nominal)
>95
1,200
2,000
1,800
50% red. cell count
50% red. Growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Freshwater Green - Chlorophyceae
Chlorella pyrenoidosa
(12-14 days1; nominal)
>95
3,200
7,200
1,800
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Fresh. Blue-Green - Cyanophyceae
Anabaena variabilis
(12-14 days; nominal)
>95
3,500
7,500
700
50% red. cell count
50% red. growth rate
50 % red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Fresh. Blue-Green - Cyanophyceae
Anabaena cylindrica
(12-14 days; nominal)
>95
8,500
5.500
4,800
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Table A-46. Degradate Deisop
ropylatrazine Nontarget Aquatic Plant Toxicity (Tier II)
Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit slope
% Response
MRID No.
Author/Year
Study Classification
Fresh. Blue-Green - Cyanophyceae
Anabaena inaequalis
(12-14 days1; nominal)
>95
2,500
7,000
9,000
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Freshwater Green - Chlorophyceae
Scenedesmus quadricauda
(12-14 days; nominal)
>95
6,900
6.500
4,000
50% red. cell count
50% red. Growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Freshwater Green - Chlorophyceae
Chlorella pyrenoidosa
(12-14 days1; nominal)
>95
> 10,000
> 10,000
3,600
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Fresh. Blue-Green - Cyanophyceae
Anabaena variabilis
(12-14 days; nominal)
>95
5,500
9,200
4,700
50% red. cell count
50% red. growth rate
50 % red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Fresh. Blue-Green - Cyanophyceae
Anabaena cylindrica
(12-14 days; nominal)
>95
> 10,000
> 10,000
9,300
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Table A-47. Degradate Diamino-Atrazine Nontarget Aquatic Plant Toxicity (Tier II)
Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit slope
% Response
MRID No.
Author/Y ear
Study Classification
Fresh. Blue-Green - Cyanophyceae
Anabaena inaequalis
(12-14 days1; nominal)
>95
7,000
>10,000
>100,000
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Freshwater Green - Chlorophyceae
Scenedesmus quadricauda
(12-14 days; nominal)
>95
4,600
10,000
>100,000
50% red. cell count
50% red. Growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Freshwater Green - Chlorophyceae
Chlorella pyrenoidosa
(12-14 days1; nominal)
>95
>10,000
>10,000
>100,000
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
87
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Table A-47. Degradate Diamino-Atrazine Nontarget Aquatic Plant Toxicity (Tier II)
Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit slope
% Response
MRID No.
Author/Y ear
Study Classification
Fresh. Blue-Green - Cyanophyceae
Anabaena variabilis
(12-14 days; nominal)
>95
>10,000
>10,000
100,000
50% red. cell count
50% red. growth rate
50 % red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Fresh. Blue-Green - Cyanophyceae
Anabaena cylindrica
(12-14 days; nominal)
>95
>10,000
>10,000
>100,000
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Table A-48. Degradate Hydroxyatrazine Nontarget Aquatic Plant Toxicity (Tier II)
Species/
Duration/Measured/nominal
% ai
Cone, (ppb)
Probit slope
% Response
MRID No.
Author/Y ear
Study Classification
Fresh. Blue-Green - Cyanophyceae
Anabaena inaequalis
(12-14 days1; nominal)
>95
>10,000
>10,000
>100,000
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Freshwater Green - Chlorophyceae
Scenedesmus quadricauda
(12-14 days; nominal)
>95
>10,000
>10,000
>100,000
50% red. cell count
50% red. Growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Freshwater Green - Chlorophyceae
Chlorella pyrenoidosa
(12-14 days1; nominal)
>95
>10,000
>10,000
>100,000
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Fresh. Blue-Green - Cyanophyceae
Anabaena variabilis
(12-14 days; nominal)
>95
>10,000
>10,000
>100,000
50% red. cell count
50% red. growth rate
50 % red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
Fresh. Blue-Green - Cyanophyceae
Anabaena cylindrica
(12-14 days; nominal)
>95
>10,000
>10,000
>100,000
50% red. cell count
50% red. growth rate
50% red. photosynthesis
Stratton 1984
Supplemental
(NOAEC and raw data
unavailable)
The Tier II results for atrazine degradates indicate that deethylatrazine is more toxic than the other
four degradates, and the most sensitive algae of the five species is generally the blue-green alga
Anabaena inaequalis with EC50 values ranging from 100 to > 100,000 ppb. Atrazine is more
toxic to these algal species than any degradate. The order of descending toxicity for these algal
species are atrazine > deethylatrazine > deisopropylatrazine > diamino-atrazine > hydroxy-
atrazine.
A.5 Effects of Environmental Factors and Life-Stage on Aquatic Atrazine Toxicity
A. 5.1 Interaction Effects on Atrazine Toxicity to Plants
Some intra-laboratory studies suggest that atrazine toxicity is affected by environmental
parameters, such as temperature, light intensity and salinity levels. Mayer etal. (1998) concluded
that a temperature difference of 1 °C will cause a difference in algal growth rate in the range of 7
to 9 percent at the typical rate increase for 10 °C temperature increase (Q10) of 2 to 2.3.
88
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In general, the toxicity of pesticides increase with increasing temperature. Mayasich, Karlander
and Terlizzi, Jr. (1986) tested two algal species in 27 combinations of temperature (15, 20 and 25
°C), light intensity (0.208, 0.780 and 1.352 mW/cm2) and atrazine concentrations of 0, 50 and 100
(j,g/L) for 7-day periods. Toxic effects of atrazine on Nannochloris oculata growth rates were
significantly (p < 0.01) dependent on both temperature and light intensity as determined by the 3-
way interactions. Atrazine toxicity increased to N. oculata with both increasing temperature and
increasing light intensity, except at 15 °C and 1.352 mW/cm2 where growth was intermediate.
Previous results yielded a similar anomaly and suggest that 15 °C is near the lower limit for
growth of this algal species. With Phaeodactylum tricornutum, growth rates were significant (p <
0.01) for light intensity and atrazine concentrations, and also significant (p < 0.05) for
temperature, but only light intensity was significantly (p < 0.01) related to an increase in atrazine
toxicity. Atrazine toxicity was highest at the lowest light intensity. "The response of P.
tricornutum to atrazine at light intensities of 0.780 and 1.352 mW/cm2 may be a reflection of
primary effects only, while at 0.208 mW/cm2, light intensity includes secondary effects"
(Mayasich et al., 1986). With respect to the insignificant effect of temperature on growth, Ukeles
(1961) and Fawley (1984) found that the growth of P. tricornutum was unchanged by
temperatures in the range of 14 to 25 °C.
Mayasich et al. (1987) repeated the above algal study with lower atrazine concentrations (0, 15,
30 and 50 [j,g/L and fewer temperatures (15 and 25 °C) and light intensities (0.208 and 1.352
mW/cm2) in unialgal and bialgal assemblages. Generally Phaeodactylum tricornutum' s presence
significantly (p < 0.01) depressed the growth of Nannochloris oculata, but it did not alter the
magnitude of the responses to temperature, light intensity or atrazine concentrations. In contrast,
the presence of N. oculata generally resulted in significant (p < 0.01) enhancement of P.
tricornutum growth. The bialgal assemblage produced magnitudes of interactions between
temperature and light intensity, and temperature and atrazine were both significantly (p <0.01)
greater for N. oculata. P. tricornutum dominated the assemblage over all concentrations of
atrazine under simultaneously low levels of temperature (15 °C) and light intensity (0.208
mW/cm2). At simultaneous high levels of temperature and light intensity and the absence of
atrazine, P. tricornutum and N. oculata tended to be co-dominant. At increased atrazine
concentrations, P. tricornutum became the dominant of the two algal species. The authors
concluded that the enhanced sensitivity of N. oculata to atrazine relative to that exhibited by P.
tricornutum posed a threat to the diversity and structure of natural phytoplankton populations.
Thus, a nutritious algal species for larval oysters (Dupry, 1973) is replaced by what is considered
to be a poor food source for larval bivalves (Walne, 1970).
Mayer et al. (1998) tested the effect of four main environmental factors on the toxicity of atrazine
to the green alga Selenastrum capricornutum in 3 day tests. The four factors tested were light
intensity (44 and 198 [xE/m2), temperature (16 and 26 °C), nitrogen source (NH4+ and NO3-) and
pH (7.6 and 8.6). Temperature influenced growth indirectly by interacting with light intensity.
Algal growth measured as the atrazine EC50 values was marginally reduced under low light
intensity at high and low temperatures (158 and 159 (J,g/L, respectively versus the atrazine control,
164 |ig/L), High light intensity at the low temperature reduced the toxicity of atrazine to the alga
by about two fold (LC50 3 00 |ig/L) while high light intensity and high temperature reduced the
89
-------
toxicity of the atrazine by about 118 fold (LC50 191 (J-g/L). Nitrogen source and pH had no
significant effect on atrazine toxicity affecting algal growth rates.
The above studies indicate that the toxicity of atrazine to plants can be affected by environmental
parameters, but differences in effects are dependant on the algal species. Hence, increases in
temperature may increase, decrease or have no effect on atrazine toxicity to algal growth. Light
intensity generally has a stronger effect on atrazine toxicity to algal growth and may, short of the
point of photo-inhibition, increase the toxicity of atrazine. Nitrogen source and pH do not have
any effect on the toxicity of atrazine to algae.
A. 5.2 Interaction Effects on Atrazine Toxicity to Aquatic Animals
A number of intra-laboratory studies suggest that atrazine toxicity to aquatic animals is affected
by environmental parameters, such as water hardness, salinity and differences in the life-stages of
organisms.
High levels of water hardness usually reduce the toxicity of pesticides. Intra-laboratory studies on
two fish species provide comparative LC50 values for two levels of water hardness (Birge, Black
and Bruser, 1979). Embryo-larval rainbow trout were exposed to atrazine for 27 days at water
hardness levels of 50 and 200 mg/L and produced LC50 values of 0.66 and 0.81 mg/L,
respectively. Channel catfish were tested at the same water hardness levels for 8 days and yielded
LC50 values of 0.22 and 0.23 mg/L. With rainbow trout embryo-larvae, the soft water increased
toxicity by about 19 percent, while the LC50 values for embryo-larval catfish were the same. It is
uncertain if the shorter exposure period, yolk sac, or differences in species sensitivity, account for
the difference in water hardness effects between embryo-larvae of channel catfish and rainbow
trout.
Salinity effects at 5, 15 and 25 g/L on the toxicity of atrazine are opposite for the estuarine fish
larvae, sheepshead minnow and the copepod nauplii, Eurytemora affinis (Ziegenfuss, Anderson,
Spittler and Leichtweis, 1994). The 96-hour LC50 values (16.2, 2.3 and 2.0 mg/L) for sheepshead
minnow consistently increased with increasing salinity. In the case of the copepod nauplii, the
96-hour LC50 values (i.e., 0.5, 2.6 and 13.3 mg/L) consistently decreased with increasing salinity.
The consistency of the two data sets suggest that salinity effects the toxicity of atrazine.
Statistical tests for both species indicate significant differences between the LC50 valves at 5 and
25 g/L, but not at 15 g/L. The authors concluded that the two species may be more
physiologically effective in metabolizing and mitigating toxic effects of atrazine at various
salinities. The increase in LC50 values for rainbow trout and sheepshead minnow are consistent
for increasing water hardness and increasing salinity.
For many pesticides, the earlier life-stages are normally more sensitive than later life-stages.
Contrary to most pesticides, the aquatic toxicity data for toad and frog tadpoles suggest that the
late stages are more sensitive to atrazine than early tadpole stages (Howe et al., 1998). The late
stage of the American toad tadpole is about 2.5 times more sensitive to atrazine than the early
90
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stage (10.7 versus 26.5 mg/L). For the northern leopard frog tadpoles, the later stage is about 3.3
times more toxic than the early tadpole stage (14.5 versus 47.6 mg/L).
The above studies suggest that decreases in water hardness and salinity can increase the toxicity
of atrazine to fish, but increasing salinity may mitigate atrazine toxicity to copepods. Life stages
show differences in sensitivity to atrazine. The later stages in frog and toad tadpole development
show an increased sensitivity to atrazine over early tadpole stages.
91
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A.6 References
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Alvarez, M. C. (2005). Significance of Environmentally Realistic Levels of Selected
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Atkins, E. L., E. A. Greywood and R. L. MacDonald. 1975. Toxicity of pesticides and
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Gefahrdungspotentials von Pflanzenschutzmitteln-Wirkstoffen fur Gewasser.
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Baturo, W., L. Lagadic and T. Caquet. 1995. Growth, fecundity and glycogen utilization
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(Bobwhite quail, mallard ducks, rainbow trout, sunfish, goldfish). Prepared by
Woodard Res. Corp.; submitted by Geigy Chemical Co., Ardsley, NY. (MRID
No. 000592-14).
Beliles, R. P. and W. J. Scott, Jr. 1965. Atrazine safety evaluation on fish and wildlife
(Bobwhite quail, mallard ducks, rainbow trout, sunfish, goldfish): Atrazine: Acute
toxicity in goldfish. Prepared by Woodard Res. Corp.; submitted by Ciba-Geigy
Corp., Greensboro, NC. (MRID No. 000247-18).
Beliles, R. P. and W. J. Scott, Jr. 1965. Atrazine safety evaluation on fish and wildlife
Bobwhite quail, mallard ducks, rainbow trout, sunfish, goldfish): Atrazine: Acute
toxicity in rainbow trout. Prepared by Woodard Res. Corp.; submitted by Ciba-
Geigy Corp., Greensboro, NC. (MRID No. 000247-16).
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(Bobwhite quail, mallard ducks, rainbow trout, sunfish, goldfish): Atrazine: Acute
toxicity in sunfish. Prepared by Woodard Res. Corp.; submitted by Ciba-Geigy
Corp., Greensboro, NC. (MRID No. 000247-17).
Bentley, R. E. and K. J. Macek. 1973. Acute toxicity of atrazine to mud crab
(Neopanope texana). Prepared by Bionomics, Inc.; Submitted by Ciba-Geigy
Corp., Greensboro, NC. (MRID No. 000247-19).
Benson, B. and G. M. Boush. 1983. Effect of pesticides and PCBs on budding rates of
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