## 1 

# Appendix B: Supporting Ecological Toxicity Data

(94 pages)

A.1 Toxicity to Birds / Reptiles ............................................................................................. .......... 4 A.1 Toxicity to Birds / Reptiles ............................................................................................. .......... 4
A.1.1 Birds: Acute Oral Studies ........................................................................................... 4 A.1.2 Birds: Subacute Dietary Studies ................................................................................. 5
A.1.3 Birds: Chronic Studies ........................................................................................................ 6 A.1.4 Birds/Reptiles: Open Literature .................................................................................. 7

A.1.4a Birds: Open Literature Data .................................................................................. 7 A.1.4b Reptiles: Open Literature Data ............................................................................. 7 A.2 Toxicity to Freshwater Animals ........................................................................................... ... 13 A.2.1 Freshwater Fish, Acute .............................................................................................. 13 A.2.2 Freshwater Fish, Chronic .......................................................................................... 16 A.2.3 Sublethal Effects, Freshw ater Fish (Open Literature) .............................................. 17 A.2.3 Sublethal Effects, Freshw ater Fish (Open Literature) ...................................................... 18

A.2.4a Sublethal Effects: Freshwater Fish: ............................................................................ 18 A.2.4c Amphibians: ................................................................................................................. 22 A.2.5 Freshwater Invertebrates, Acute ....................................................................................... 22 A.2.6 Freshwater Invertebrate, Chronic .............................................................................. 25
A.2.8b Freshwater Field Studies (New Open Literature Data) ............................................... 28 A.3 Toxicity to Estuarine and Marine Animals ............................................................................. 29 A.3.1 Estuarine and Marine Fish, Acute ............................................................................. 29 A.3.2 Estuarine and Marine Fish, Acute (Open Literature 2006 Review) ......................... 30 A.3.2 Estuarine and Marine Fish, Chronic ......................................................................... 30
A.3.3a Sublethal Effects: Estuarine/Marine Fish ........................................................... 31
A.3.4 Estuarine and Marine Invertebrates, Acute ............................................................... 32

452083-03 & ................................................................................................................... ................ 32

452277-11 ..................................................................................................................... .................. 32 Hall, Ziegenfuss, Anderson, Spittler & Leichtweis 1994 ............................................................... 32
A.3.5 Estuarine and Marine Invertebrate, Chronic ............................................................. 34

A.3.7a Estuarine and Marine Fiel d Studies (2003 IRED Data) ......................................... 36
A.3.7b Estuarine and Marine Field Studi es (New Open Literature Data) .............................. 40 A.4 Toxicity to Plants .................................................................................................................... 42
A.4.1 Terrestrial Plants .................................................................................................... ... 42 A.4.2 Aquatic Plants ................................................................................................................... 44 A.5 Toxicity to Terrestrial Invertebrates .................................................................................... ... 55 A.6 Effects of Environmental Factors and Life-Stage on A quatic Atrazine Toxicity ................... 56 A.6.1 Interaction Effects on Atrazine Toxicity to Plants .................................................... 57 A.6.2 Interaction Effects on Atrazine Toxicity to Aquatic Animals ................................. 58 A.7 Pesticide Toxicity Interactions .......................................................................................... ...... 59

A.7.1 Plants ........................................................................................................................ ...... 59

A.7.2 Animals ....................................................................................................................... ... 61

## 2 

# A.8. References .............................................................................................................................. 65

## 3 

# This appendix presents available submitted and open literature studies available on atrazine. Studies that are submitted to the Agency in support of pesticide registration or re-registration are categorized as either acceptable, supplemental, or invalid. Acceptable m eans that all essential information was reported, the data are scientifically valid, and the study was performed according to recommended protocols. Studies in the “ acceptable” category fulfill the corresponding data requirement in 40 CFR Part 158 and are appropriate for use in risk assessment. Supplemental studies are also scientifically valid; however, they were either performed under conditions that deviate from recommended guideline protocols or certain data nece ssary for complete verification are missing. Supplemental studies may be used quan titatively in the risk assessment and can, at the Agency’s discretion, fulfill the corresponding data requirement in 40 CFR Part 158. Invalid studies are not scientifically va lid, or deviate substantially from recommended protocols such that they are not useful for risk assessment. I nvalid studies do not fulfill the corresponding data requirement in 40 CFR Part 158.

With respect to the open literature, studies may be classified as either qualit ative, quantitative, or invalid. The degree to which open literature data are quantitatively or qua litatively characterized is dependent on whether the information is releva nt to the assessment endpoints (i.e., maintenance of the survival, reproduction, and growth of the assessed listed species) identified in the problem formulation. Open literature studies classified as qualitative are not appropriate for quantitative use but are of good quality, addre ss issues of concern to the risk assessement, and, when appropriate, are discussed qualitatively in th e risk characterization discussion. Those open literature studies that are classified as quantitat ive are appropriate for quantitative use in the risk assessment including calculation of RQs. It s hould be noted that this appendix includes all
relevant data taken from the 2003 IRED atr azine effects appendix. In addition, ECOTOX information was obtained on October 16, 2011. The May 2007 ECOTOX search included all open literature data for atrazine (i.e., pre- and post-IRED). Da ta that pass the ECOTOX screen
described in Section 4.1 of the assessement are ev aluated along with the registrant-submitted data, and may be incorporated qualitati vely or quantitatively into this endangered species assessment. In general, effects data in the open literature th at are more conservative than the registrantsubmitted data are considered for quantitative use.

Citations for all open literature not considered as part of this assessmen t because it was either rejected by the ECOTOX screen or accepted by EC OTOX but not used (e.g., the endpoint is less sensitive and/or not appropriate fo r use in this assessment) is in cluded in Appendix G. Appendix G also includes a rationale for rejection of thos e studies that did not pa ss the ECOTOX screen and those that were not evaluated as part of this e ndangered species assessment. Further detail on the ECOTOX exclusion categories is provided in the Agency’s Guidance of the Evaluation Criteria for Ecological Toxicity Data in the Open Literature (U.S. EPA, 2004)

.

## 4 

# A.1 Toxicity to Birds / Reptiles

Given limited ecotoxicity data for reptiles, av ian 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 reptile s including turtles (red-eared slider [ Trachemys scripta elegans ] and snapping turtles [ Chelydra serpentine]) and American alligators ( Alligator mississippiensis ). Ecotoxicity data for birds and reptile s are discussed in Sections A.1.1 through

A.1.4.

A.1.1 Birds: Acute Oral Studies

An acute oral toxicity study us ing the technical grade of the ac tive ingredient (T GAI) is required to establish the toxicity of atr azine to birds. The preferred te st species is either mallard duck ( Anas platyrhynchos ; a waterfowl) or bobwhite quail ( Colinus virginianus ; an upland gamebird).
Results of these studies are su mmarized below in Table A-1.

Table A-1. Avian Acute Oral Toxicity: Technical Grade and Formulations

Surrogate Species % ai LD 50 (mg/kg) Probit Slope Toxicity Category MRID No.

Author/Year Study

Classification 1

Northern bobwhite quail ( Colinus virginianus ) 14-day old chicks; 8-day test Tech. 783 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 (only 3 birds) (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)



# Degradates : Minor atrazine degradates include deethylatrazine (DEA), deisopropylatrazine (DIA) and diaminochlorotriazine. Acute mammalian LD 50 

# values available for deethylatrazine and deisopropylatrazine are both more sensitive than the parent atrazine. Th erefore, a special (70- 1) acute oral toxicity test with the upland game bird (preferably northern bobwhite) are required to address the concern for these degradates. Acute avian LD 50 

# data for the atrazine degrdates, deethylatrazine (DEA) and deisopropylatrazine (DIA), and hydroxyatrazine (HA) are summarized
in Table A-2.

## 5 Table A-2. Avian Acute Oral Toxicity: Degradates

Surrogate Species Degradate
% ai LD 50 (mg/kgbw) Probit Slope Toxicity Category MRID No.

Author/Year Study

Classification 1

Northern bobwhite quail ( Colinus virginianus ) 18-week old chicks; 14-day test Deisopropyl
atrazine (DIA)

96% > 2,000 slope none Practically non-toxic 465000-07 Stafford, 2005a Acceptable

Northern bobwhite quail ( Colinus virginianus ) 17-week old chicks; 14-day test Hyrdroxy

atrazine (HA)

97.1% > 2,000 slope none Practically non-toxic 465000-08 Stafford, 2005b Acceptable

Northern bobwhite quail ( Colinus virginianus )

16-week old chicks; 14-day test Desethyl

Atrazine (DEA)

96% 768

Slope = 6.21

(95% CI = 3.19 – 9.27) Slightly toxic 465000-09 Stafford, 2005c Acceptable



# A.1.2 Birds: Subacute Dietary Studies

Two subacute dietary studies using the TGAI are required to establis h the toxicity of atrazine to birds. The preferred test speci es are mallard duck and bobwhite qua il. Results of these tests are
tabulated below in Table A-3.

Table A-3. Avian Subacute Dietary Toxicity

Surrogate Species % ai 5-Day LC 50 (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 et al . 1975 Supplemental (no control raw data)

Northern bobwhite ( Colinus 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 et al . 1975 Supplemental (no control raw data)

Japanese Quail ( Coturnix. japonica ) 7-days old chicks 99.0 > 5,000 (7 % mortality at 5,000 ppm) Practically non-toxic 000229-23

Hill et al . 1975 Supplemental (no control raw data; 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 et al . 1975 Supplemental (no control raw data)

1 Test organisms observed an additiona l three days while on untreated feed.

## 6 

# Subacute dietary studies using a typical end-us e product (TEP) may be required on a case-by-case basis to establish the toxicity of atrazine formul ations to birds. The preferred test species are mallard duck and bobwhite quail. Results of th ese tests are summarized below in Table A-4.

Table A-4. Formulation Avian Subacute Dietary Toxicity

Surrogate Species % ai

Form 5-Day LC 50 (ppm ai) 1
Probit Slope Toxicity Category MRID No.

Author/Year Study

Classification

Northern bobwhite ( Colinus virginianus ) (6-weeks old) 76

80 WP 5,760

slope 3.252 Practically non-toxic 000592-14
Beliles & Scott 1965 Supplemental (birds too old)

Mallard duck ( Anas platyrhynchos ) 76

80 WP 19,560 slope 1.807 Practically non-toxic 000592-14
Beliles & Scott 1965 Acceptable for

80W
formulation

1 Test organisms observed an additiona l three days while on untreated feed.



# A.1.3 Birds: Chronic Studies

Avian reproduction studies usi ng the TGAI are required for at razine, because the following conditions are met: (1) birds may be subject to re peated or continuous expos ure to the pesticide, especially preceding or during the breeding season, (2) the pesticide is stable in the environment to the extent that potentially t oxic amounts may persist in animal fe ed, (3) the pesticide is stored or accumulated in plant or animal tissues, and/or, (4) information derived from mammalian reproduction studies indicates reprod uction in terrestrial vertebrate s may be adversely affected by the anticipated use of the product. The prefer red test species are ma llard duck and bobwhite quail. Results of these tests are provided below in Table A-5.

Table A-5. Avian Reproduction

Surrogate Species/ Study Duration % ai NOAEC/

LOAEC (ppm ai) MRID No.

Author/Year Study

Classification

Northern bobwhite ( Colinus virginianus )

20 weeks 97.1 NOAEC 225

LOAEC 675 (based on egg producti on and embryo viability) 425471-02
Pedersen &

DuCharme 1992 Acceptable

Mallard duck ( Anas platyrhynchos )

20 weeks 97.1 NOAEC <75

LOAEC 75 (based on egg production a nd hatchability, adult food consumption and male wt; hatchling wt at 75 ppm), 425471-01
Pedersen &

DuCharme

1992 Acceptable

Japanese quail ( Coturnix. japonica )

8 days Tech Reduced food consumption and body wt; sescation of egg production; LC50 >10000 ppm 00024723

Sachsse and Ullman

1975 Supplemental (not chronic exposure)

## 7 

# A.1.4 Birds/Reptiles: Open Literature

A.1.4a Birds: Open Literature Data

Three studies were located in the open literature th at evaluated the potential for atrazine to affect endpoints including growth, sexual maturity, liver effects, and endocrine effects in birds (summarized in Table A-6).

Table A-6. Avian Reproduction/Growth Effects Tests 

## from Open Literature (2007 Review)

Study type/

Test material Test Organism (Common and Scientific Name) and

Age and/or Size Test Design Endpoint Concentration Citation (EcoRef. #) Rationale for Use in Risk

Assessment

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: 42547101 produced a

more sensitive LOAEC

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,
but not at lower levels. Wilhelms et al., 2006a (82035) Qual: 42547101 produced a

more sensitive LOAEC.

Reproduction toxicity study in

birds Japanese quail Birds were exposed to atrazine in the diet at concentrations that ranged from 0.001

p pm to 1000 ppm.. No effects on body weight, food intake, mortality, circulating corticosterone levels, or weights of liver, ovaries, or oviducts. N OAEC: 1000 ppm Wilhelms et al., 2006b (85511) QUAL. No effects were observed in the study.

Subchronic toxicity study in

birds Japanese quail Male birds orally dosed at concentrations of 10-500 mg/kg-bw for 45 days. Feed consumption reduced at 100-500 ppm and reduced body weight at 25-500 ppm;

at ≥ 50 ppm decrease in leukocyte

counts N OAEC: 10 ppm (based on body wt) Hussain et al.

2011 (153875_ Qual: Formulation used, uncertain if registered in U.S.;



# A.1.4b Reptiles: Open Literature Data

Open literature studies for reptiles exposed to atrazine are summarized below in Table A-7.

## 8 Table A-7. Reptilian Toxicity 

## Tests from Open Literature (2007 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

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 conc = 0.64

p pm) and 13.2 lb ai/A (measured conc = 8.1 ppm)
and control.
- 3 replicates (with 23-24 eggs/replication)/treatment

group. - Incubator temp = 25 o (+ 1 o 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

p roduced in atrazinetreated 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:

3 PAHs detected at nontoxic levels in control soil, but not analyzed
for in the atrazine
treatment groups
- low incidence of

intersex or females

p recluded ability to
differentiate between a

low incidence caused by atrazine exposure and random sampling

error

Chronic lab (duration NR) / Atrazine (99 % ai) American alligator ( Alligator mississippiensis ) eggs at stage 21 in embryonic development, j ust 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 μl of 95% ethanol.
- 5 eggs/treatment were incubated at temperatures to produce either 100% males (33 o C) or 100% females (30 o C).
- Endpoints: gonadal histology and hepatic steroidogenic activity NOAEC = 14 ppm

All atrazine treated eggs
incubated at female- and male-determining temps

p roduced female and male hatchlings, respectively. No differences in gonadal histology (Mullerian duct epithelial cell height and medullary regression) and hepatic aromatase activity was
noted between atrazine treated groups and

controls. Crain et al., 1999 (70208) QUAL: No treatmentrelated effects occurred in the study (study did not provide a sensitive endpoint for use in risk assessment).

Relevance of exposure

p athway (direct application to eggs) to

the current assessment is questionable.

10 day study /
Atrazine 97% American alligator ( Alligator mississippiensis )
and red-eared slider turtle ( Trachemys scripta elegans Eggs -eggs placed in nests treated w/0, 10, 50, 100 and 500 ppb via nest

saturation of solution for 10 days

-Incubated at 27.3 and

32.8° for turtles and alligators, respectively
-Endpoints:hatchability, embryo mortality and
sexual differentiation NOAEC=500 ppb

N o adverse effects found; no atrazine detected in embryonic
fluids Gross 2001 (MRID 45545302 and 03) QUAL: No treatmentrelated effects occurred in the study (study did not provide a sensitive endpoint for use in risk assessment).

Subchronic (10 day) / atrazine (%

N S) Caiman (Caiman latirostris) Stage 20 - 0.2 ppm atrazine (ethanol used as solvent) topically applied to eggs once;
-Incubated at 33°C (male temp) for 10 days
-Endpoints: histopathology and testosterone analysis N OAEC <0.2 ppm

Altered
histoarchitecture of the
testis Rey et al. 2009 (153844) QUAL:

Relevance of exposure

p athway (direct application to eggs) to
the current assessment is questionable.

## 9 Table A-7. Reptilian Toxicity 

## Tests from Open Literature (2007 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

Subchronic (10 day) / atrazine (%

N S) Caiman (Caiman latirostris) Stage 20 - 0.2 ppm atrazine (ethanol used as solvent) topically applied to eggs once;
-Incubated at 30°C (female temp) or 33°C (male temp) for 10 days
-Endpoints: sex determination, histopathology and hormone analysis N OAEC <0.2 ppm

Changes in Type III germ cells in ovaries;

decrease testosterone compared to control in
females Rey et al. 2009 (153844) QUAL:

Relevance of exposure

p athway (direct application to eggs) to

the current assessment is questionable.

Chronic (11 months) / Atrazine (>99%) 

### Map turtles (Graptemys

ouachitensis and G.

p seudogeographica)/

eggs -Incubated ggs exposed to sand containing 0, 0.1, 1.0 and 100 ppb atrazine applied once and sand rehydrated sand with
control water

-Reared subset of neonates

for 11 months under
control conditions.

-Endpoints: 

### hatching success, incubation time, external morphological abnormalities, gonadal sex, three measures of
b ody size, righting time, and swimming time for all turtles; subset- nest escape behavior, time to first foraging event, time to capture prey, growth, and survival - N OAEC = not dose responsive.

At hatch, no significant

effects but abnormalities
decreased with increasing conc. however, % still lower
than control

For subset, turtles at 0.1

p pb had inhibited nes t escape behavior and reduced post hatch

survival N euman-Lee &

Janzen 2011 (153816) Qual: no raw data,; negative control not used; results for both species presented as one

dataset



# NR = Not reported.

A. 1.5. Toxicity to Mammals

Acute Exposure (Mortality) Studies

Acute oral toxicity data ar e summ arized in Table A.8.

## 1 0 

# Table A-8. Summary of Acute Toxicity of Atrazine and its Degradates of Concern

Chemical Acute Mammal LD50 (mg/kg-bw)

Atrazine 1869 (MRID 00024709)

HA Not available

DEA 1240 (MRID 43013201)

DIA 1111 (MRID 43013202)

DACT Not available

Reproduction Toxicity

A 2-generation reproduction study wi th parent atrazine is availabl e. Reproduction studies are not available on the degradates of concern. However, the degradates have been tested in prenatal developmental studies in rodents. These studies are summarized belo w. Results for atrazine from the same guideline study are also presented for comparison. Additional repr oduction toxicity data is available for amphibians and will be evaluated for possible inclusion in the risk assessment.

Table A-.9. Toxicity Reference Values for Atra zine and Degradates of Concern in Guideline Prenatal Developmental Toxicity Studies a nd the 2-generation Reproduction study for

Parent Atrazine Chemical Study type/Data

Source Study Summary

2-generation reduction study in
rate / 40431306 LOAEL = 500 mg/kg-diet (39 mg/kg/day in males, 43 mg/kg/day in females) based on decreased body weights, body weight gains, and food consumption. NOAEL = 50 ppm (3.8 mg/kg/day in males, 3.7 mg/kg/day in females).

Atrazine Prenatal developmental in
rodents / 40566302 Maternal NOAEL = 10 mg/kg/day. Maternal LOAEL = 70 mg/kg/day, based on reduced body weight gain

Developmental NOAEL = 10 mg/kg/day Developmental LOAEL = 70 mg/kg/day based on delayed or no
ossification at several sites

MRID 41065201 Maternal NOAEL = 25 mg/kg/day. Maternal LOAEL = 100 mg/kg/day based on reduced body weight gain and food consumption.

Developmental NOAEL = 25 mg/kg/day. Developmental LOAEL = 100 mg/kg/day based on increased incidence of delayed ossification of skull bones.

HA Prenatal developmental in
rodents / MRID 41065202 Maternal NOAEL = 25 mg/kg/day Maternal LOAEL = 125 mg/kg/day based on decreased food consumption during the dosing period and enlarged and mottled kidneys. Developmental NOAEL = 25 mg/kg/day.

## 1 1 Developmental LOAEL = 125 mg/kg/day based on increased incidence of partially ossified interparietal and hyoid bones and decreased fetal body weight.

DIA Prenatal developmental in
rodents / MRID 43013209 Maternal NOAEL = 5 mg/kg/day Maternal LOAEL = 25 mg/kg/day based decreased food consumption, body weight/weight gain and food consumption

Developmental NOAEL = 25 mg/kg/day Developmental LOAEL = 100 mg/kg/day based on increased fetal
and litter incidences of fused sternebrae 1 and 2 and increased fetal incidence of poor ossification of the proximal phalanx of posterior digit 5 (a skeletal variation)

DEA Prenatal developmental in
rodents / MRID 43013208 Maternal NOAEL= 5 mg/kg/day Matenal LOAEL= 25 mg/kg/day based on decreased body weight gain and food consumption

Developmental NOAEL= 5 mg/kg/day Developmental LOAEL 25 mg/kg/day based on . increased fetal and
litter incidences of fused sternebrae 1 and 2

DACT Prenatal developmental toxicity in rodents /

MRID 41392402 Maternal NOAEL = 25 mg/kg/day Maternal LOAEL = 75 mg/kg/day, based on decreased body weight gain during dosing. Developmental NOAEL is 2.5 mg/kg/day. Developmental LOAEL = 25 mg/kg/day, based on increases in incidences of incompletely ossified parietals, interparietals and unossified hyoids.



# Metabolism in Mammals

Metabolism data are summarized in Table A-10.

Table A.10. Summary of Available Metab olism Data for Atrazine in Rodents

Guideline No./

Study Type MRID No. (year) /Doses Results

870.7485
Metabolism and pharmacokinetics 40431304 (1987) 0, 1, and 100 mg/kg for a single dose given through oral gavage. 1.0 mg/kg/day for 15 days by oral gavage. Distribution, accumulation Distribution was dose-dependent and independent of sex. Distribution appeared to follow first-order kinetics and the
half-life in the tissues was 31.3 hours.

Excretion Approximately 95% of the atrazi ne excreted within 7 days of dosing. Urinary route acc outed for about 75% of the
excretion feces accounted for 20 %. Route of excretion did not seem to vary among sexes or with dose.

870.7485 MRID 40431305 (1987) Distribution, accumulation

## 1 2 Guideline No./

Study Type MRID No. (year) /Doses Results

Metabolism and pharmacokinetics The animals were dosed daily for 10 days through a stomach tube with dose levels of 0, 1, 3, 7, 10, 50 or 100 mg/kg/day. Distribution was highest in the red blood cell, followed by the liver, ovary and kidney. Wh en the dose increased the
amount distributed in the tissues increased. The distributionappeared to follow first-order kinetics and the tissue halflife was 38.6 hours. This indicates that atrazine, with possible exception of the red blood cell, does not
bioaccumulate.

870.7485
Metabolism and pharmacokinetics MRID 40431306 (1987) Rats were given test 100 mg/kg article was given through the stomach tube in a single oral dose. Other rats were given 16.18 and 19.64 mg/kg and urine
was collected over a 24 hr period. The urine was analyzed for metabolites. Excretion In the rats given 100 mg/kg greater than 100% of the administered radioactivity was recovered within 3 days of dosing. Urine was found to contain 47.3% of the radioactivity and the feces 49.3%. The tissues contained
5.75% and 1.4% was found in the blood.

Metabolism Metabolites indicate that dechlorination of the triazine ring and N-dealkylation are the major metabolic pathways for atrazine in rats. Oxidation of the alky substituents of atrazine appears to be of minor metabolic importance.

870.7485
Metabolism and pharmacokinetics MRID 42165503 (1993) Fecal and urinary samples from rats exposed in a seprate metabolism study (MRID 40431304) were obtained and analyzed to determine metabolism profiles. Metabolic profile

No sex differences in metabolic profiles were evident. The major fecal metabolite was DACT which accounted for 40% of the total fecal radioactivity.

870.7485
Metabolism and pharmacokinetics MRID 44713802 (1993) single oral dose of 1 or 100 mg/kg through oral gavage Distribution, accumulation Time to maximum blood concentration (t

cmax 

## ) was 2 hours and 24 hours for the low and high dose groups, respectively. With exception of red blood cells, whole blood, and skeletal muscle, tissue burden for an y specific tissue or organ represented less than 1% of the administered dose by 14 days post dosing

Excretion Urinary excretion was 64.72% of the total administered low dose over a 48-hour period and 66.16% of the total administered high dose over a 168-hour period. Within 48 hours urinary excretion was 100% and 94% complete for the low-dose and high-dose group, respectively.
Fecal elimination accounted for 10.80% and 19.69% of the total dose for the low and high dose groups, respectively.

## 1 3 

# A.2 Toxicity to Freshwater Animals

A.2.1 Freshwater Fish, Acute

Two freshwater fish toxicity st udies using the T GAI 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). Toxcity data for TGAI are
summarized below in Table A-8.

Table A-8. Freshwater Fish Acute Toxicity (TGAI)

Surrogate Species/

Static or Flow-through test % a.i. 96-hour LC 50 (ppb) (measured/nominal) Probit Slope Toxicity Category MRID No.

Author/Year Study

Classification

Rainbow trout ( Oncorhynchus mykiss )
Static test 98.8 5,300 (nominal) slope - 2.723 moderately toxic 000247-16
Beliles & Scott 1965 Acceptable; water quality other than temp not reported

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 CaCO 3 ; 22 Ε C 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 CaCO 3 : 22 Ε C 96 9,370 (not specified) moderately toxic 452029-11 Hussein, El-Nasser &

Ahmed 1996 Supplemental (non-native sp.; 38-gram fish; no raw data) Silver catfish (2-4 g) (

Rhamdia quelen ) Static, Form (250 g/L both ais) 10,200 (atrazine only) 10,500 (atrazine & simizine) (Nominal) moderately toxic E11938

Kreutz et al. 2008 Supplemental (nonnative sp., no raw data, unknown formulation used)

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 CaCO 3 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; 10 Ε C; 11 mg/L CaCO 3 NR 27,000 (nominal) slightly toxic 452029-09 Grande, Anderson & Berge 1994 Supplemental (no raw data; slight aeration & purity unknown)

## 1 4 Table A-8. Freshwater Fish Acute Toxicity (TGAI)

Surrogate Species/

Static or Flow-through test % a.i. 96-hour LC 50 (ppb) (measured/nominal) Probit Slope Toxicity Category MRID No.

Author/Year Study

Classification

Zebrafish ( Brachydanio rerio ) NR 37,000 (NR) slightly toxic MRID # NR
Korte & Greim 1981 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)



# Table A-9 presents fish and amphibian t oxicity data for formulated products.

Table A-9. Freshwater Fish Acute Toxicity (Formulated Products)

Surrogate Species/ Flow-through or Static % ai

formul. 96-hour LC 50 (ppb) (measured/nominal) Toxicity Category MRID No.

Author/Year Study

Classification

Black Bass - fry ( Micropterus salmoides ) Static test; 20 Ε C 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; 23.3-25.8 Ε C 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; 25-27 Ε C 78 mg/L hardness 80

80 W 20,000 (nominal) no slope slightly toxic 452277-17

R. O. Jones 1962 Supplemental (limited raw data)

Coho Salmon ( Oncorhynchus kisutch ) Renewal daily; 144 hr 40.8*

AAtrex Liquid > 18,000 25 % mortality

(measured) slightly toxic 452051-07

Lorz et al . 1979 Supplemental (no LC 50 value & 12-17 months old)

Rainbow trout ( Onchorhynchus 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 ( Oncorhynchus 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 descri ption as a liquid formulation, AAtrex.

## 1 5 

# Acute toxicity data that did not produce a more sensitive LC50 value were reported in the 2008 ECOTOX summary and that data is presented below in Table A-9a.

Acute Studies identified in 2008 ECOTOX search whic h have a less sensitive toxicity value than most
sensitive value in Table A8. Endpoint Common Name Toxicity Value Reference (ECOTOX #) 48- h LC50 Bluegill fish ca. 26 mg/L Beliles et al. 1965 (80976) 96- h LC50 Brown trout 27 mg/L Grande et al. 1994 (62367)

48-96 h LC50 Goldfish >56-60 mg/L Beliles et al. 1965 (80976)

48-96 h LC50 Rainbow trout 4.5-10 mg/L (same study as above for 000247-16 4.5 study
authors value but
recalculation results in

value above 

## Beliles et al. 1965 (80976)

120 hours post
fertilization Zebrafish 7.5 uM Muncke et al. 2007 (95954)

96 hours post
fertilization LC50
and EC50 Zebrafish LC50 = 1255 uM

EC50 = 440 uM Ton et al. 2006 (93401)

48 and 96 hours post
fertilization NOAEL

& LOAEL Zebrafish 48 hpf NOAEL = 500 uM;
96 NOAEL/LOAEC
= 200/500 uM Ton et al 2006 (93401)

12-48 hr LOEC
12-48 h NOEC Zebrafish LOEC = 5-40 mg/L NOEC = 5-30 mg/L Wiegand et al. 2001 (62460)

96-h NOEC Fathead minnow 4.1 mg/L Jop 1991 (81782) 72 h Non-lethal Guppy 106.1 mg/L Bogacka et al 1990 (61069)

96-h
NOAEL/LOAEL Mummichog 0.05/0.5 mg/L Fortin et al. 2008 (103349)

72-h LOAEL Nile tilapia 0.00610 mg/L De campos et al. 2008 (103350) 12-h LOAEL Spotted seatrout 0.1 uM Thomas and Sweatman 208 (103377) 24-48 LC50 (unsure of test substance) Harlequin fish, red
rasbora 0.55-0.5 mg/L Alabaster 1969 (542)



# Degradates Acute fish testing with bluegill and rainbow trout are required to address degradate concerns for hydroxyatrazine (HA). Acute studies in rainbow trout have also been submitted for DACT and DIA degradates. Table A- 10 presents freshwater fish toxicity data for HA, DIA, and

DACT.

Table A-10. Freshwater Fish Acute Toxicity

Surrogate Species/ Flow-through or Static % ai

formul. 96-hour LC 50 (ppb) (measured/nominal) Toxicity Category MRID No.

Author/Year Study

Classification


### HA Bluegill sunfish ( Lepomis macrochirus ); 1.15 g Static test; 20.8 – 21.6 o C 125 mg/L hardness 98 >3,800 (measured dissolved) moderately toxic* 465000-05 Peither, 2005b Acceptable

## 1 6 Table A-10. Freshwater Fish Acute Toxicity

Surrogate Species/ Flow-through or Static % ai

formul. 96-hour LC 50 (ppb) (measured/nominal) Toxicity Category MRID No.

Author/Year Study

Classification

Rainbow trout ( Oncorhynchus mykiss ); 0.75 g Static test; 13.2 – 14.1 o C 125 mg/L hardness 98 >3,000 (measured dissolved) moderately toxic* 465000-04 Peither, 2005a Acceptable

DIA
Rainbow trout ( Oncorhynchus mykiss ); 1.5 grams Static test; 14 o C 165 mg/L hardness Not reported 17,000 (measured dissolved) Slightly toxic 47046103 Vial, 1991a Supplemental

DACT
Rainbow trout ( Oncorhynchus mykiss ); 1.5 g Static test; 14 o C 164 mg/L hardness Not reported >100,000 (measured dissolved) Practically non-toxic 47046104 Vial, 1991b Supplemental

* 

# B iological 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 an d rainbow trout 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 wate r from the intended use site, and the following conditions are met: the pesticide is intended for use su ch that its presence in water is likely to be continuous and recurrent; the pest icide is persistent in water ( i.e. , half-life greater than 4 days). Table A-11 presents the chronic toxi city data for freshwater fish.

Table A-11. 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/Year Study

Classification

Rainbow trout ( Oncorhynchus mykiss ) 86 days, flow-through 50 mg/L CaCO 3 Tech. NOAEC 410 LOAEC 1,100 (measured) sign. delays in hatching @ 1,100 and 3,800 μg/L sign. red. wet wt. at 30 & 58 days @ 1,100 & 3,800 μg/L sign. red. dry wt. @ 3,800 μg/L 58.8 % mortality @ 3,800 μg/L at swim-up 452083-04
Whale et al

. 1994 Supplemental (DMSO used as solvent, no raw
data for statistical analyses)

Rainbow trout embryo-larvae ( Oncorhynchus mykiss ) 27 days; flow-through 80

WP Hardness 50 mg/L:

LC50 660

LC01 29 Slope 1.2 Hardness 200 mg/L:

LC50 810

LC01 77 Slope 1.38 % normal survival 50/200 mg/L 19 μg/L - 94 98

54 - 88 90
54 ** - 68 74 5,020 ** - 10 9 50,900 ** - 0 0 452029-02 Birge, Black

& Bruser

1979 Supplemental (short test;
no raw data for

statistical analyses)

## 1 7 Table A-11. 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/Year Study

Classification

Channel catfish embryo-larvae ( Ictalurus punctatus ) 8 days; flow-through 80

WP Hardness 50 mg/L:

LC50 220 Slope 0.977 Hardness 200 mg/L:

LC50 230 Slope 0.84 highly teratogenic in all tests;
no results for soft water

420 μg/L - 16% terata 830 μg/L - 47 % terata 46,700 μg/L - 86 % terata 452029-02 Birge, Black

&

Bruser 1979 Supplemental (short test;
no raw data for

statistical analyses)

Zebrafish ( Brachydanio rerio ) 35 Days; pH 8; 27+ 1 Ε C Flow-through test Hardness 24 mg/L 98 NOAEC 300 LOAEC 1,300

(measured) 35-Day LC50 890

Slope 1.25 2 - 3 % sign. incr. in edema 45-62 % mortality 452029-08 Gorge & Nagel 1990 Supplemental (no raw data for
statistical analyses)



# 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 reproduc tive physiology of fish may be affected. Results of four fish life-cycle tests are tabulate d below in Table A-12.

Table A-12. Freshwater Fish Life-Cycle Toxicity

Surrogate Species/ Study Duration/ Flow-through or

Static Renewal %

ai NOAEC/LOAEC μg/L (ppb) (measured or nominal) Statistically sign. (p< 0.05) Endpoints Affected MRID No.

Author/Year 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 00024377
Macek et al . 1976 Supplemental (used DMSO,
treated fish for
disease after

allocation to tanks, no SC)

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. 00024377
Macek et al . 1976 Supplemental (Low survival for fry (F1) in the controls)

Fathead minnow ( Pimephales promelas ) 39 weeks; flow-through 97.1 NOAEC < 150

LOAEC 150 (measured) 6.7 % red. in F 1 length
22 % red. in F 1 body wt. (sign. diff. from neg. control) 42547103
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)

## 1 8 

# A.2.3 Sublethal Effects, Freshw ater Fish (Open Literature)

A.2.4a Sublethal Effects: Freshwater Fish:

A number of open literature studies were review ed as part of the 2003 IRED. Additional studies were reviewed for the California Red-legged frog assessment and results from the 2007 are presented. A number of studies were also located in the October, 2008 ecotox search that evaluated effects to fish. Most studies located in the open literature search reported endpoints that were not more sensitive than the currently available data. Additional studies, evaluating biochemical or molecular endpoints, were identif ied in the 2011 ECOTOX query and the studies are presented below.

Table A-15. Freshwater Fish Sublethal Effects Tests 

## (from 2003 IRED and 2007 ECOTOX Review)

Study type/

Test material Test Organism (Common and Scientific Name) and Age and/or

Size Test Design Endpoint Concentration in ppb Citation (MRID/ EcoRef. #) Classification

Hormone evaluation Adult largemouth bass ( Micropterus
salmoides ) Fish exposed to 0, 25, 35, 50, 75,and 100 ppb for 20 days ↑ plasma estradiol (sign) and ↑ vitellogenin (37x) in female bass at 100 ppb formulation; sign. ↓ plasma

11-ketotestosterone in male fish at 100 ppb form; plasma 11-ketotestosterone sign ↓ in fish at >35 ppb Wieser and

Gross 2002 (MRID 45622304) Supplemental (high variability)

Behavior (refuge) Zebrafish ( Brachydanio

rerio ) Fish exposed to 5 to 3125

p pb for 4 week Exposed fish showed a sign.

p reference for dark part of aquaria at 5-3125 ppb but no
difference between atrazine treatments; after 4 weeks 15- 24% more fish preferred dark
habitats Steinberg et al 1995 (MRID 45204910) Supplemental

Behavior (grouping, surfacing, swimming) Goldfish Fish exposed to 0.5, 5 and 50 ppb for 24 hours

Skin extract added to tanks Significant reduction in groups at 5 and 50 ppb; other significant behaviors
observed but not doseresponsive – surfacing at 5

p pb, burst swimming at 0.5 and 50 ppb, reduced sheltering and groups at 5 ppb

after skin extract added Saglio and Trijase 1998 (MRID 45202914) Supplemental

Kidney function Rainbow trout 28 day exposure to 5, 10 and 20, 40 and 80 ppb;
96 hour exposures Chronic exposure at 5 ppb and higher altered kidney

structure Fischer-Scherl
et al 1991 (45202907) Supplemental

Growth and

p roperties of various
tissues Rainbow trout;

Galaxias maculates
and Pseudaphritis

urvillii Fish exposed to 0.9, 3.0, 10, 50 and 340 ppb for 10 days Consistent significant effects observed at 10 ppb for G.

maculates ( ↑ muscle RNA/DNA); Glutathione in liver significantly reduced at 50 ppb in P. urvillii ; Reduction in muscle protein significant in rainbow trout; other effects (not consistent) observed at 0.9 and 3.0 ppb Davies et al 1994 (MRID 45202904)

## 1 9 Gill physiology Gnathonemus

p etersii Fish exposed to 500 and 5000 ppb for 6 hours Breaks in gill epithelium at 500 ppb with deep pits at 5000 ppb Alazemi et al 1996 (MRID 45202901) Supplemental

Behavior (breathing, swimming, feeding, equilibrium) and

survival N ile river fish

Oreochromis

niloticus and Chrysichthyes

auratus Fish exposed to 3000 and 6000 ppb for 28 days. Significant decrease in red blood cells, hemoglobin and
hematocrit
Significant changes in corpuscular endpoints and
AChE levels
Clinical signs such as rapid respiration and increase gill cover movement, slower reflexes and swimming, reduced feeding, equilibrium and death (more pronounced

in C. auratus ) reported Hussein et al 1996 (MRID 45202911) Supplemental – not all endpoints could be confirmed with data presented

Enzyme activity Carp Fish exposed to 1500, 3000 and 6000 ppb Serum alkaline phosphatase level sign. higher at all atrazine levels; effects on glutamic-oxaloacetic in liver and kidney; histopathological effects to gills, liver, kidney and intestine at 6000ppb N eskovic et al 1993 (MRID 45202913) Supplemental

Olfactory function Atlantic salmon
(Salmo slar) Adult male fish exposed to 0.5, 5, 10, 20 ppb (measured 0.04,3.6,,6.0, and 14 ppb) Effects on olfactory function at 0.5-20 ppb Moore and Waring 1998 (MRID 45204906) Supplemental (uncertainty in measured conc due to possible degredation)

Gill physiology and

survival after transfer to full salinity

seawater. Salmon smolts Fish were exposed to atrazine for 7 days at
atrazine concentrations of 1

–

23 ug/L under flow through conditions. Endpoints evaluated included gill physiology

and survival after transfer to full salinity sea water.

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 higher. Effects included altered Na K ATPase activity, increased sodium levels, and increased potassium levels.

Transfer of fish exposed to
atrazine in freshwater at 1ug/L and higher into atrazinefree full salinity seawater resulted in mortality; 43% mortality was observed at 5 ug/L and higher after 24
hours. Waring and

Moore 2004 (72625) Qual 

## : Relevance of

environmental conditions used in this study to the assessed species is questionable because the assessed fish species do not
enter full salinity seawater 

## ; an unacceptable solvent was used (industrial methylated spirits)

Olfactory detection o f female priming

p heromone,

p rotogandin F2 α in FW
fish

30 min exposure

Simazine, Atrazine,

and Simazine/ Atrazine mixtures (% a.i. NR) Mature male
Atlantic salmon ( Salmo salar L.)

p arr; length = 140 mm; weight = 34.2 g)

source: Environment Agency, Cynrig hatchery, Wales Skin and cartilage removed to expose olfactory rosettes

Olfactory epithelium

p erfused with control waterfor 30 min, then to atrazinetreated water at nominal concentrations of 0.1, 0.5, and 2.0 ug/l 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

p riming response of male
salmon to PGF 2 α (increased levels of expressible milt not

p resent following exposure to PGF) was observed at 0.5 ug/L. Moore, A., and

N . Lower,

2001 (67727) Qual: A solvent control, but no negative control, was used; therefore, potential solvent effects cannot be evaluated;

Study conducted on olfactory epithelium; therefore it is unclear whether response to
chemical stimuli would impair similar responses in
intact fish.

Relationship between the magnitude of effects on the endpoints evaluated and reproduction or survival has
not been established.

## 2 0 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 LC 50 (combined mortality + terata incidences) in rainbow trout was 870 ug/L. Birge et al., 1983. , (19124) Qual: No control responses were reported; limited water quality parameters were

p rovided; use of a solvent is uncertain; Reported toxicity
value is less sensitive than the available life-cycle NOAEC of 65 ppb. LC 50 s were based
on combination of abnormalities and mortality.

Olfactory-mediated
behavioral and neurophysiological response in FW fish

30 min exposure

Atrazine (97.4% ai) Juvenile rainbow trout (mass = 32.7

+ 1.2 g, length =

14.7 + 0.18 cm)

Source: Sun Valley Trout Farm (Mission BC) Fish statically exposed to 1, 10, and 100 ug/L atrazine (6

p er treatment level) and
acetone control.

Preference/avoidance to 10 -7

L-histidine was measured. Electro-olfactograms (EOGs) of olfactory rosettes were measured (EOGs measure electrical responses of olfactory neurons)

N = 6 fish/treatment level

and solvent control

Filtered, dechlorinated municipal water used.

p H = 6.8
Hardness = 6.12 CaCO3 Oxygen = > 90% sat. Light/Dark = 12:12 h Food: salmon pellets ad

libitum Temp: 12 o C Exposure to 10 ppb atrazine
resulted in L-histidine
avoidance

Hyperactivity observed at

atrazine concentrations of 1 and 10 ppb.

L-histidine evoked EOGs were significantly reduced at 10 ppb Tierney et al.,

2007 (89625) Qual: A solvent control, but no negative control, was used; therefore, potential solvent
effects cannot be evaluated.

Study conducted on olfactory epithelium; therefore it is unclear whether response would impair similar responses in intact fish.

Relationship between the magnitude of effects on the endpoints evaluated and reproduction, survival, or growth has not been

established.



## Studies from 2008 ECOTOX Endpoint Common name Toxicity Value Ref#

Reference

7-21 d
LOEC/NOAEC Goldfish 0.859 mg/L/0.19-0.9 mg/L Spano et al. 2004 (73297)

8-8.5 d nonlethal

4.5 d nonlethal Channel catfish 4.5, 8-8.5 d: 46.7 mg/L Birge et al. 1979 (563)

23 d non-lethal Rainbow trout 50 mg/L Birge et al 1979 (563)

12-h

N OAEL/LOAEC Atlantic
croaker 1-10 uM Thomas and Sweatman 2008 (103377)

81-d NOAEL

5-d NOAEL Atlantic
salmon 81-d: 0.0005/0.005 mg/L 5-d: 0.000495 mg/L Moore et al. 2007 (103267)

10-21-d

N OAEL/LOAEL Atlantic
salmon 0.0085-0.084 / 0.084 N ieves-Puigdoller et al. 2007 (93473)

10-d LC50 Blennie

Common j ollytail

Rainbow trout >0.34 mg/L Davies et al. 1994 (4442)

10-d

N OEC/LOEC Blennie 0.003-0.01 mg/L Davies et al. 1994 (4442)

10-d NOEC/

LOEC Rainbow trout 0.05- >0.34/ 0.34 mg/L Davies et al. 1994 (4442)

## 2 1 8-d

N OAEL/LOAEL Red drum 0.06-0.08 / 0.033 - 0.037 mg/L McCarthy and Fuiman 2008 (103059)

27-d LC01 Channel catfish 0.029-0.0772 mg/L Birge et al. 1979 (563)

Studies from 2011 ECOTOX

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Yan X;Zha J;Shi B;Wang D;Wang Z;Tang H. 2010. In Vivo Toxicity of Nano

‐ C60 Aggregates Complex with Atrazine to Aquatic Organisms. Chin. Sci. Bull.55(4/5): 339

‐ 345E153821

## 2 2 Matsumoto J;Hosmer AJ;Van Der Kraak G. 2010. Survival and Iono

‐ Regulatory Performance in Atlantic Salmon Smolts is not Affected by Atrazine
Exposure
Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol.152(3): 379

‐ 384. E153822

Xing H;Han Y;Li S;Wang J;Wang X;Xu S. 2010. Alterations in mRNA Expression of Acetylcholinesterase in Brain and Muscle of Common Carp Exposed to Atrazine and Chlorpyrifos

Ecotoxicol. Environ. Saf.73(7): 1666

‐ 1670. E153823

Yang L;Zha J;Zhang X;Li W;Li Z;Wang Z. 2010. Alterations in mRNA Expression of Steroid Receptors and Heat Shock Proteins in the Liver of Rare

Minnow (Grobiocypris rarus) Exposed to Atrazine and p,p'

‐ DDE Aquat. Toxicol.98(4): 381

‐ 387. E153830

Nwani CD;Nagpure NS;Kumar R;Kushwaha B;Kumar P;Lakra WS. 2011. Mutagenic and Genotoxic Assessment of Atrazine

‐ Based Herbicide to

Freshwater Fish Channa punctatus (Bloch) Using Micronucleus Test and Single Cell Gel Electrophoresis Environ. Toxicol. Pharmacol.31(2): 314

‐ 322. E153833

Nwani CD;Nagpure NS;Kumar R;Kushwaha B;Kumar P;Lakra WS. 2010. Lethal Concentration and Toxicity Stress of Carbosulfan, Glyphosate and

Atrazine to Freshwater Air Breathing Fish Channa punctatus (Bloch) Int. Aquat. Res.2(2): 105

‐ 111. E153834

Zhu L;Zhu LS;Shao B;Song Y;Xie H;Wang J;Wang JH;Liu W;Hou XX. 2011. DNA Damage and Effects on Antioxidative Enzymes in Zebra Fish (Danio rerio) Induced by Atrazine. Toxicol. Mech. Methods21(1): 31

‐ 36. E153842

Cavas T. 2011. In Vivo Genotoxicity Evaluation of Atrazine and Atrazine

‐ Based Herbicide on Fish Carassius auratus Using the Micronucleus Test

and the Comet Assay. Food Chem. Toxicol.49(6): 1431

‐ 1435. E153855
Wang X;Xing H;Li X;Xu S;Wang X. 2011. Effects of Atrazine and Chlorpyrifos on the mRNA Levels of IL

‐ 1 and IFN

‐ [Gamma]2b in Immune Organs

of Common Carp Fish Shellfish Immunol.31(1): 126

‐ 133. E153872.



# A.2.4c Amphibians:

Toxicity Data for amphibians in presented in Appendix C.

A.2.5 Freshwater Invertebrates, Acute

A freshwater aquatic inv ertebrate toxicity test usi ng the TGAI is required to establish the toxicity of atrazine to aquatic invertebrate s. Results of this test and ot hers are summarized below in Table

A-18.

Table A-18. Freshwater Invertebrate Acute Toxicity

Surrogate Species/

Static or Flow-through % ai 48 or 96-hour LC 50 /EC 50 μg/L (ppb) (measured/nominal) Toxicity Category MRID No. or

ECOTOX No.

Author/Year Study Classification

Midge ( Chironomus tentans )
Static test 94 720 (nominal) highly toxic 000243-77
Macek et al . 1976 Supplemental (48-hour LC50 & raw data are missing)

Midge ( Chironomus riparius ) 85.5 1,000 (unknown) highly toxic 450874-13
Johnson 1986 Supplemental (raw data are missing)

Scud ≤ 7-d old (Hyalella azteca)
Static renewal 98 1,500 (measured, unknown if used in calc) Moderately
toxic E 118745 Ralston-Hooper 2009 Qualitative (raw data missing)

Waterflea ( Daphnia magna ) 85.5 3,500 (unknown) moderately

toxic 450874-13
Johnson 1986 Supplemental (raw data are missing)

## 2 3 Table A-18. Freshwater Invertebrate Acute Toxicity

Waterflea < 24-hours old ( Daphnia magna )
26-Hour static test ?? 3,600 (unknown) at least moderately
toxic 000028-75 Frear & Boyd 1967 Supplemental (unknown ai, 26-hour test & no raw data)

Scud ( Gammarus fasciatus )

Static test 94 5,700 (nominal) moderately
toxic 000243-77
Macek et al . 1976 Supplemental (48-hour LC50 & raw data are missing)

Stonefly (nymph) ( Acroneuria sp.) Flow-through test 67.4 mg/L CaCO 3 98.5 6,700 (measured) moderately

toxic Brooke 1991 Supplemental (raw data are missing)

Waterflea ( Daphnia magna )
Static test 94 6,900 (nominal) moderately

toxic 000243-77
Macek et al . 1976 Supplemental (raw data are missing)

Shrimp (1-1.5 cm) (Paratya australiensis)
Static-renewal-48 hr 97 9,700-9,900 (water only) 6,500-6,800 (water & sediment) (measured-initial) Moderately
toxic E76267 Phyu et al. 2005 Qualitative (raw data missing, non-native spp.,

Scud juvenile ( Hyalella azteca ) Flow-through test 67.4 mg/L Ca CO 3 98.5 14,700 (measured) slightly toxic Brooke 1991 Supplemental (raw data are missing)

Scud juvenile ( Gammarus pulex ) Static-renewal - daily ?? 14,900 (measured) 4.4 @ 10 days slightly toxic 452029-17 Taylor, Maund &

Pascoe 1991 Supplemental (raw data are missing)

Cladoceran ( Pseudosida ramosa )

static 99 17,100 (mean of 20 trials) (nominal) Slightly
toxic E153837
Freitas et al 2011 Qualitative (raw data missing)

Waterflea (Daphnia carinata)

Static 22,400-24,600 (water only) 25,300-26,700 (water & sediment) (measured –initial) Slightly
toxic E74233 Phyu et al 2004 Qualitative (raw data missing)

Scud adult (Diporeia spp.)

Static renewal 98 >3,000 (measured unknown if used in calc) unknown Ralston-Hooper et al.

2009

E118745 Qualitative (raw data missing)

Waterflea ( Ceriodaphnia dubia )

48-Hour static test 97 > 4,900 (measured) Slope - no mortality unknown 452083-09 Jop 1991 Supplemental ( EC50 value not determined)

Leech ( Glossiphonia complanata ) Static-renewal weekly 99.2 > 16,000 ( measured) 6,300 μ g/L @ 28 days slightly toxic 452029-16
Streit & Peter 1978 Supplemental (raw data are missing)

Leech ( Helobdella stagnalis ) Static-renewal weekly 99.2 > 16,000 (measured)

9,900 

# μ g/L @ 27 days slightly toxic 452029-16
Streit & Peter 1978 Supplemental (raw data are missing)

Snail ( Ancylus fluviatilis ) Static-renewal weekly 99.2 >16,000 (measured) > 16, 000 μ g/L @ 40 days (35 % mortality) slightly toxic 452083-05 Oris, Winner &

Moore 1991 Supplemental (raw data are missing)

Waterflea <12 hr old ( Ceriodaphnia dubia )

Static 48-hour test 57 mg/L CaCO 3 > 99 > 30,000 (measured) Slope - no data slightly toxic 452029-17 Taylor, Maund &

Pascoe 1991 Supplemental (raw data are missing)

## 2 4 Table A-18. Freshwater Invertebrate Acute Toxicity
Mussel (glochidia and juveniles) (Lampsilis siliquoidea)
static 98 >30,000 (both life stages) (measured, results based on nominal) unknown E99469 Bringolf et al. 2007 Qualitative (raw data missing)

Freshwater mussel

A. imbecillis juvenile and mature organisms 97 >60,000 in both juvenile

and mature A. imbecillis. Unknown Johnson et al. 1993 (50679) Qualitative (no raw data, uncertain about reported high concentrations)

Midge ( Chironomus riparius ) Static-renewal - daily 10-Day test ?? > 33,000 (measured) 18,900 μ g/L @ 10 days slightly toxic 000272-04
Drake 1976 Supplemental (raw data are missing) (EC 50 115 ppm exceeds water solubility (33 ppm)

Midge ( Chironomus tentans ) Flow-through 10-Day test; water-spiked

exposure 98.5 Mortality :

LC 50 > 24,000

(measured) (37% mortality) NOAEC = 16,000 LOAEC = 24,000 Growth (dry weight) :

EC 50 = 8,300 (measured) NOAEC <3,200 LOAEC = 3,200 slightly toxic 459040-01 Putt, 2002 Supplemental (does not fulfill any currently-approved U.S. EPA SEP guideline)

Midge ( Chironomus temtans )
Static-renewal – to maintain water quality10-Day test; sedimentspiked exposures 98.5 Mortality (measured conc):

SED NOAEC = 130,000 SED LOAEC = 270,000 Pore Water (PW) NOAEC = 26,000 PW LOAEC = 29,000 (14% mortality)

PW LC 50 >30,000 Growth: Dry Weight (measured conc):

SED NOAEC = 24,000 SED LOAEC = 60,000 PW NOAEC = 4,000 PW LOAEC = 21,500 slightly toxic 459040-02 Putt, 2003 Supplemental (does not fulfill any currently-approved U.S. EPA SEP guideline)

Formulations % ai

Product

Waterflea ( Daphnia magna ) Flow-through test 79.6

80 WP >31,000 (higher concs. than 31,000

υ g/L were cloudy; 40% immobilization at 31 ppm) (measured) slope 2.433 slightly toxic 420414-01

Putt 1991 Supplemental

for formulation (EC 50 was not identified due to insolubility)

Waterflea ( Daphnia pulex ) Static test; 15 Ε C 282 mg/L hardness
With & without sediment 40.8

4 L 36,500 (nominal)

46,500 (with sediment) slightly toxic 452277-12
Hartman & Martin

1985 Supplemental

for formulation (EC50 exceeds water solublity and low temp.)

Mussel (glochidia and juveniles) (Lampsilis siliquoidea)
static 40.8

4L >30,000 (both life stages) (measured, results based on nominal) unknown E99469 Bringolf et al. 2007 Qualitative (raw data missing)

Freshwater mussel

Utterbackia imbecillis
Glochidia
static 40.8

4L LC 50 = 241,000 Practically
non-toxic Conners and Black,

2004 (74236) Qual: Study tested a formulated product at
concentrations considerably higher than the solubility limit of atrazine of 33 mg/L.

## 2 5 

# Degradates : Table A-19 presents freshwater invert ebrate toxicity data for four degradates.

Table A-19. Freshwater Invertebrate Acute Toxicity

Surrogate Species/ Flow-through or Static % ai

formul. 48-hour EC 50 (ppb) (measured/nominal) Toxicity Category MRID No.

Author/Year Study

Classification

HA

Waterflea ( Daphnia magna ); 1 st instar (6-24 h old)
Static test 98 >4,100 (measured dissolved) moderately toxic* 465000-01 Peither, 2005c Acceptable

DIA

Waterflea ( Daphnia magna ); 1 st instar (6-24 h old)
Static test Not reported >100,000 (measured dissolved) Practically non-toxic 47046101 Vial, 1991c Supplemental

Amphipod (Hyalella azteca) 98 96-hr LC50 7,200 (measured unknown if used in calc) Moderately toxic Ralson-Hooper et al

2009

E118745 Qualitative – no raw data,

Amphipod (Diporeia spp) 98 96-hr LC50 >3,000 (measured unknown if used in calc) Moderately toxic Ralson-Hooper et al

2009

E118745 Qualitative – no raw data,

DACT

Waterflea ( Daphnia magna ); 1 st instar (6-24 h old)

Static test Not reported >100,000 (measured dissolved) Practically non-toxic 47046102 Vial, 1991d Supplemental

DEA Amphipod (Hyalella azteca) 98 96-hr LC50 7,200 (measured unknown if used in calc) Moderately toxic Ralson-Hooper et al

2009

E118745 Qualitative – no raw data,

Amphipod (Diporeia spp) 98 96-hr LC50 >3,000 (measured unknown if used in calc) Moderately toxic Ralson-Hooper et al

2009

E118745 Qualitative – no raw data,

* 

# B iological 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.



# Although the freshwater invertebrate EC 50 

# value (>4,100 ppb) for the de gradate, hydroxyatrazine, is within the range classifying it as moderately toxic, the biological resu lts for the study were based on dissolved (filtered) mean-measured con centrations 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 us ing the TGAI is required for atrazine since the end-use product is expected to be transporte d 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 aquati c acute LC50 is less than 1 mg/L; and the pesticide is persistent

## 2 6 

# 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/ Study Duration/ Flow-through or

Static Renewal % ai NOAEC/LOAEC μg/L (ppb) (measured or nominal) Statistically sign. (p=0.05) Endpoints Affected MRID No.

Author/Year Study

Classification

Scud ( Gammarus fasciatus ) 30 days / flow-through 94 NOAEC 60

LOAEC 140 (measured) Based on reduced development of

F 1 to seventh instar. 000243-77
Macek et al

. 1976 Supplemental (used DMSO, no SC, 64-74%
control survival, reproduction in 1 of 2 reps in control)

Midge ( Chironomus tentans ) 38 days / flow-through 94 NOAEC 110

LOAEC 230 (measured) Based on reduced adult emergence 000243-77
Macek et al

. 1976 Supplemental (used DMSO, no SC)

Waterflea ( Daphnia magna ) 21 days / flow-through 94 NOAEC 140

LOAEC 250 (measured) Based on reduced production of young/female in F 0 generation 000243-77
Macek et al

. 1976 Supplemental (used DMSO, no SC, control survival 61%)

Waterflea ( Daphnia pulex ) 28-Day static-renewal

70-Day static-renewal test 99.2 NOAEC 1,000 LOAEC 2,000 (nominal) Based on reduction in young/female 452029-15
Schober & Lampert 1977 Supplemental (no raw data for
statistical analyses)

Waterflea - 6 generations ( Daphnia magna )
Static-renewal test NR Cups:

NOAEC 200 LOAEC 2,000 (unknown) 4 L aquarium:

NOAEC ??

LOAEC ?? (water from treated corrals) Based on reduction in # of young in generations 4, 5, & 6. Kaushik, Solomon, Stephenson and Day 1985 Supplemental (methods and
raw data are not reported)

Leech ( Helobdella stagnalis ) 40 Days Static-Renewal weekly 99.2 NOAEC <1,000 LOAEC 1,000 (measured) Based on reduced % hatch 452029-16
Streit & Peter

1978 Supplemental (no raw data for

statistical analyses)

Waterflea < 12 hr. old ( Ceriodaphnia dubia ) Two 7-Day static-renewal tests; Renewed M, W, & F

57 CaCO 3 ; Temp. 25 Ε C > 99 NOAEC 2,500 LOAEC 5,000 NOAEC 2,500 LOAEC 5,000 (measured) Based on reduction in mean total number of young per living female (3 broods) 452083-05 Oris, Winner and

Moore 1991 Supplemental (no raw data for analyses)

Green hydra (normal) ( Chlorohydra viridissima ) 21-Day Static test NR NOAEC <5,000 LOAEC 5,000 (nominal) Based on reduction in budding

rates 452029-01
Benson & Boush

1983 Supplemental (no raw data for analyses)

Waterflea 3-day old adult ( Ceriodaphnia dubia ) Two 4-Day static-renewal tests; Renewed M & W

57 CaCO 3 ; Temp. 25 Ε C > 99 NOAEC 5,000 LOAEC 10,000 NOAEC 10,000 LOAEC 20,000 (measured) Based on reduction in mean total number of young per living female (3 broods) 452083-05 Oris, Winner and

Moore 1991 Supplemental (no raw data for analyses)

## 2 7 Table A-20. Freshwater Aquatic Invertebrate Life-Cycle Toxicity

Freshwater Snail ( Ancylus fluviatilis ) 40 Days Static-Renewal weekly 99.2 1,000 4,000

16,000 (measured) 38-39% red. in egg capsules & eggs in April/May 56-57% red. in eggs in April/May 15-16% red. in eggs in July/Aug. 68-73% red. in eggs in April/May 65-71% red. in eggs in July/Aug. 452029-16
Streit & Peter

1978 Supplemental (no raw data for

statistical analyses)

Leech ( Glossiphonia complanata ) 27-Days Static-Renewal weekly 99.2 1,000 4,000 16,000 (measured) no reduction in egg production 17 % higher mortality 33 % higher mortality 67 % higher mortality 452029-16
Streit & Peter

1978 Supplemental (no raw data for

statistical analyses)

Ancylus fluviatilis (river limpet)

, Glossiphonia complanata (leech), Helobdella stagnalis (leech)

40-Day lab 99.2 Effects observed

at all
concentrations (1000, 4000 and 16000 ) Evaluated for food ingestion, growth, and egg production. Streit and Peter,

1978 QUAL.

NOAEC was not achieved;
no information was reported on experimental conditions (e.g.,
temperature, pH, DO);
limited
information on study design
parameters
such as no. of replicates, no.
of animals treated per concentration, etc.) was reported.

Numerous invertebrates including annelids, arthropods, and mollusks.

8 wks NS No effects were observed at any

concentration.

NOAEC = 670 ppb evaluated for abundance of various

taxa EG&G, 1979 Invalid:
Possible control contamination;; unacceptable solvent; a solvent control, but no negative
control was

used

Lymnaea palustris

12-week mesocosm study 97.8 No effects occurred at any
concentration for any of the
parameters
evaluated..The

NOAEC was 125 ug/L (nominal). Endpoints evaluated included mortality, growth, fecundity, and biochemical parameters (glycogen content, polysaccharide hydrolysis Baturo et a. (1995) QUAL. Replicate

mesocosms were not used

per concentration;
atrazine

concentrations

were not analytically confirmed; no
water/sediment quality data were provided.

Mussel (1.475 mm)

(Lampsilis siliquoidea)

21 –d static renewal (95% every 48 or 72 hours) 98
and

40.8 Tech 21-d EC50 = 10,100

Form 21-d EC50 = 3,100

(measured based

on Based on immobility of organisms; no effect on growth E99469

Bringolf et al

2007 Qualitative: no
raw data reported water quality and
control mortality was provided

## 2 8 

# A.2.8b Freshwater Field Studies (Ne w Open Literature Data)

Based on the results of the 2003 IRED for atrazine , potential adverse eff ects on sensitive aquatic plants and non-target aquatic organisms includi ng their populations and communities, are likely to be greatest when atrazine concentrations in water equal or exceed approximately 10 to 20 μ 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 μ g/L aquatic-community effect level we re considered. In addition, data for taxa that are directly relavent to the endange red 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 revi ew of new open literature, all
of the available studies show eff ects levels to freshwater fish a nd invertebrates at concentrations greater than 10 μ g/L.

One open literature artifical stream mesocosm study was reviewed because it provides data on freshwater snails, which may be used as surroga te for endangered mussels. The results of this study, which are summarized as pa rt of Table A-25, show potential indirect effects to grazing behavior (i.e., increased searching ve locity and movement patterns) at 15 μ 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 qua ntity, resulting in increased searching speed when the biomass of periphyton decreases. However, it is not possible to make a quantitative link between increased searching velocity in snails and the assessment endpoints of survival, fecundity or growth; therefore, data from th is study is not used to derive RQs in the risk assessment.

Table A-25. Freshwater Mesocosm Study 

## from Open Literature (2007 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

N R) Freshwater snails ( Physa

acuta and Ancylus f luviatilis ) - 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

p onds, 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

p atterns 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 snails to survival, growth and reproductive success is
uncertain

## 2 9 (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.



# 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. Re sults of these tests are summarized in Table A26

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/Year Study

Classification

Sheepshead Minnow

larvae < 48-hours old ( Cyprinodon variegatus ) Static test, T - 20 Ε C 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; fed fish during study)

Spot ( Leiostomus xanthurus )

Static test Salinity - 12 g/L; T - 22+ 1 Ε C 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 - 22-23 Ε C 97.1 13,400 (measured) Slope 4.377 slightly toxic 433449-01
Machado 1994 Acceptable

Spot (juvenile) ( Leiostomus xanthurus ) Flow-through test Salinity - 29 g/L; T - 28 Ε C 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 LC 50 

# 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 endpoi nt for estuarine/marine fish is based on the

LC 50 

# value of 2,000 ppb for sheepshea d minnow at a salinity of 25 o 

# /

oo 

# (MRID 452083-03 and 452277-11).

## 3 0 

# A.3.2 Estuarine and Marine Fish, Ac ute (Open Literatu re 2006 Review)

A.3.3. Acute Marine/Estuarine Toxicity Data - Degradates

A special acute estuarine fish test (72-3) is re quired to address concerns for the toxicity of atrazine degradates to estuarine fish (prefe rably sheepshead minnow). Table A-27 presents
estuarine/marine fish toxic ity data for hydroxyatrazine.

Table A-27. Marine/Estuarine Invertebrate Acute Toxicity (Hydroxyatrazine)

Surrogate Species/ Flow-through or Static % ai

formul. 96-hour LEC 50 (ppb) (measured/nominal) Toxicity Category MRID No.
Author/Year Study

Classification

Sheepshead minnow ( Cyprinodon variegatus ) Static test; T = 21-24 o C Salinity = 32‰ 97.1 >1,900 (no mortality) (measured) moderately toxic* 465000-06 Sayers, 2005a Acceptable

* 

# B iological results for the study were based on the m ean-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 LC 50 

# value (>1,900 ppb) for the degradate, hydroxyatrazine, is within the range classifying it as moderately toxic, the biological resu lts for the study were
based on mean-measured concentrations of hydroxyatrazine, which remained constant (> 90% recovery of nominal concentratio ns) at the limit of its water sol ubility (~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 direc tly to the estuarine/marine environment or is expected to be transported to this environmen t from the intended use site, and the following conditions are met: the pesticide is intended for use su ch that its presence in water is likely to be continuous; an aquatic acute LC 50 

# or EC 50 

# is less than 1 mg/L; and the pesticide is persistent in water ( i.e. , half-life greater than 4 days). The pref erred test species is sheepshead minnow.
Results of this test are summarized below in Table A-28.

## 3 1 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

μ g/L (ppb) (measured or nominal) Statistically sign. (p=0.05) Endpoints Affected MRID No.

Author/Year Study

Classification

Sheepshead Minnow ( Cyprinodon variegatus) Study duration - unknown Flow-through test Salinity -13g/L; T 30+ 1 Ε C 97.4 NOAEC 1,900 LOAEC 3,400

(measured) 89 % red. in juvenile

survival 452029-20
Ward &
Ballantine 1985 Supplemental (no raw data for
statistical analyses)

Sheepshead Minnow ( Cyprinodon variegatus) Study duration – 28 days PH Flow-through test Salinity = 29 – 31 o /

oo T = 24 – 27 o C 97.1 NOAEC = 1,100

LOAEC = 2,200

(measured 17% reduction in mean length; 46% reduction in mean wet weight 466482-03, 49526- 06, and 469526-04

Cafarella, 2005a Acceptable



# In the 2003 atrazine IRED, chroni c 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/mari ne fish data from Caferalla, 2005a (MRID # 466482-03) show that juvenile growth may be a mo re sensitive endpoint than survival. Although no effect on pre- or post-hatch survival was obs erved 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, rela tive 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 a ssociated with estuarine/marine fish exposure to atrazine are based on re spective NOAEC and LOAEC values of 1,100 and 2,200 ppb (MRID # 466482-03).

A.3.3a Sublethal Effects: Estuarine/Marine Fish

Biagianti-Risbourg and Bastide (1995) exposed juvenile gray mullets ( Liza ramada ) to 170 μ g/L atrazine for 9, 20, and 29 days in static te sts and for 11 days followed by 18 days of decontamination; and then measured the sublethal effects on the liver. At 170 μ g/L, 10, 25 and 60 percent mortality occurred fo llowing 9-, 20- and 29-day expos ures, respectively; control mortality was a constant 10 pe rcent throughout the test. Treated mullets showed normal behavior until Day 20 after which they stopped feed ing and rapidly died; whic h 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 hepato cytes had particularly large lipofuscin granul es (MRID # 452049-02).

Alvarez (2005; ECOTOX No. 81672) investigated effects of exposure to atrazine for up to 8 days on growth, swimming behavior, predator re sponse, and respiration rate in red drum

## 3 2 

# ( Sciaenops ocellatus ) larvae. This study reported effects including reduced growth rate at 80 ppb and changes in swimming speed and respiration at 40 ppb. However, a negative control was not used in the evaluation of these endpoints; ther efore, potential solven t effects could not be evaluated. Also, mortality in the control and treated groups was high for an exposure study that was approximately 1 week (23% mortality, survival for each test group was not reported). Also, it is uncertain if any relationshi p between the respiration and beha vioral effects observed in this study and reduced survival and reproduction exists. Therefore, these data were not used in derivation of risk quotients.

A.3.4 Estuarine and Marine Invertebrates, Acute

Acute toxicity testing with estuar ine/marine invertebrate s 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. Results of these tests for the TGAI a nd 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 LC 50 /EC 50 μ g/L (ppb) (measured/nominal) Probit Slope Toxicity Category MRID

No./ECOTOX No.

Author/Year Study

Classification

Mysid shrimp (Neomysis integer)Juvenile 4-7 mm/ Static renewal – 48 hrs / 5 psu ; T15oC 98-99 48 (measured) Very highly toxic E103334 / Noppe et
al. /2007 Qualitative (no raw data)

Copepod ( Acartia tonsa ) Static-renewal - daily Salinity - 31 g/L; T 22 o C 70
Tech. 88.9 (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 o C 97.4 94 (nominal) Slope - none very highly toxic 452029-20
Ward &

Ballantine 1985 Supplemental (no raw data)

Copepod (Tigriopus brevicornis) various life stages / static / 35 ppt; T: 20 99 121-153 (nominal) Slope no data Highly toxic E19281 / Forget et
al. / 1998 Qualitative (no raw data; uncertainty in

CIs Copepod (Eurytemora affinis) / static / 2 ppt; T: 18 Tech 125 (nominal) Slope no data Highly toxic E80951/ Forget et
al. / 2005 Qualitative (no raw data)

Copepod ( Acartia tonsa ) Static-renewal - daily Salinity - 31-32 g/L; T 22 o 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 affinis ) Static test; T – 20 o 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)

## 3 3 Table A-30. Estuarine/Marine Invertebrate Acute Toxicity

Surrogate Species/ Static or Flow-through/ Salinity & Temperature % ai. 96-hour LC 50 /EC 50 μ g/L (ppb) (measured/nominal) Probit Slope Toxicity Category MRID

No./ECOTOX No.

Author/Year Study

Classification

Mysid Shrimp ( Americamysis bahia ) Flow-through test Salinity 26 g/L; T 22+ 1 o C 97.4 1,000 (Measured) Slope - none highly toxic 452029-20
Ward & Ballantine

1985 Supplemental (no raw data)

Brown Shrimp (juvenile) ( Penaeus aztecus ) Flow-through test Salinity - 30 g/L; T 27 o C 99.7 1,000 (nominal) Slope - none at least highly
toxic 402284-01 Mayer 1986 Supplemental (48-hr LC 50 & no raw data)

Copepod - 17 days old ( Acartia tonsa ) Flow-through test Salinity - 31-33 /L, T – 20 o C 97.1 4,300 (measured) Slope - 2.467 moderately toxic 452083-08

McNamara 1991 Supplemental (cloudy with no

0.45 μ m filter
of undissolved test material)

Mysid Shrimp ( Americamysis bahia ) Flow-through test Salinity -32 g/L; T 25-26 o C 97.1 5,400 (measured) Slope 4.513 moderately toxic 433449-02
Machado 1994 Acceptable

Pink Shrimp ( Penaeus duorarum )

Static test Salinity 26 g/L; T 22+ 1 ο C 97.4 6,900 (nominal) Slope - none moderately toxic 452029-20
Ward &

Ballantine 1985 Supplemental (no raw data)

Copepod ( Acartia clausii ) Static-renewal - daily Salinity - 31 g/L; T 6-6.2 o C 70
Tech. 7,900 (nominal) Slope 0.958 moderately toxic 452029-18 Thursby et al . 1990

memo Acceptable

Grass Shrimp ( Palaemonetes pugio )
Static test Salinity - 26 g/L; T 22+ 1 o C 97.4 9,000 (nominal) Slope - none moderately toxic 452029-20
Ward &
Ballantine 1985 Supplemental (no raw data)

Eastern oyster (juvenile) ( Crassostrea virginica ) (Shell deposition) Flow-through test Salinity - 28 g/L; T – 28 o C 99.7 > 1,000
no effect (nominal) Slope - none unknown 40228-01 Mayer 1986 Supplemental (EC 50 has not
been identified & no raw data)

Eastern oyster (juvenile) ( Crassostrea virginica ) (Shell deposition) Flow-through test Salinity 31-32 g/L; T =20-21

o C 97.1 > 1,7 00
no effect (measured)

Slope - none unknown 466482-01

Caferalla, 2005b Acceptable

Mud Crab ( Neopanope texana )

Static test Salinity & T - unknown Tech. > 1,000 (nominal) Slope - none slightly toxic 000247-19 Bentley & Macek

1973 Supplemental (LC 50 exceeds

water solubility)



### Table A-31. Estuarine/Marine Invertebrate Acute Toxicity - Formulations

## 3 4 Surrogate Species/

Static or Flow-through % ai.

Product 96-hour LC50/EC50

μ g/L (ppb) (measured/nominal) Probit Slope Toxicity Category MRID No.

Author/Year Study

Classification

Eastern Oyster ( Crassostrea virginica ) (Shell deposition) Flow-through test Salinity -11.8 mg/L; T 21 Ε C 79.6

80 WP > 800
no effect (nominal) Slope - none unknown 000247-20 Wright & Beliles

1966 Supplemental (EC 50 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; 15 Ε C ??

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; 15 Ε C ??

WP > 100,000 (nominal) no slope practically
non-toxic 452277-28

Portmann 1972 Supplemental (only 48 hours;

LC 50 exceeds water solubility & no raw data)

Fiddler Crab ( Uca pugilator )
Static test Salinity - 30 g/L; T 19 Ε C 79.6

80 WP 198,000 (nominal) Slope - none unknown 000243-95
Union Carbide Corp. 1975 Supplemental (LC 50 exceeds water solubility)

Fiddler Crab ( Uca pugilator )

Static test Salinity - 30 g/L; T 19 Ε C Unknown

4-1-3-1

WDL 239,000 (nominal) Slope - none unknown 000243-95
Union Carbide Corp. 1975 Supplemental (LC 50 exceeds water solubility)



# Degradates : Estuarine invertebrate acu te 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 invertebra te toxicity data for hydroxyatrazine.

Table A-32. Estuarine/Marine Invertebrate Acute Toxicity (Hydroxyatrazine)

Surrogate Species/ Flow-through or Static % ai
formul. 96-hour LEC 50 (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 o 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, hydroxyatr azine is not toxic to mysids on an acute basis at the limit of its water solubility.



# A.3.5 Estuarine and Marine Invertebrate, Chronic

## 3 5 

# An estuarine/marine invertebrate life-cycle toxicity test using th e TGAI is required for atrazine because the end-use product may be applied direc tly to the estuarine/marine environment or is expected to be transported to this environmen t from the intended use site, and the following conditions are met: the pesticide is intended for use su ch that its presence in water is likely to be continuous and recurrent; an aquatic acute EC 50 

# is less than 1 mg/L; and the pesticide is persistent in water ( e.g. , half-life greater than 4 days). Resu lts of this test are summarized below
in Table A-33.

Table A-33. Estuarine/Marine Invertebrate Life-Cycle Toxicity

Species/

Duration/ Flow-through/ Static-renewal % ai NOAEC/LOAEC

μ g/L (ppb) (measured/noml) Statistically sign. (P=0.05) Endpoints Affected MRID No. /

ECOTOX no.

Author/Year Study

Classification

Copepod (Eurytemora affinis) / ca. 21 days / static renewal (every 3 days) Tech 

### A 10-day

NOAEC (survival)= 25 LOAEC = 49 ; 30-day LOAEC =

25 Delayed metamorphosis E 19281 / Forget

et al. 2005 Qualitative (no raw data,
limited data

on test conc.



### Copepod Amphiascus

tenuiremis 98 

### NOAEC < 2.5

LOAEC = 2.5 Decrease in viable offpring production 

### E73333 / Bejarano and
Chandler / 2003 QUAL. Negative
control was not used; therefore, potential
solvent

effects could
not be
evaluated

Mysid (Americamysis bahia)
Duration of test – unknown Flow-though test Salinity 20 g/L; T 25+ 1 o C 97.4 NOAEC 80

LOAEC 190 (measured) 37 % red. In adult survival 452029-20
Ward &

Ballantine 1985 Supplemental (no raw data

for statistical analyses)

Mysid (Americamysis bahia) Study Duration = 28 days Flow-though test

Salinity 19-21 g/L; T 26+ 2 o C 97.1 NOAEC 260

LOAEC 500

(measured) 9.8% red. In male length

11% red. In male dry weight

8.5% red. In female dry weight 466482-02, 469526-01, and

469526-02

Cafarella, 2005c Acceptable

Mysid shrimp (Neomysis integer) <24 hrs old / ca. 3 weeks / design not specified / 98-99 NOAEC 1

LOAEC >1

(measured) none E103334 / Noppe

et al./ 2007 Supplemental (no raw data, missing test design info)

## 3 6 

# A.3.7a Estuarine and Marine Field Studies (2003 IRED Data)

A summary of all the estuarine/marine aquatic microcosm and mesocosm fi eld studies that were summarized as part of the 2003 IRED is incl uded in Tables A-35 and A-36, respectively. Similar to the freshwater field studies, the estuarine/marine microcosm and mesocosm field studies were classified as supplemental in the 2003 IRED because this information is used to provide context to the effects data seen in indi vidual organism toxicitytests. Data from nonguideline microcosm/mesocosm tests are typically not used quantitatively to derive RQs in the Agency’s ecological risk assessments, but rather to provide qualitatative information regarding potential aquatic community-level effects of atrazine.

Table A-35. Marine/Est uarine Microcosm Tests

Application rate (lb ai/A)

Nominal/Measured Conc. Concentration affecting endpoint (time to effect) o Affected species and life stage Narrative of Study Trends MRID No.

Author/Year

Estuarine microcosm: Wild celery

Vallisneria Americana

1 treatment Nominal concentrations of 4, 8,16, 32, and 64 ppb NOAEC < 4 ppb

4 ppb (reproductive season) o sign. 16% reduction in
tuber formation
o 55% reduction in
biomass 8 ppb (reproductive season)
o 21% reduction in tuber

formation 16 ppb (mid season and reproductive season)
o 60% reduction in tuber
formation
o 27% reduction in tuber weight o sign. reduction in leaf growth, biomass, and female

flowers 64 ppb (reproductive season)
o 75% reduction in tubers

o reduction in female
flowers Laboratory microcosms were used to grow Vallisneria

americana through entire seasons (divided into th ree periods: early- , mid-, and reproductive). The aquaria were dosed one time at
nominal concentrations after a 14-day acclimation period. With respect to leaf growth, atrazine caused the plants to be shorter and more fragile. With respect to flowering and rhizome production, effects were generally first noted at the 16 to 32 ppb range. Tuber formation appeared to be the most sensitive endpoint, with production in terms of numbers significantly reduced at the 4 ppb level. 450200-01 Cohn, 1985

Supplemental

## 3 7 Table A-35. Marine/Est uarine Microcosm Tests

Application rate (lb ai/A)

Nominal/Measured Conc. Concentration affecting endpoint (time to effect) o Affected species and life stage Narrative of Study Trends MRID No.

Author/Year

Estuarine lab microcosm: 7-day exposure Nominal concentrations of 22, 220, and 2200 ppb

Estuarine field microcosm 108-days duration Single exposure Nominal applications of 0.4, 1.4, 4.5, and 45 lb ai/A “NOAEC” = 10 ppb (based on author’s use of a 10-fold safety

factor from the I 1 level = 100 ppb)

200 ppb (1 week) o significant (0.05 level)
reduction in cell # of Thalassiosira fluviatilis o significant reduction in photosynthesis of T. fluviatilis
and Nitzschia sigma

2200 ppb (1 week) o significant reduction in cell #, photosysnthesis, and chlorophyll content for both algae

1.4 lb ai/A (effect up to 5 days) o significant reduction in surface chlorophyll and primary production (85-89%) 1.4 lb ai/A (effect up to 8 and 17 days) o significant reduction in carbon fixation (52-73%) 0.4/4.5 lb ai/A (effect at 16 days, but not 26 days) o significant reduction in
carbon fixation 45 lb ai/A (42 days) o significant reduction in
carbon fixation

No statistical effects found in
atrazine treatments on: o 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”
o indirect effects on function

or taxonomic composition of benthic community structure Laboratory studies were
conducted with the salt marsh edaphic diatoms Thalassiosira fluviatilis and Nitzschia sigma

. The I 50 for both species combined was reported to be 939 ppb. The

I 1 was reported to be 100 ppb, and by applying a 10-fold safety factor, the acceptable level (NOAEC) was reported to be 10 ppb. Subsequently, studies were conducted in greenhouse microcosms (1.4 lb ai/A) and in two field studies (1.4 lb ai/A or 0.4, 4.5, and 45 lb ai/A) on the
beach wherein enclosures were sunk into the sand and exposed to

a tidal action. Atrazine treatment also appeared to cause a shift to a

Navicula sp. Dominated system. Field results with higher rates of atrazine were expected, with carbon fixation reduced for up to 16 days at the 2 lower rates and up to 42 days at the highest rate. 450874-06

Plumley and Davis, 1980

Supplemental

## 3 8 Table A-35. Marine/Est uarine Microcosm Tests

Application rate (lb ai/A)

Nominal/Measured Conc. Concentration affecting endpoint (time to effect) o Affected species and life stage Narrative of Study Trends MRID No.

Author/Year

Estuarine microcosm:
5 weeks 3 weekly applications followed by 2 weeks observation.
Mean-measured concentration at approx. mid-point of Spartina test were 30, 280, and 3100 ppb
and in the Juncus test were 30, 250, and 3800 ppb 30, 280, and 3100 ppb (5 weeks): o sign. (0.05 level) increase in peroxidase activity in Spartina alterniflora

30, 250 and 3100 ppb (5 weeks) o sign. (0.05 level) reduction in chlorophyll a (Chl-a)

and Chl- a/Chl-b ration in Juncus

roemerianus

250 and 3800 ppb (5 weeks) o sign. red. in Chl-b in J.

roemerianus

3100 ppb (1 week) o sign. red. in growth of S. alterniflora

3800 ppb (1 week) o sign. red. in growth of J.

roemerianus o sign. increase in oxidized lipids in J. roemerianus

250 ppb (1 year) o partial recovery in J.

roemerianus

3800 ppb (1 year) o practically no survival of

J. roemerianus Two aquatic estuarine plants were exposed to atrazine in greenhouse microcosms. The plants were exposed to atrazine by placing treated sand on the surface of the pots three times (once a week for the first 3 weeks of the study) followed by 2 more

weeks for a total of 5 weeks. The water samples were taken after the third application. The pots were also tidally-exposed (i.e., low tide during the day and high tide at night). S. alternifloraplants demonstrated a doseresponse increase in peroxidase activity. In contrast, J.

roemerianus plants demonstrated a dose-responsive reduction in chlorophyll and increase in the amount of oxidized lipids. The
authors state that atrazine “should pose no significant adverse

effects on S. alternaiflora . In contrast, if chronic levels of atrazine persist in the range of 250 ppb or greater, J.

roemerianus most likely will exhibit die off or decline that may
lead to loss of th is species within

the habitat. 450874-05 Lytle and Lytle, 1998

Supplemental

Estuarine microcosm: Duration not reported Nominal concentrations of 0, 50, and 100 ppb Both Nannochloris oculata and Phaeodactylum tricornutum were significantly (mostly at the 0.01 level) affected by changes in light, temperature, and atrazine

concentration A 3x3x3 factorial design
examined the effect of temperature, light, and atrazine concentration on two species of estuarine algae. N oculata was significantly affected by all variables, and the three two-way and one three-way interactions wer also significant. P.

tricornutum was affected by the main variables and the only significant interaction was light by atrazine Mayasich et al., 1986

Supplemental

Estuarine microcosm Duration not reported Nominal concentrations of 0, 15, 30, and 50 ppb The above mentioned algae were tested together and this variable also caused a significant (0.01 level) effect on N. oculata growth

rate. An extension of the above described study. In addition to separate culture, the two estuarine algae were cultured together. The
end result was that P. tricornutum
dominated the cultures due to the
stress of atrazine N. oculata under optimum growth conditions. Mayasich et al., 1987

Supplemental

## 3 9 Table A-35. Marine/Est uarine Microcosm Tests

Application rate (lb ai/A)

Nominal/Measured Conc. Concentration affecting endpoint (time to effect) o Affected species and life stage Narrative of Study Trends MRID No.

Author/Year

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): o no photosynthesis

130 ppb (Weeks 2-4) o 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

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): o sign. (p < 0.05) red. average ratio of # or ramets (branches): initial # or ramets

30,000 ppb (Day 22 or 23): o sign. (p < 0.05) 46-58% reduction in total above-ground
biomass o 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 -2 s -1 ), 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

## 4 0 Table A-36. Marine/Estuarine Mesocosm Tests

Application rate (lb ai/A)
Nominal/Measured

Conc. 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) o sign. lower pH levels (Days 5-14);

indicative of reduced photosynthesis o higher dissolved organic nitrogen (DON) (Days 6-11) o up to 50% red. primary production (Days 3-11) o up to 60% red. particulate carbohydrates (Days 5-15) o up to 70% red. chlorophyll (Days 4- 15) 0.56 ppb o sign. lower pH levels (Days 5-13) o incr. total dis. organic phosphate (DOP) (Days 3-14) o higher DON (Days 5-15) o up to 50 % red. primary production (Day 3-13) o up to 85% red. particulate carbohydrate (Days 5-15) o up to 80% red. chlorophyll (Days 4- 15) 5.8 ppb o sign. lower pH levels (Days 5-11) o up to 200% increase in total DOP (Days 3-14) o up to 200 % increase in total DON (Days 2-15) o up to 50% red. in primary productivity (Days 3-7) o up to 60% red. in partic. carbohydrates (Days 5-15) o up to 30% red. in chlorophyll conc. (Days 4-15) Mesocosms (2 m 2 ) 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 dissolve d organic nitrogen and phosphorus. “Excretions” may be caused by atrazine stress on cells or lysis of cells. 450200-21

Bester et al ., 1995

Supplemental

Nominal applications of 0.4, 4.5, or 45 lb ai/A Salt marsh edaphic alage Elaboration of Plumley et al ., 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

Supplemental



# A.3.7b Estuarine and Marine Field St udies (Ne w Open Literature Data)

As previously discussed, the 2003 IRED identified 10-20 μ 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 in cluding their populations and communities. As such, estuarine/marine field data from the ope n literature were considered only when the relevant endpoints were less than or more sensitive than the 10 μ g/L aquatic-community effect level. In addition, data for taxa that are directly relavent to the endangered species

## 4 1 

# evaluated as part of this assessment were al so 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 μ 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 pote ntial food item and source of habitat for sea turtles (M acinnis-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 aff ect the chlorophyll a concentration of estuarine/marine sea grasses at exposure concentrations ranging from 10 to 100 ppb. Data from this study are not used to quantitatively calculate RQs for estu arine/marine macrophytes because a more sensitive endpoint of 4 ppb was reported for wild celery (Cohn, 1985).

Table A-37. Estuarine/Marine Field Study 

## from Open Literature (2007 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

N R) Seagrass ( Zostera capricorni ) - open-bottom cylindrical containers enclosed grasses within a seagrass meadow; salinity = 35 ppt; temp = 25

+ 1 o 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

N o 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

p eriod. 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.

(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.

## 4 2 

# A.4 Toxicity to Plants

A.4.1 Terrestrial Plants

Terrestrial plant testing (seedling emergen ce and vegetative vigor) is required for
herbicides that have terrestri al non-residential outdoor use patterns a nd that may move off the application site through volatili zation (vapor pressure >1.0 x 10 -5 

# mm Hg at 25 o 

# C) or drift (aerial or irrigation) a nd/or that may have endangered or threatened plant species associated with the application site.

For seedling emergence and vegetative vi gor 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 r oot 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 an y pesticide showing a negative response equal to or grea ter than 25% in Tier I tests. Tier II tests measure the response of plants, relative to a control, and five or more test concentr ations at a test level that is equal to the highest us e rate (expressed as lbs ai/A). Results of Tier II seedling emergence and vegetative vigor t oxicity 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 excep tion of corn in the seedling emergence and vegetative vigor tests and ryegrass in the ve getative vigor test, exhi bited adverse effects following exposure to atrazine.

Table A-38. Nontarget Terrestrial Plant Seedling Emergence Toxicity (Tier II)

Surrogate Species % ai EC 25 / NOAEC (lbs ai/A) Probit Slope Endpoint Affected MRID No.

Author/Year Study

Classification

Monocot - Corn ( Zea mays ) 97.7 > 4.0 / > 4.0 No effect 420414-03
Chetram 1989 Acceptable

Monocot - Oat ( Avena sativa ) 97.7 0.004 / 0.0025 red. in dry weight 420414-03
Chetram 1989 Acceptable

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 / 0.0025 red. in dry weight 420414-03
Chetram 1989 Acceptable

Dicot - Soybean ( Glycine max ) 97.7 0.19 / 0.025 red. in dry weight 420414-03
Chetram 1989 Acceptable

Dicot - Lettuce ( Lactuca sativa ) 97.7 0.005 / 0.005 red. in dry weight 420414-03
Chetram 1989 Acceptable

Dicot - Cabbage 97.7 0.014 / 0.01 red. in dry weight 420414-03 Acceptable

## 4 3 ( Brassica oleracea alba ) Chetram 1989

Dicot - Tomato ( Lycopersicon esculentum ) 97.7 0.034 / 0.01 red. in dry weight 420414-03
Chetram 1989 Acceptable

Dicot - Cucumber ( Cucumis sativus ) 97.7 0.013 / 0.005 red. in dry weight 420414-03
Chetram 1989 Acceptable



### Table A-39. Nontarget Terrestrial Plant Vegetative Vigor Toxicity (Tier II)

Surrogate Species % ai EC25 / NOAEC (lbs ai/A) Endpoint Affected MRID No.

Author/Year Study Classification

Monocot - Corn ( Zea mays ) 97.7 > 4.0 / > 4.0 No effect 420414-02
Chetram 1989 Acceptable

Monocot - Oat ( Avena sativa ) 97.7 2.4 / 2.0 red. in dry weight 420414-02
Chetram 1989 Acceptable

Monocot - Onion ( Allium cepa ) 97.7 0.61 / 0.5 red. in dry weight 420414-02
Chetram 1989 Acceptable

Monocot - Ryegrass ( Lolium perenne ) 97.7 > 4.0 / > 4.0 No effect 420414-02
Chetram 1989 Acceptable

Dicot - Root Crop - Carrot ( Daucus carota ) 97.7 1.7 / 2.0 red. in plant height 420414-02
Chetram 1989 Acceptable

Dicot - Soybean ( Glycine max ) 97.7 0.026 / 0.02 red. in dry weight 420414-02
Chetram 1989 Acceptable

Dicot - Lettuce ( Lactuca sativa ) 97.7 0.33 / 0.25 red. in dry weight 420414-02
Chetram 1989 Acceptable

Dicot - Cabbage ( Brassica oleracea alba ) 97.7 0.014 / 0.005 red. in dry weight 420414-02
Chetram 1989 Acceptable

Dicot - Tomato ( Lycopersicon esculentum ) 97.7 0.72 / 0.5 red. in plant height 420414-02
Chetram 1989 Acceptable

Dicot - Cucumber ( Cucumis sativus ) 97.7 0.008 / 0.005 red. in dry weight 420414-02
Chetram 1989 Acceptable



# A summary of safety studies evaluating phytoxicity of atrazi ne to woody plants (target species) was submitted to the Agency in 2006 (Wall, 2006). A total of 35 species were tested in 13 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% General b

Black pine 2 Chlorosis (IS-1) a

Boxwood 2 3.8% General

## 4 4 Table A-39b. Summary of woody plant safety study (Hall, 2006).

Species Application Rate (lbs a.i./Acre) Phytotoxicity (%)
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 c

Pinus strobus 2 5% General

Pinus virginiana 1.5 90% General c

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 b

Spruce 2 0

Tilia 2 0

a IS rating grades chlorosis severity (norma l 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 c leaned. 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 ou tdoor 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).

## 4 5 

# Aquatic Tier II studies are required for al l 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. Aqua tic plant testing is required for atrazine because it is applied on crops outdoor s and appears to be mobile with a water solubility value of 33 ppm.

Results of Tier II toxicity testing on tec hnical grade and typical end-use products (TEP) are tabulated below. The data are presente d in four toxicity tables separating the
freshwater data from the marine data and th e short, 7-day or less tests from the longer
tests. Tables A-40 and A-41 summarize freshwat er plant toxicity for short-term (i.e., < 7 days exposure) and longer-term tests. Ta bles A-42 and A-43 summarize short-term (< 10 days exposure) estuarine/marine plant toxic ity for technical grade and formulations of atrazine, respectively. Toxicity data fo r longer-term exposure of atrazine to estuarine/marine plants ar e provided in Table A-44.

Field studies involving at razine toxicity to freshwater a nd estuarine/marine aquatic plants are summarized as part of Sec tions 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 % ai Conc. (ppb) Probit Slope % Response MRID No.

Author/Year Study Classification

Vascular Plants:

Duckweed ( Lemna gibba ) 5-Day test; Static-Renewal 97 170 (nominal) Slope 3.93 50% red. in growth 410652-03d Hughes 1986 Supplemental (5 days, not 14 days)

Duckweed ( Lemna gibba ) 7-Day test; Static-Renewal 97 170 (measured) Slope 2.2 50% red. in growth 420414-04 Hoberg 1991 Supplemental (7 days, not 14 days)

Non-Vascular Plants:

Cyanophyceae

Oscillatoria lutea (1week; 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 Torres and O’Flaherty

1976 Supplemental (raw data unavailable)

## 4 6 production

Ciliate Tetrahymena pyriformis (24 hrs) NR 5.83 50% red. In survival Toth &
Tomasovicova

1979 Supplemental

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)

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, 450200-15

Larsen et al

. 1986 Supplemental (raw data unavailable)

## 4 7 respect.

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)

Euglenoid Euglena gracilis (7 days) NR 496 50% inhibition of photosynthesis ThuillierBruston et al.

1996 Supplemental



### Table A-41. Longer Term, Nontarget Freshwater Plant Toxicity

Surrogate Species/

Duration/

Measured/nominal % ai Conc. (ppb) Probit Slope % Response MRID No.

Author/Year Study Classification

Vascular Plants:

Broad Waterweed

Elodea Canadensis (14 days; measured) 96 0.001 50% reduction in dry
root biomass McGregor et al.

2008 Supplemental

Broad Waterweed

Elodea canadensis (20 days; measured) Unk. NOAEC 2

LOAEC 10 200% incr. dark respiration
33% incr. net photosynthesis 452277-14
Hofmann and
Winkler 1990 Supplemental (raw data unavailable)

Pondweed Potamogeton perfoliatus (4 weeks; initial conc. nominal, terminal conc. measured) Unk 30
Week 3: LOAEC 5

NOAEC < 5
4 Weeks: LOAEC 50

NOAEC 5 50% red. O 2 product. sign. red. O 2 product.

sign. red. O 2 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) Unk. 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) Unk. 91

NOAEC 5

LOAEC 50 50% red, O 2 product.

Sign. red. O 2 product. Kemp et al

. 1985 Supplemental (raw data unavailable)

## 4 8 Non-Vascular Plants:

36 freshwater algal strains (2 weeks; nominal) 99.0 10

1,000 growth <than control

strong growth red. Butler et al

. 1975 Supplemental (raw data unavailable)

Chlorophyceae Chlorella vulgaris (11 days; nominal) 99.9 25 50% red. cell growth 452277-03
Burrell et al.

1985 Supplemental (raw data unavailable)

Cyanophyceae Anabaena inaequalis (12-14 days 1 ; nominal) >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 days 1 ; 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 days 1 ; nominal) > 95 300 1,000

500 50% red. cell count 50% red. growth rate
50% red. photosynthesis Stratton 1984 Supplemental (NOAEC and raw data unavailable)

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 Conc. (ppb) Probit Slope % Response MRID No.

Author/Year Study Classification

Vascular Plants:

Fontinalis sp. (24 hours; measured) NR NOAEC 2

LOAEC 10 red. net O 2 production Supplemental (raw data unavailable)

Pondweed (Estuarine) Potamogeton perfoliatus (2 hours; nominal) NR 77 50% red. O 2 evolution 452277-18
Jones and
Winchell 1984 Supplemental (Insufficient duration; raw data unavailable)

Pondweed Potamogeton perfoliatus (2 hours; nominal) NR 80

650 50% red. O 2 product.

87% red. O 2 product.. 452277-18

Jones et al

. 1986 Supplemental (Insufficient duration; raw data unavailable)

Zannichellia palustris (2 hours; nominal) NR 91 50% red. O 2 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) NR 102 50% red. O 2 evolution 452277-19 Supplemental

## 4 9 Ruppia maritima (2 hours; nominal Jones and
Winchell 1984 (Insufficient duration; raw data unavailable)

Non-Vascular Plants :

Prasinophyte

Nephroselmis pyriformis (4 hours) NR 14.2 μg/L LOAEC = 1.1 μg/L 50% photoinhibition Magnussun et

al. 2010 Supplemental

Blue-green - Cyanophyceae

Oscillatoria lutea (1week; 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)

Xanthophyceae

Tribonema sp. (1 week; nominal) 76

80 W 1

1,000 42% red. chlorophyll production 75% red. chlorophyll production 000235-44
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 prod. 000235-44
Torres and O’Flaherty

1976 Supplemental (raw data unavailable)

Dinoflagelate Amphidinium operculatum (96 hrs) NR 17.9

NOAEC <12.5 50% red. In total

biovolume DeLorenzo et
al. 2004 Supplemental

Marine Diatom Bellerochea polymorpha (48 hr) NR 19.4 50% reduction in population growth Walsh et al.

1988 Supplemental

Chrysophyceae Isochrysis galbana (120 hours; nominal) 97.4 22

NOAEC < 13

LOAEC 13 Slope 3.065 50% red. growth

30% red. growth 410652-04
Parrish 1978 Supplemental (NOAEC, method & raw data unavailable)

Cryptomonad Storeatula major
SW 96 hrs. NR

22.17

NOAEC < 12.5 50% reduction in
abundance DeLorenzo et
al. 2004 Supplemental

Marine Diatom

Skeletonema costatum (120 hours; nominal) 97.4 24

NOAEC < 13

LOAEC 13 Slope 3.343 50% red. growth

14% red. growth 410652-04
Parrish 1978 Supplemental (NOAEC, method & raw data unavailable)

Coccolithophorad Isochrysis galbana (SW 72 hrs) NR 30 μg/L 50% growth inhibition Debelius et al.

2008 Supplemental

Marine Diatom
Skeletonema costatum (120 hours; measured) 97.1 53

NOAEC 14 Slope 2.798 50% red. cell growth 430748-01 Hoberg 1993 Acceptable

Marine Green - Chlorophyceae Chlamydomonas sp. (72 hours; nominal); Salinity 30 g/L 99.7 60 50% red. O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Golden Algae Monochrysis lutheri (1 hr) NR 77 50% red. O 2 production Hollister and

Walsh 1973 Supplemental (Short duration)

Marine Yellow - Chrysophyceae 99.7 77 50% red. O 2 production 402284-01 Supplemental

## 5 0 Monochrysis lutheri ( 72 hours; nominal); Salinity 30 g/L Mayer 1986 (72 hrs & endpoint)

Marine Red - Rhodophyceae Porphyridium cruentum (72 hours; nominal); Salinity 30 g/L 99.7 79 50% red. in O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Green - Chlorophyceae

Neochloris sp. (72 hours; nominal); Salinity 30 g/L 99.7 82 50% red. in O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Bacillariophyceae Cyclotella nana (72 hours; nominal); Salinity 30 g/L 99.7 84 50% red. in O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Bacillariophyceae Achnanthes brevipes (72 hours; nominal); Salinity 30 g/L 99.7 93 50% red. in O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Yellow - Chrysophyceae Isochrysis galbana (240 hours; nominal); Salinity 30 g/L 99.7 100 50% red. cell growth 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

Marine Green - Chlorophyceae

Chlorococcum sp. (240 hours; nominal); Salinity 30 g/L 99.7 100 50% red. cell growth 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

Marine Green - Chlorophyceae Platymonas sp. (72 hours; nominal); Salinity 30 g/L 99.7 100 50% red. O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Bacillariophyceae Thalassiosira fluviatilis (72 hours; nominal); Salinity 30 g/L 99.7 110 50% red. O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Bacillariophyceae Stauroneis amphoroides (72 hours; nominal); Salinity 30 g/L 99.7 110 50% red. O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Algae Microcystis aeruginosa (120 hours - nominal) 97.4 129

NOAEC 65 Slope 3.162 50% red. growth 7% red. growth 410652-04
Parrish 1978 Supplemental (NOAEC, method & raw data unavailable)

Marine Green - Chlorophyceae

Chlorella sp. (72 hours; nominal); Salinity 30 g/L 99.7 140 50% red. O 2 production 402284-01 Mayer 1986 Supplemental (NOAEC unavailable

Blue-green - 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)

Marine green - Chlorophyceae

Dunaliella tertiolecta (120 hours; nominal) 97 180

NOAEC < 100

LOAEC 100 Slope 1.95 50% red. growth

34% red. growth 410652-03 Hughes 1986 Supplemental (NOAEC unavailable)

Marine Yellow-Green NR 185 50% total fluorescence
inhibition Debelius et al.

2008 Supplemental

## 5 1 Nannochloropsis gaditana

(72 hrs)

Marine Yellow - Chrysophyceae Phaeodactylum tricornutum (240 hours; nominal); Salinity 30 g/L 99.7 200 50% red. cell growth 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

Marine Bacillariophyceae
Nitzschia closterium (72 hours; nominal); Salinity 30 g/L 99.7 290 50% red. O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Bacillariophyceae Amphora exigua (72 hours; nominal); Salinity 30 g/L 99.7 300 50% red. O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

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)

Marine Red - Rhodophyceae Porphyridium cruentum (120 hours) 97.4 308

NOAEC <130

LOAEC 130 Slope 2.449 50% red. growth

16% red. growth 410652-04
Parrish 1978 Supplemental (NOAEC, method & raw data unavailable)

Marine Bacillariophyceae
Nitzschia (Ind. 684) (72 hours; nominal); Salinity 30 g/L 99.7 430 50% red. O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)

Marine Green -Chlorophyceae Kirchneria subcapitata (120 hours; nominal) 97.4 431

NOAEC 200 Slope 4.217 5% red. in growth 4% red. in growth 410652-04
Parrish 1978 Supplemental (NOAEC, method & raw data unavailable)

Marine Bacillariophyceae

Navicula inserta (72 hours; nominal); Salinity 30 g/L 99.7 460 50% red. in O 2 production 402284-01 Mayer 1986 Supplemental (72 hrs & endpoint)



### Table A-43. Formulation Nontarget Marine/Estuarine Algal Toxicity (Tier II)

Species/

Duration/Measured/nominal % ai Conc. (ppb) Probit slope % Response MRID No.

Author/Year Study Classification

Mar. Yellow - Chrysophyceae Isochrysis galbana (nominal); Salinity 30 g/L 76

80 WP 100 (240 hrs)

200 (2 hrs) 50% red. cell growth

50% red. O 2 production 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

Mar. Yellow Chlorophyceae

Chlorococcum sp. (nominal); Salinity 30 g/L 76

80 WP 100 (240 hrs)

400 (2 hrs) 50% red. cell growth

50% red. O 2 production 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

Mar. Yellow - Chrysophyceae Phaeodactylum tricornutum (nominal); Salinity 30 g/L 76

80 WP 200 (240 hrs)

200 (2 hrs) 50% red. cell growth

50% red. O 2 production 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

Mar. Green - Chlorophyceae

Dunaliella tertiolecta (nominal); Salinity 30 g/L 76

80 WP 400 (240 hrs)

600 (2 hrs) 50% red. cell growth

50% red. O 2 production 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

## 5 2 Table A-44. Longer-term (> 10 days exposure) Nontarget Marine/Estuarine Plant Toxicity

Surrogate Species/

Duration/Measured/nominal % ai Conc. (ppb) Probit Slope % Response MRID No.

Author/Year Study Classification

Vascular Plants:

Sago Pondweed (Estuarine) Potamogeton pectinatus (28 days; measured/nominal) NR Salinity 12 ppt:

NOAEC 7.5

LOAEC 14.3 Salinity 1 & 6 ppt:

NOAEC 14.3

LOAEC 30 sign. red. dry weight

sign. red. dry weight 450882--31 Chesapeake Bay Program 1998 Supplemental (raw data unavailable)

Estuarine rush

Juncus roemerianus (5 weeks - 1 year; measured 97.1 LOAEC 30

NOAEC 30

NOAEC < 30

250 ppb 3, 800 ppb sign. red. chlorophyll a in
5 weeks

(1 year) partial recovery (1 yr) practically no survival 450874-05 Lytle & Lytle

1998 Supplemental (raw data unavailable)

Pondweed Potamogeton perfoliatus (4 weeks; initial conc. nominal, terminal conc. measured) NR 30
Week 3: LOAEC

5

NOAEC < 5
4 weeks: LOAEC

50

NOAEC 5 50% red. O 2 product. sign. red. O 2 product.

sign. red. O 2 product. 452277-20 Kemp et al

. 1985 Supplemental (raw data unavailable)

Pondweed (Estuarine) Potamogeton perfoliatus (3 weeks; nominal) NR 53 50% red. ???? 450874-10 Forney and
Davis 1981 Supplemental (raw data unavailable)

Eelgrass (Estuarine)

Zostera marina (10 days; measured) NR est. 69

50

80 50% red. leaf growth 25% red. leaf growth 62% red. leaf growth 452277-29
Schwarzschild

et al . 1994 Supplemental (raw data unavailable)

Estuarine Eelgrass

Zostera marina (21 days; nominal) NR 100

NOAEC 10 21-day LC50 red. production 452277-05 Delistraty and

Hershner 1984 Supplemental (raw data unavailable)

Wild Celery (Estuarine)

Vallisneria americana (6 weeks; nominal) NR 163 50% red. shoot length no difference at 0, 3, or 6 parts/thousand 450874-10 Forney and

Davis 1981 Supplemental (raw data unavailable)

Seagrass (Estuarine) Halodule wrightii (22 - 23 days; measured) Atrazi

ne 4L 30,000 46-58% red. total aboveground biomass 452051-01
Mitchell 1987 Supplemental (raw data unavailable)

Non-Vascular Plants:

Marine Brown macroalgae Laminaria hyperborea (18 days; nominal) NR NOAEC < 1

LOAEC = 10 sign. red. growth rate; delayed sporophyte

formation Hopkin &Kain

1978 Supplemental (raw data unavailable)

Marine Yellow - Chrysophyceae Isochrysis galbana (240 hours; nominal); Salinity 30 g/L 99.7 100 50% red. cell growth 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

Marine Green - Chlorophyceae

Chlorococcum sp. (240 hours; nominal); Salinity 30 g/L 99.7 100 50% red. cell growth 402284-01 Mayer 1986 Supplemental (NOAEC unavailable)

Marine Yellow - 99.7 200 50% red. cell growth 402284-01 Supplemental

## 5 3 Chrysophyceae Phaeodactylum tricornutum (240 hours; nominal); Salinity 30 g/L Mayer 1986 (NOAEC unavailable)

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)



# Degradates : Special tests are required for alga l 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 Conc. (ppb) Probit slope % Response MRID No.

Author/Year Study Classification

Fresh. Blue-Green - Cyanophyceae Anabaena inaequalis (12-14 days 1 ; nominal) > 95 1,000 4,000 2,500 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 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 days 1 ; 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 Deisopropylatrazine Nontarget Aquatic Plant Toxicity (Tier II)

Species/

Duration/Measured/nominal % ai Conc. (ppb) Probit slope % Response MRID No.

Author/Year Study Classification

Fresh. Blue-Green - Cyanophyceae Anabaena inaequalis (12-14 days 1 ; 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 days 1 ; 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)

## 5 4 Table A-46. Degradate Deisopropylatrazine Nontarget Aquatic Plant Toxicity (Tier II)

Species/

Duration/Measured/nominal % ai Conc. (ppb) Probit slope % Response MRID No.

Author/Year Study Classification

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)

Freshwater Green Algae ( Scenedesmus subspicatus

(72 hours; nominal) NR 1,300 (slope

= 2.18

+ 0.377) 50% red. cell density 470461-05

Vial, 1991e Supplemental (study
duration not sufficient
to be classified as Tier II study)

230 5% red. cell density

370 NOAEC for red. cell density

1,500 (slope

= 2.36+ 0.382) 50% red. biomass

290 5% red. biomass

370 NOAEC for red. biomass



### Table A-47. Degradate Diamino-Atrazine Nontarget Aquatic Plant Toxicity (Tier II)

Species/

Duration/Measured/nominal % ai Conc. (ppb) Probit slope % Response MRID No.

Author/Year Study Classification

Fresh. Blue-Green - Cyanophyceae Anabaena inaequalis (12-14 days 1 ; 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 days 1 ; 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)



### Table A-48. Degradate Hydroxyatrazine Nontarget Aquatic Plant Toxicity (Tier II)

Species/

Duration/Measured/nominal % ai Conc. (ppb) Probit slope % Response MRID No.

Author/Year Study Classification

Fresh. Blue-Green - Cyanophyceae Anabaena inaequalis (12-14 days 1 ; 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)

## 5 5 Table A-48. Degradate Hydroxyatrazine Nontarget Aquatic Plant Toxicity (Tier II)

Species/

Duration/Measured/nominal % ai Conc. (ppb) Probit slope % Response MRID No.

Author/Year Study Classification

Freshwater Green - Chlorophyceae Chlorella pyrenoidosa (12-14 days 1 ; 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)



# A.5 Toxicity to Terrestrial Invertebrates

The available open liter ature data for the toxicity of atrazine to terrestrial invertebrates is
summarized in Table A-49.

Atrazine is practically no n-toxic to honey bees (LD 50 

# : 97 μg/bee). It also did not cause
adverse effects in fruit flie s exposed to 15 μg/fly. LC 50 

# values in earthworms ranged from 273 to 926 ppm soil (Mosleh et al., 2003; Haque and Ebing, 1983) . Atrazine did not produce statistically signifi cant (p>0.05) adverse effects in studies on several beetle species at any level tested, which ranged fr om application rates of approximately 1 lb
a.i./Acre to 8 lbs a.i./Acr e (Kegel, 1989; Brust, 1990; Samsoe-Petersen, 1995).

The most sensitive terrestrial invertebra te species tested was the springtail ( Onychiurus apuanicus and O. armatus ). Exposure to O. apuanicus at 2.5 ppm resulted in 18% mortality, and exposure to O. armatus at 20 ppm resulted in 51% mortality (Mola et al., 1987); lower levels were not tested. These so il concentrations are associated with an application rate of approximately 1 lb a.i./A cre and 7 lbs a.i./Acre, respectively, assuming a soil density of 1.3 grams/cm 3 

# and a soil depth of 3 cm. Additional details on these studies may be found in Appendix A.

Several field studies reported reduced abundance in terres trial invertebrates (Fox, 1964; Fretello et al., 1985). However, in these st udies, reduced abundance could have been caused either by direct effects or by indirect effects caused by reduced vegetation of the
herbicide. Available studies are summarized in Table A-49.

Table A-49. Toxixcity of Atrazine to Terrestrial Invertebrates Species Dose Comment Citation Honey bees LD50: >97 g/bee 5% mortality occurred at the highest dose tested (97 ug/bee) MRID 00036935

Ground Beetles (Poecilus)
P. versicolor NOAEC: 8 lbs/Acre Spiked soil st udy; no effects occurred at any
level tested. Kegel, 1989

Ecotox No. 64007

P. cupreus NOAEC: 0.8 lbs/Acre Spiked soil study; no effects occurred at any

## 5 6 level tested. P. lepidus NOAEC: 0.8 lbs/Acre Spiked soil study; a 25% reduction in survival was observed at the highest level tested that was not significant at the p<0.05 level. 5 species of
carabid beetles NOAEC: 2 lbs/Acre Beetles were dipped in atrazine solution then placed in treated soil (2 .24 kg a.i./Acre); a transient repellency effect occurred for 6 days

after treatment. Brust, 1990

Ecotox No. 70406

Rove beetle NOAEC: The single
level tested was intended to approximate practical application

rates. Exposure occurred via sprayed sand; “no measurable effect” occurred at any concentration tested; dose level was reported to approximate a practical field application rate. Samsoe-Petersen,

1995

Ecotox No. 63490

Onychiuridae Onychiurus apuanicus NOAEC: <2.5 ppm soil (<approx. 1 lbs/Acre)a Exposure occurred by treated soil; 18% mortality occurred at 2. 5 ppm compared to 0%
in controls. Mola et al., 1987.

Ecotox No. 71417

O. armatus NOAEC: <20 ppm soil (<approx. 7 lbs/Acre)a Exposure occurred by treated soil; 51% mortality occurred at 20 ppm compared to 0% in
controls. Micro arthropods NOAEC: 0.9 lbs/Acre Field application of 1 kg/ha; atrazine was not
associated with adverse effects. Cortet et al., 2002

Ecotox No. 75784 Microarthopods NOAEL = 2 kg/ha (1.05 ppm) LOAEC = 6 kg/ha (3.15 ppm) Field study testing several species of microarthopods. It could not be determined if reduced abundance was caused by migration (repellency), by toxic effects, or both. Fratello et. al., 1985

Ecotox No.

59428

Fruit flies Drosphilia NOAEC: 15 ug/fly No increased mortality occurred in groups exposed to atrazine alone relative to controls. Lichtenstein et al.,

1973

Ecotox No. 2939

Earthworm Aporrectodea caliginosa 28-day LC50: 381 ppm Spiked soil study, Slope: 8.37 Mosleh et al., 2003

Ecotox No. 77549

Earthworm
Eisenia fetida 14-Day LC50: 273 mg/kg soil (ppm) – 926 ppm Spiked soil study; endpoints evaluated included mortality and biomass. Haque and Ebing,

1983

Ecotox No.

40493

Earthworm
Eisenia fetida 4-Day LC50: 2.9 ug/cm2 Filter paper study Lydy and Linck,

2003

Ecotox No. 71459 Earthworm LOAEC = 8 lb/acre Field study examining the impacts of several herbicides on soil invertebrate populations. The endpoint measured was abundance of several species. Study authors suggested that reduced abundance was likely caused by repellency and not direct toxicity. Fox, 1964

Ecotox No. 36668 Wire worm Springtail

An estimation of application rates was made from soil concentration by assuming a soil depth of 3 cm and a soil density of 1.3 g/cm3.



# A.6 Effects of Environmental Factors and Life-Stage on Aquati c Atraz ine Toxicity

## 5 7 

# A.6.1 Interaction Effects on Atrazine Toxicity to Plants

Some intra-laboratory studies s uggest that atrazine toxicity is affected by environmental parameters, such as temperature, ligh t intensity and salinity levels. Mayer et al . (1998) concluded that a temper ature difference of 1 o 

# C will cause a difference in algal growth rate in the range of 7 to 9 percen t at the typical rate increase for 10 o 

# C temperature increase (Q 10 

# ) of 2 to 2.3.

In general, the toxicity of pesticides incr ease with increasing temperature. Mayasich, Karlander and Terlizzi, Jr. (1986) tested two algal spec ies in 27 combinations of temperature (15, 20 and 25 o 

# C), light intensity (0.208, 0.780 and 1.352 mW/cm 2 

# ) and atrazine concentrations of 0, 50 and 100 μ 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 determ ined by the 3-way interactions. Atrazine toxicity increased to N. oculata with both increasing temper ature and increasing light intensity, except at 15 o 

# C and 1.352 mW/cm 2 

# where growth was intermediate. Previous results yielded a similar anomaly and suggest that 15 o 

# 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 c oncentrations, and also significant (p < 0.05) for temperature, but only light inte nsity was significantly (p < 0.01) related to an increase in atrazine toxicity. Atrazine toxicity was highest at the lo west light intensity. ”The response of P. tricornutum to atrazine at light in tensities of 0.780 and 1.352 mW/cm 2


# may be a reflection of primary effects only, while at 0.208 mW/cm 2 

# , 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 o 

# C.

Mayasich et al . (1987) repeated the above algal study with lower at razine concentrations (0, 15, 30 and 50 μ g/L and fewer temperatures (15 and 25 o 

# C) and light intensities (0.208
and 1.352 mW/cm 2 

# ) 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 magn itude of the responses to te mperature, 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 betw een temperature and light intensity, and temperature and atrazine we re 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 o 

# C) and light intensity (0.208 mW/cm 2 

# ). At simultaneous high levels of temperature and li ght 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 divers ity and structure of natural phytoplankton populations. Thus, a nutritious algal species for la rval oysters (Dupry, 1973) is replaced by what is considered to be a poor food source for larval bivalves (Walne, 1970).

## 5 8 

# Mayer et al . (1998) tested the effect of four main environmental f actors 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 μ E/m 2 

# ), temperature (16 and 26 o 

# C), nitrogen source (NH 4 

# + and NO 3 

# -) and pH (7.6 and 8.6). Temperatur e influenced growth indirectly by interacting with light intensity. Alga l growth measured as the atrazine EC 50 

# values was marginally reduced under low light intens ity at high and low te mperatures (158 and

159 μ g/L, respectively versus the atrazine control, 164 μ g/L). High light intensity at the low temperature reduced the toxicity of atr azine to the alga by about two fold (LC 50 

# 300

μ g/L) while high light intensity and high temperature reduced the toxicity of the atrazine by about 118 fold (LC 50 

# 191 μ g/L). Nitrogen source and pH had no significant effect on atrazine toxicity affec ting 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 increa se, decrease or have no effect on atrazine toxicity to algal growth. Li ght 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 a ny effect on the toxicity of atrazine to algae.

A.6.2 Interaction Effects on Atrazine Toxicity to Aquatic Animals

A number of intra-laboratory st udies suggest that atrazine t oxicity 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 us ually reduce the toxicity of pe sticides. Intra-laboratory studies on two fish species provide comparative LC 50 

# values for two levels of water hardness (Birge, Black and Bruser, 1979). Em bryo-larval rainbow tr out were exposed to atrazine for 27 days at water hardness levels of 50 and 200 mg/L and produced LC 50 

# values of 0.66 and 0.81 mg/L, respectively. Ch annel catfish were tested at the same water hardness levels for 8 days and yielde d LC50 values of 0.22 and 0.23 mg/L. With rainbow trout embryo-larvae, the soft water in creased toxicity by about 19 percent, while
the LC 50 

# 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-larv ae of channel catfish and rainbow trout.

Salinity effects at 5, 15 and 25 g/L on the to xicity of atrazine are opposite for the estuarine fish larvae, sheepshead minnow and the copepod nauplii, Eurytemora affinis (Ziegenfuss, Anderson, Spittler a nd Leichtweis, 1994). The 96-hour LC 50 

# values (16.2, 2.3 and 2.0 mg/L) for sheepshead minnow consistently increased with in creasing salinity. In the case of the copepod nauplii, the 96-hour LC 50 

# values (i.e., 0.5, 2.6 and 13.3 mg/L) consistently decreased with increasing salin ity. The consistency of the two data sets suggest that salinity effects th e toxicity of atrazine. Stat istical tests for both species

## 5 9 

# indicate significant differences between the LC 50 

# 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 LC 50 

# values for rainbow trout and sheepshead minnow are consistent for increasing water hardness and increasing salinity.

For many pesticides, the earlier life-stages ar e normally more sensitive than later lifestages. Contrary to most pesticides, the a quatic toxicity data for toad and frog tadpoles suggest that the late stages ar e more sensitive to atrazine th an early tadpole stages (Howe

et al ., 1998). The late stage of the Ameri can toad tadpole is about 2.5 times more sensitive to atrazine than the early stag e (10.7 versus 26.5 mg/L). For the northern leopard frog tadpoles, the later stage is about 3.3 times more toxic th an the early tadpole stage (14.5 versus 47.6 mg/L).

The above studies suggest that decreases in water hardness and salin ity can increase the toxicity of atrazine to fish, but increasing salinity may mitigate atrazine toxicity to copepods. Life stages show diffe rences in sensitivity to atrazi ne. The later stages in frog and toad tadpole development s how an increased sensitivity to atrazine over early tadpole
stages.

A.7 Pesticide Toxicity Interactions

A num ber of authors have reported toxic inte ractions between atra zine, its dealkylated degradates and other pesticides. Synergis m between atrazine and a number of other pesticides has also been reported in aquatic organisms, particularly with organophosphate insecticides, a carbamate insecticide and other herbicides including metolachlor.

A.7.1 Plants

In 1974, Putnam and Penner reported on the e ffects of interactions of herbicides on higher plants. Atrazine was c ited in test combinations with 5 herbicides, 2 insecticides and a fungicide. Synergistic effects (i.e., in creased toxicity higher than additivity) was
identified in 6 out of the 8 te st combinations. Atrazine was synergistic with 4 herbicides (i.e., 2, 4-D (oil), paraquat, EPTC, and al achlor) and 2 in secticides (i.e., diazinon and fensulfothion). Atrazine test combinations with da lapon, a herbicide, and dexon, a fungicide, showed antag onistic interactions.

Torres and O’Flaherty (1976) re port additive toxicity of at razine with simazine at concentrations of 1.0 ug/L and 1 mg/L for Chlorella vulgaris

, Stigeoclonium tenue

, Tribonema sp., Vaucheria geminata , and Oscillatoria lutea . Additive toxicity of
malathion with atrazine was found in Chlorella vulgaris , but could not be assessed with other species, because malathion produced total inhibition of chlorophyll production at 1 ug/L or greater concentrations . At 1 and 1,000 ug/L, pest icides mixtures increased toxicity from 2.4 to 100 percent ove r the toxic levels of atrazine alone. Mixtures of these

## 6 0 

# pesticides at concentrations of 0.1 and 0.5 ug/L usually enhanced the production of chlorophyll.

Stratton (1984) also tested the most sensitive algal species, Anabaena inaequalis , with
mixtures of atrazine and its two most toxic degradates, deethylatrazine and deisopropylatrazine. Cell count results indicate that combinations of atrazine/deethylatrazine (1.8) and atrazine/deisopropylatrazi ne (1.3) are sy nergistic and deethylatrazine/deisopropyl-atrazine mixtures are additive (1.03). For photosynthesis, results after 3 hour exposures indicate that all mixture combinations for these three chemicals are antagonistic (0.8, 0.86, and 0.89).

Burrell et al . (1985) reported 11-day interactions between algal populations and between algal populations and pesticides. Population interactions showed that Chlorella vulgaris inhibited population growth of Ankistrodesmus braunii by 32 percent. The addition of the bacterium, Chromobacterium violaceum , added to the algal mixture further inhibited population growth of A

. braunii by an additional 17% and bacterial growth was stimulated, but the bacterium had no effect on Chlorella populations. The combined effect of the mixtures of atrazine (60 μ g/L) and sodium pentachlorophenate (Na-PCP) (0, 300, 800, 1,000 and 1,200 μ g/L) and atrazine (40 and 100 μ g/L) with Na-PCP (700 and
1,200 μ g/L) on A. braunii populations were additive over a wide range of concentrations. Similar results of atrazine (10 and 100 μ g/L) and Na-PCP (300 and 1,200 μ g/L) were
obtained with C. vulgaris. In mixed algal cultures tested with atrazine (40 and 100 g/L),
cell numbers of A

. braunii were reduced 50 and 80 percen t, respectively, which was not significantly different than effects when test ed alone. In the same mixed culture test, atrazine inhibited growth of C. vulgaris by 79 and 85 percent, respectively, which showed a significant growth inhibition only at the lower test concentration (40 μ g/L).

The authors concluded that the high atrazine co ncentration (100 μ g/L) did not alter the established population relationship between the two algal species , but at the lower concentration (40 μ g/L), A

. braunii increased the susceptibility of C

. vulgaris to atrazine. When mixed cultures of algae were treated with both atrazine (60 μ g/L) and Na-PCP (300, 800, 1,000 and 1,200 μ g/L), ch emical antagonism wa s observed. The addition of the bacterium, C

. violaceum , to the microcosm, had no effe ct on the level of antagonism

for A

. braunii

. C

. violaceum modified the antagonism of atrazine toxicity to C

. vulgaris by about 40 percent, but the antagonistic effect was not el iminated. The net atrazine toxicity decreased as the Na-PCP concentr ation increased. The authors found no reason for the modification of atrazine effects by C

. violaceum

.

Carder and Hoagland (1998) repor ted that pesticide interactio ns of atrazine (0, 12 and

150 μ g/L) and alachlor (0, 5, 90 μ g/L) on benthic algal communities in artificial recirculating streams showed significant interaction (i.e., antagonism) only in the first week in the combination of high alachlor a nd low atrazine test concentrations. The
authors concluded that the interaction is most likely anomalous and the lack of significant synergistic effects may be attribut ed to different modes of action.

## 6 1 

# A.7.2 Animals

A number of authors have reported synergistic effects of atrazine with terrestrial and aquatic animals with one or more of the follo wing pesticides: (i.e., alachlor, chlorpyrifos, DDT, diazinon, malathion, methyl parathion, parathion, metol achlor, and trichlorfon).

Liang and Lichtenstein (1975) also found at razine synergism between soil residues of both DDT and parathion using fruit flies, Drosophila melanogaster and measured lethal effects versus the age of the pe sticide residues in soil. Ten grams of Plainfield sand (1.2 % organic matter) or Plano silt loam (4.7% organic matter) was mixed with parathion (2.3 μ g/10 g of soil = 0.23 ppm) or DDT (30 μ g/10 g of soil = 3 ppm), then was mixed with 10 g of the same soil type, which cont ained increasing atrazi ne levels (40 to 1000

μ g/10 g of soil = 4 to 100 ppm) or controls. Fifty fruit flies were placed in 120 ml test jars for 24 hours with the 10-g portions of air-dri ed soil untreated or treated with atrazine, parathion, DDT or combinations thereof. The resulting 24-hour fruit fly LD 50 

# values for constant soil levels of para thion (2.3 ppm) and DDT (3.0 ppm ) were as follows: parathion (6.2 ppm atrazine in sand and 92 ppm in lo am) and DDT (8.5 ppm atrazine in sand and 68 ppm in loam). Synergistic effects were a pparent in all test combinations of soil and pesticides yielding a dose-response effect on fly mortality with in creasing atrazine soil concentrations. Fruit fly mortality levels with both parathion and DDT in soils also clearly indicate a strong reduc tion in toxicity with the silt loam soil with a higher percentage of organic matter (4.7% ) compared to sandy soil (1.2%).

Additional loam soil toxicity tests were c onducted daily for 4 days, with aged-atrazine soil with an initial 50 ppm aged in the dark at 22 Ε C and both fresh and aging-parathion soil levels (0.35 ppm). In the test with fresh parathion soils and aged-atrazine soils, toxicity to fruit flies decreased linearly fr om 95% mortality on Day 0 to 43.3% over four days. By the fourth day, atrazine levels had declined to 19 ppm, which was barely enough to synergize parathion in loam soils. In another toxi city test, parathion-treated soils were aged under the same conditions as above and added it daily to the initial 10 g of atrazine-treated soil (50 ppm). In this test, the toxicity to fruit flies decreased logarithmically from about 68% on Day 0 to 10% mortality on Day 4. The measured concentrations of aging parath ion in the silt loam soil decrea sed at a rate paralleling the logarithmic toxicity curve. The fina l parathion level on Day 4 was 0.24 ppm.

Liang and Lichtenstein (1975) found atrazine to be synergistic with parathion in 24-hour aquatic tests with third-instar mosquito larvae, Aeddes aegypti and also assessed the
effects of sand and loam soils on their indi vidual and combined t oxicity in 20 ml of pesticide-treated wate r. Atrazine at 10,000 μ g/L showed no toxicity to the mosquito larvae; alone, parathion (15 μ g/L) killed 20 + 7 percent of the larvae; and at these concentrations, the combination of the two pesticides produced significantly (p = 0.01) higher mortality (73 + 18 %). Addition of 5 g of Pl ainfield sand (1.2% organic matter)

with 15 μ g/L parathion reduced the toxi city of parathion from 20 + 7% to 18 + 4%, but when sand was mixed into the water, mortality drop to 5%. Plano silt loam soil (4.7% organic matter) without mixing redu ced parathion toxicity from 20 + 7% to 5 + 4% and when the loam soil was mixed into the water, no mosquito larvae di ed. When these two

## 6 2 

# soils were added to the same combination of atrazine and para thion, sand reduced the mortality from 73 + 14% to 71 + 14% (unmixed) and to 18 + 4 % when mixed into the water; loam soil reduced the mortality from 73% to 64 + 4% (unmixed) and to no mortality with mixing. The combination of at razine and parathion was significantly (p = 0.01) more toxic than the toxicity of parathion or atrazine alone.

The above toxicity test method was repeated us ing 1 and 5 grams of sand or silt loam to
measure the effect of different amounts of soil on toxicity following 24-hour exposures. Atrazine (10 ppm) produced no mortality in 24 hours to mosquito larvae. Parathion (0.015 ppm) produced 24 + 7% mortality (no soil), 16 + 7% (1g of sand), 2 + 2% (5 g of sand), 7 + 0% (1 g of loam soil) and 0% (5 g of loam soil). The combination of atrazine (10 ppm) and parathion (0.015 ppm) showed synergistic effects on mosquito larvae mortality: 62 + 8% (no soil), 42 + 10% (1 g of sand), 2 + 2% (5 g of sand), 22 + 4% (1 g of loam soil) and 0% (5 g of loam soil). This test format was repeated using higher pesticide concentrations and again the mortality levels were increased with a mixture of atrazine (20 ppm) and parathi on (0.30), but the synergistic increase was much lower than in the previous test. The 24- hour results indicated that atra zine alone was not toxic to mosquito larvae; 0.30 ppm parathion (93 + 6% mortality with on soil), 62 + 8% with 5 g of sand and no mortality with silt loam soil. The mixture of 20 ppm atrazine and 0.30 ppm parathion produced 98 + 4% mortality with no soil, 76 + 4% dead when shaken with 5 g of sand, and 38 + 10% lethality when shaken with 5 g of silt loam soil. These studies demonstrate that atrazine is synergistic with parathion and, like single toxicants, organic matter in soils and sediments will modify toxici ty of pesticide mixtur es, especially if the organic matter is suspended in the water. While this particular study has limited value for risk assessment, because the atrazine levels (10 and 20 ppm) exceed the normal environmental range of atrazine exposures, the study suggests that sy nergism of atrazine and parathion may occur at lo wer concentrations, possibly in the range of environment
levels of atrazine.

Pape-Lindstrom and Lydy (1997) tested atra zine with 6 pesticides for chemical interactions using 4 th 

# instar midges ( Chironomus tentans ). The 96-hour test results for the pesticide mixtures indicat ed that atrazine was syne rgistic with the phosphonate insecticide, trichlorfon, (0.26 toxic units) and 3 phosphorothioate insecticides (i.e., malathion (0.36 TU), chlorpyrifos (0.58 TU ) and methyl parathion (0.59 TU). The atrazine-mevinophos (a phosphate) mixture was less than additive (1.34 TU), while methoxychlor, a organochlorine insecticide mixture was also less than additive (1.67 TU). The results from these tests are ques tionable, since DMSO was used as a solvent with atrazine. These tests were repeated by Belden and Lydy (2000) without DMSO and with lower atrazine concentrations (0, 10, 40, 80, and 200 μ g/L). Acute 96-hour tests
with Chironomus tentans were conducted with each pesticide and EC 1 

# , EC 5 

# , EC 15 

# and

EC 50 

# values were determined based on inability of the midge to swim when prodded with forceps. Chemical interactions were tested at each of these EC levels with atrazine levels of 0, 10, 40, 80 and 200 μ g/L using 5 replicates of 10 mi dges each. Atrazine increased the toxicity of chlorpyrifos, diazinon and parathion, but not malathion. The authors
concluded that “Interaction terms were not si gnificant for atrazine + methyl parathion and atrazine + diazinon; however, a significant interaction was found for the atrazine +

## 6 3 

# chlorpyrifos test (p = 0.002, df - 12, F = 2.94) .” Synergistic ratios were reported as follows: chlorpyrifos, 1.83 at 40 μ g/L and 4.00 at 200 μ g/L atrazine; at 200 μ g/L diazinon the SR was 2.71 and for methyl pa rathion, the SR was 1.94. The variety of chemical interactions produced by atrazine mixt ures indicates that th e effect of atrazine on an organism is dependent on the species, cocontaminant, and the concentration of

atrazine. Additional tests with 200 μ g/L atrazine and chlorpyrif os showed that atrazine increased the uptake of chlorpyrifos by 42 pe rcent, and that the atrazine induction of cytochrome-P450 increased the formation of th e O-analog which increased the toxicity of chlorpyrifos at enviromental ly relevant concentrations.

Anderson and Lydy (2002) demonstrated that atrazine concentrations as low as 80 μ g/L significantly increased the acute t oxicity of diazinon to the amphipod Hyallela azteca

. Using larvae of the mide Chironomus tentans , Belden and Lydy (2000) demonstrated a significant increase in diazinon toxicity when simultaneous exposure to 40 μ g/L of
atrazine occurred. In anothe r study by Banks et al (2005), th e study authors demonstrated

that atrazine concentrations as low as 5.0 μ g/L in combination with diazinon resulted in significant (P < 0.05) increases in the 48-h toxicity of diazinon to Ceriodaphnia dubia

. Atrazine induces cytochrome P450 and general es terase activities in in sects (Kao et al., 1995). It is possible that induc tion of the P450 system by atrazi ne may either increase or decrease the toxicity of other chemicals, depending on whether metabolites of the chemical in question are more or less toxic than the parent compound itself. Syngeristic effects of organophosphates and atrazine obs erved by Pape-Lindstrom and Lydy (1997) for the midge C. tentans suggest that pr ocesses involved with oxidation of some organophosphate molecules to more toxic oxon metabolites may be enhanced in the presence of atrazine.

Howe et al . 1998 reported synergism between atrazine and alachlor, a herbicide, in tests with young rainbow trout, channel catfish and early and late tadpole stages of the northern frog and the American toad. The results are presented in the table below. (MRID # 452029-10).

Species (stage) Time (hour) Atrazine LC50 ( 95% CI) mg/L Alachlor LC50 (95% CI) mg/L Atrazine-Alachlor LC50 a (95% CI) mg/L Additive Index b (95% CI)

Rainbow trout (0.8-1.0-gram juveniles) 24

96 31.6 (28.2 - 35.4) 20.5 (18.3 - 22.9) 10.6 (9.5 - 11.7) 9.1 (9.0 - 9.2) 9.5 (8.3 - 10.9) 6.5 (5.7 - 7.7) -0.20 (-0.53-0.059) -0.03 (-0.28-0.15)

Channel catfish (0.9-1.1-gram juveniles) 24

96 51.3 (44.6 - 59.0) 23.8 (22.3 - 25.5) 23.8 (22.7 - 25.0) 16.7 (15.1 - 18.4) 11.1 ( 9.6 - 12.4) 7.5 (5.3 - 8.4) 0.29 (0.067-0.55) c 0.31 (0.072-0.57) c

Northern leopard frog (0.7-0.9-gr early larvae) 24

96 69.7 (63.1 - 77.2) 47.6 (41.4 - 54.8) 14.9 (13.3 - 16.6) 11.5 (10.1 - 13.2) 12.1 (11.0 - 12.9) 6.5 ( 5.7 - 7.7) 0.015 (-0.17-0.24) 0.43 (0.054-0.87) c

Northern leopard frog (1.4-1.9-gr late larvae) 24

96 45.3 (42.3 - 48.5) 14.5 (11.9 - 17.5) 7.3 ( 6.6 - 8.0) 3.5 ( 3.1 - 3.8) 5.9 ( 5.5 - 6.4) 2.1 ( 2.0 - 2.3) 0.07 (-0.12-0.25) 0.34 (0.069-0.56) c

American Toad (0.1-0.2-gr early larvae) 24

96 66.4 (58.9 - 74.9) 26.5 (23.0 - 30.5) 5.7 ( 4.7 - 5.8) 3.9 ( 3.7 - 4.2) 4.4 ( 4.2 - 4.6) 1.8 ( 1.7 - 1.9) 0.19 (-0.057-0.28) 0.89 (0.68 - 1.2 ) c

American Toad (0.4-0.5-gr late larvae) 24

96 15.8 (13.5 - 18.4) 10.7 ( 9.2 - 12.5) 4.3 ( 3.8 - 4.8) 3.3 ( 2.8 - 3.6) 2.9 ( 2.6 - 3.3) 1.5 ( 1.4 - 1.6) 0.17 (0.11 - 0.46) c 0.68 (0.34 - 1.0 ) c

a 50:50 mixture of atrazine 4L (40. 8% ai.) and alachlor EC (43.0% ai.).

b An additive index great er than zero indicates great er than additive toxicity.

## 6 4 c Significant chemical synergy interaction between atrazine and alachlor.



# Boone and Bridges-Britton (2006) examined the single and interactive effects of atrazine, carbaryl, and ammonium nitrat e fertilizer on metamorphosis of tadpoles of the gray treefrog ( Hyla versicolor ). Tadpoles were reared in mesocosms from hatching through metamorphosis and were exposed to the presen ce or absence of the three contaminants. The results of the study indicated that th e presence of multiple, sublethal chemical
stressors with different modes of action may not be more determintal than that of one
chemical factor alone.

As previously discussed in Section A.2.4d a nd summarized in Table A-16, Hayes et al. (2006) assessed the effect of three different mixtures containing atrazine, to mortality, growth and development, gonadal development, thymus histology, and disease rates (i.e., immune function) in larval leopard frogs ( R. pipiens ). The three mixtures included
atrazine and S-metalachlor at 0.1 an d 10 ppb, Bicep II Magnum (reported as 33.3% atrazine, 0.7% atrazine-related products, 26.1% TGAI of S- metolachlor, and 40.2% inert ingredients), and a mixture of the 9-pesticides (atrazine, metolachlor, alchlor, nicosulfuron, cyfluthrin, cyha lothrin, tebupirimphos, methala xyl, and propiconizole all at 0.1 pbb). The specifics of the study, includi ng uncertainties, which preclude the use of this data quantitatively, are di scussed as part of Section A .2.4d. In summary, many of the confounding effects identified in previous st udies by the FIFRA SAP limit the utility of this study.

Thymus histology was completed (to m easure immunocompetence) after the study authors noted that animals exposed to th e 9-compound pesticide mixture experienced
increased incidence of bacterial infection with Chryseobacterium (Flavobacterium) menigosepticum . Animals exposed to the 9-com pound pesticide mixture at 0.1 ppb had significantly longer larval periods. All mixtur es resulted in reduced growth (SVL and BW), as compared to the solvent control, with the atrazine and S-metolachlor mixture having the greatest negative effect. With respect to gonadal development, the gonads and gametes were underdeveloped in both the cont rol and treatment groups; therefore, it was not possible for the study authors to assess th e affects of mixtures on sex differentiation. Exposure to the Bicep mixture (atrazine a nd S-metolachlor) and the 9-compound mixture resulted in damage to the thymus as measured by thymic plaques; however, the ecological relevance of thymic plaques is not discussed. Given the increased incidence of disease and evidence of histological effect s on the thymus in animals exposed to the mixtures, the study authors suggest that exposure to pesticide mixtures renders amphibians more susceptible to diseas e as a result of immunosuppression.

As part of the same study, Hayes et al. ( 2006) also examined the effects of the 9- compound mixture on plasma corticosterone levels (stress hormone) in adult male African clawed frogs ( X. laevis ). Five males were treated with the 9-compound pesticide mixture (including atrazine at 0.1 ppb) and fi ve males were exposed to ethanol only (no negative control was tested). Following th e 27 day exposure period, blood was collected by cardiac puncture. The study au thors report a clea r effect on corticosterone levels in male African clawed frogs with corticostero ne levels increasing 4-fold in pesticide-

## 6 5 

# exposed males. However, there are se veral flaws in the study design, which are discussed in Section A.2.4d, that add a high degree of uncertainty to the results.

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## 6 9 

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