Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Mufioz, 1., M. Real, H. Guasch, E. Navarro, and S. Sabater. 2001. Effects of atrazine on

periphyton under grazing pressure. Aquatic Toxicology. 55: 239-249. (MRID 48261125)

Purpose ofReview:

Determine potential effects of atrazine on an aquatic community. Endpoints from this study

contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant communities

and associated determination of a Level ofConcern (LOC).

Date of Review: October 5, 2009

Purpose of Study:

This study examines the potential effects of atrazine on periphyton, in the presence and absence

ofsnails (Physella acuta), in indoor experimental flow-through microcosms.

Summary of Study Findings:

Experiments including snails will be mentioned, although in limited detail, in order to focus on

the periphyton community experiments conducted in the presence and absence of atrazine alone.

Methods:

Twelve indoor, once-through Perspex artificial channels (170 cm long, 10 cm wide, 10 cm deep)

were continuously supplied with water to yield a flow rate of 1 cm/second (90 L/hour) and water

depth of 1.8-2.2 cm within the channels (microcosms). The source water was a well located 50

m from the test facility. The microcosms were maintained on a 8:16 hour lightdark cycle using

500W metal halide lamps with irradiance of 140 IlE/m2/s at the water surface. The floor of each

channel (microcosm) was homogenously covered with 20 x 10 cm sections ofsand-blasted glass

to serve as surface substrates for periphyton, with smaller 1.2 x 1.2 cm sections ofthe glass

substrate interspersed between the larger sections to serve as sampling units.

To collect periphyton, sections of glass substrate were left in a stream (L'Avenc6) for 2 weeks;

no additional details were provided. According to the study authors, this stream was

"unpolluted". The colonized sections of glass substrate were then introduced into the

- 1 -

1••••1.111

2087814
microcosms and allowed to acclimate for 12 days prior to atrazine treatment. Snails (Physella

acuta) were collected from another local stream (Congost, NE Spain) and transported to the test

facility in aerated containers. Selected snails (shell length 7-9 mm) were introduced into

microcosms 48 hours prior to treatment (20 snails per microcosm, tissue dry wt. 0.9 g/m2

,

equivalent density 118 snails/m2). A net (mesh size not specified) was placed at each microcosm

out-flow to retain the snails.

The experimental design consisted offour treatment groups as follows: 1) untreated microcosms

without snails (control), 2) atrazine treated microcosms without snails, 3) untreated microcosms

with snails, and 4) atrazine treated microcosms with snails; with all groups replicated in

triplicate. No solvent controls were utilized.

The treatment solution was prepared by dissolving powdered atrazine (2:90% purity, Makhteshim

S.A., personal communicationl) in well water (1 L, concentration not specified) with acetone

(0.5 mL, co-solvent final concentration 0.05%, v:v) and sonication for 10-15 minutes*. Six

microcosms were continuously treated (flow rate 4 mL/minute) at the microcosm in-put with

atrazine, at a nominal concentration of 14 flg/L, using a peristaltic pump.

Following treatment, oxygen saturation, temperature, conductivity and pH were measured in the

microcosm water daily for up to 18 days in September, 1998. No additional experiment dates

were specified. Soluble reactive phosphorus, nitrate (N-N03-), nitrite (N-N02-) and ammonium

(N-NH4+) were measured in the water at three unspecified intervals.

At five unspecified intervals, water samples (volume not specified) from treated and control

microcosms were analyzed for atrazine using the EnviroGard Triazine QuantiTube Test Kit

(Millipore), with a detection limit of 0.04 flg/L. Analysis methods were not further described.

Photosynthetic activity (carbon incorporation). Nine 1.2 x 1.2 cm sampling units with

periphyton were randomly selected from each microcosm at days 0, 1,3,6, 12 and 18

posttreatment and separately placed in vials with filtered (glass fiber, Whatman type GF/F) 4 mL

ofmicrocosm water. For each nine-sample unit set, five samples were used for light incubations,

two for dark incubations and two were formaldehyde-killed to serve as controls. Samples were

pre-incubated for 1 hour in an agitator under the same temperature and light conditions as the

microcosms, then e4

C]bicarbonate (Hl4C03-) solution (specific activity 1 mCi/mL) was added at

1 flCi/vial and incubation continued under the same conditions for 1 hour. Formaldehyde (2%,

v:v) ceased e4

C]-incorporation, then HCl (pH 2-3) was added and the samples evaporated for 8

hours on a hot-plate. Dried residues were treated with 1 mL dimethylsulfoxide and analyzed for

total radioactivity using LSC.

1 Information obtained via personal communication with Dr. Munoz on October 20, 2009 is provided in Attachment

1.

- 2 -
Chlorophyll a. Five 1.2 x 1.2 cm sampling units with peiiphyton were randomly selected from

each microcosm at the same intervals as for the photosynthetic activity tests. Samples were

extracted with 5 mL of90% acetone using sonication for 7-10 minutes in an ice-bath, then

analyzed spectrophotometrically (method ofJeffrey and Humphrey, 1975).

At study termination, extracted pigments were analyzed using reverse-phase HPLC (Hypersil

ODS column, 250 mm, 5 flm particle size, i.d. not specified) with fluorescence detection

(Kontron SFM detector). Chromatographic conditions were referenced (Mantoura and

Llwewllyn, 1983), but not described. Chlorophylls and carotenes were identified through

comparison ofspecific extinction coefficients, retention times and absorbance characteristics and

against reference standards (Sigma) of chlorophylls a and band u- and ~-carotenes (Foppen,

1971, Mantoura and Llewellyn, 1983, Rowan, 1989). Chlorophyll derivatives phaeophytin a and

b, chlorophyllide a and b, and pheophorbides were also prepared (Sartory, 1985, Leavitt et al.,

1989).

Species composition. Two 1.2 x 1.2 cm sampling units with periphyton were randomly selected

from each microcosm at days 0, 3 and 18 posttreatment and preserved in 4% formaldehyde

solution. Periphyton was detached via sonication for microscopic examination at a

magnification of 600. Periphyton taxa were assigned to four classes, crustose, prostrate, stalked

or filamentous, based on physiognomy. Bio-volume was determined according to the method of

Lowe and Laliberte (1996).

Snails. Initial average dry weight (70°C until constant weight) was determined for a subsset of

snails from the same source as the treatment organisms. Individual dry weights were determined

for each treatment organism at study termination. Snail number in each microcosm was

monitored daily. Dead snails were replaced with snails ofthe same shell length. Snails found on

the net at the out-flow were removed and returned to the respective microcosm input. At study

termination, number oflaid egg masses and number of eggs per mass were determined. No

juveniles developed during the study.

Statistical analyses. Periphyton response values were analyzed by three-way analysis of variance

(ANOVA) to detect differences between treatments with a repeated measures design (with time

as a repeated factor). When statistically significant (p<0.05), response values were analyzed

using the Tukey Honest significant difference (HSD) test. Differences in periphyton pigment

composition between treatments were analyzed by two-way ANOVA, while snail biomass

changes were analyzed by one-way ANOVA.

*Atrazine was detected in the source stream in a prior study (see Guasch et al. 2001).

- 3 -
Results:

Over the 18-day study, atrazine concentrations in treated microcosms averaged (n = 22) 14.18 ±

5.93 f.lg/L; individual results were not presented. Atrazine was not detected «0.04 f.lg/L) in

untreated control microcosms.

Physico-chemical parameters of the microcosm water remained constant throughout the study,

with an average water temperature of23 ± 0.05°C, pH range of 7.4-7.6 and conductivity of 1,900

f.lS/cm. Mean phosphate (P-P04-), nitrate and nitrite were 2.4 ± 1.4 f.lg/L, 15.5 ± 0.83 mg/L and

0.22 ± 0.07 mg/L, respectively, while ammonia was not detected «0.01 mg/L).

Microcosms without snails. Periphyton bio-volume increased significantly (p<0.05) in both

control (untreated) and atrazine treated microcosms over the 18-day study, but was decreased

~40% in atrazine treated microcosms, as compared to the controls, at study termination (Figure

1). There were no significant differences in pigment composition between atrazine treated

microcosms and the untreated controls at study termination (Table 1). Chlorophyll a

concentration, photosynthetic activity (carbon incorporation) and photosynthetic efficiency

(carbon incorporation per unit chlorophyll a) were decreased ~15% (day 12), ~15% (day 12) and

~40% (day 6), respectively, at intervals during the study (Figures 1 and 2). However, there

were no significant differences over time between atrazine treated microcosms and the untreated

controls.

The periphyton community colonizing the microcosms was dominated by the diatoms

Achnanthes lanceolata, Achnanthes minutissima and Nitzschia linearis, plus basal cells of the

green alga Stigeoclonium tenue (Table 2). At study termination, filamentous form periphyton in

atrazine treated microcosms were decreased ~90%, as compared to controls, crustose forms

~40%, prostrate forms 10% and stalked forms ~5%. However, decreases were significant

(p=0.02) only for the filamentous forms (Figure 3, Table 3). Total periphyton abundance was

decreased ~30% in atrazine treated microcosms at 18 days, as compared to controls (Table 3,

attached Worksheet). The densities of two species, Cocconeis placentula and S. tenue (cells and

filaments) were reported as significantly (p=0.02-0.04) increased in the atrazine treated

microcosms (Table 2).

Microcosms with snails. Snail grazing alone resulted in decreases in periphyton chlorophyll a

concentration (p<O.OOI), photosynthetic activity (p<O.OOOI), photosynthetic efficiency (p=0.047)

and inhibition in the rate of bio-volume growth, with the addition of atrazine further enhancing

those effects (Figures 1 and 2) Grazing and atrazine exposure combined produced more-thanadditive effects with respect to carbon incorporation (p=0.02). Pigment composition was not

significantly affected by grazing either in the presence or absence of atrazine (Table 1).

Snail grazing significantly reduced the abundance of stalked (p=0.01), prostrate (p=0.008) and

crustose (p=0.02) forms of periphyton with abundances of all forms, including filamentous,

- 4 -
further decreased in the presence of atrazine (Figure 3). Grazing resulted in significant

decreases in densities of A. minutissima (p=0.002), Amphora sp. (p=0.02) and C. placentula

(p=0.04); whereas, grazing plus atrazine resulted in significantly lower densities for A.

lanceolata (p=0.0l), A. minutissima (p=0.04), Amphora sp. (p=0.04) and Nitzschia fonticola

(p=0.002). At study termination (18 days), total periphyton abundances were decreased 56% and

77% in microcosms containing snails alone and snails plus atrazine, respectively, as compared to

untreated controls (no snails, no atrazine; Table 2, attached Worksheet).

There were no significant differences between microcosms treated with atrazine and untreated

controls regarding snail biomass (p=0.9), number of escaped snails (p=0.70), number of egg

masses (p=0.73) or number of eggs per mass (p=0.66). Mortality was near 10% for all

microcosms, with no significant differences (p=0.09) between treatments.

- 5 -
40

A

C'\I 30 I

E

(.)

co

...!.. 20

..c

()

0)

::J.

10

o

__ 200 B -v- Atrazine

M ---b- Atrazine+Snails

E Snails

::J.

ex:> 150 • Control

0

T"""

X

---

(J) 100 E

::;,

0

>

0 50

CD

0

0 2 4 6 8 10 12 14 16 18

days

Figure 1. Periphyton chlorophyll a (A) and bio-volume (B); mean values from three microcosms per

treatment with standard deviation (vertical bars). Figure 1 from Munoz et al., 2001.

- 6 -
24 A

T""'

I

C\lI 16

E

c.>

()

0)

:::L 8

o

6 B -0-- Atrazine

T""' -b- Atrazine+Snails

I

Snails T""'

I • Control

co

I 4

()

0)

:::L

() 2

0)

:::L

0

0 2 4 6 8 10 12 14 16 18

days

Figure 2. Photosynthetic activity (A) and photosynthetic efficiency (B); mean values from three microcosms

per treatment with standard deviation (vertical bars). Figure 2 from Munoz et al., 2001.

- 7 -
_ Filamentous

- I I Stalked

W##1 Prostrate

Crustose -

-

-

-

-

, T'"

-

J -::iF I ; I I

40

...- 35

C\II

E

0

(/) 30

(i)

0

L{) 25 0

x

--(/) 20 E

0

-.2 15 E

0

c

C)

0 10 "en

>.

.s:::.

a.. 5

o

o

A AS S

3

C A AS S

18

C Treatment

Days

Figure 3. Abundance of periphyton physiognomic classifications; mean values from three microcosms per

treatment with standard deviation (vertical bars). A = atrazine treatment, AS = atrazine + snail, S =snail

treatment, C = control (no atrazine, no snail). Figure 3 from Munoz et al., 2001.

Table 1. Effect of atrazine (14 IlglL), on periphyton pigment composition in the presence and absence of

grazing pressure after 18 days of exposure, expressed as percentage of recovered (mean ± s.d., n = 3; from

Table 1 ofstudy).

Control Atrazine Snail

Pigment (no atrazine, no snail) (no snail) (no atrazine) Atrazine + Snail

Carotenoids 32.8 ± 0.5 33.4 ± 2.3 32.2 ± 0.8 31.9 ± 6.6

Chlorophyll a 56.8 ± 3.4 57.4 ± 4.8 51.8 ± 6.5 50.8 ± 6.7

Chlorophyll b 0.6 ± 0.2 0.9 ± 0.7 5.1±5.1 6.3 ± 5.4

Chlorophyll c 2.7 ± 0.4 2.3 ± 0.3 2.2 ± 0.7 1.9 ± 0.7

Degraded chlorophyll 10.1 ± 7.9 6 ± 3.1 8.6 ± 2.5 9.3 ± 5.2

- 8 -
Table 2. Effect of atrazine (14 /!gIL) on periphyton species abundance in the presence and absence of grazing pressure, expressed as density x 103

cells/cm2 (mean ± s.d., n = 3; from Table 2 ofstudy).

Day 3 Day 18

Atrazine + Atrazine +

Taxa Day 0 Control Atrazine Snail Snail Control Atrazine Snail Snail

Achnanthes Ianceolata Breb. 469 ± 321 316±390 32 ± 20 152 ± 92 127 ± 63 584±318 647 ± 230 578 ± 196 427 ± 450

Achnanthes minutissima Klitz. 1,224 ± 1,034 646 ± 858 817 ± 454 364 ± 470 196± 103 2,274 ± 1,689 203 ± 806 598 ± 750 385 ± 345

Amphora sp. 68 ± 52 142 ± 152 21 ± 13 28 ± 18 16 ±.28 158±151 117 ± 47 5±9 12 ± 21

Ceratoneis sp. 17 ± 35 0 0 0 0 0 0 0 0

Chroococcus sp. 60 ± 120 0 0 0 0 0 0 23 ±40 0

Cocconeis placentula Ehr. 41 ± 20 31 ± 35 25 ± 33 20 ± 7 7±3 39 ± 35 70± 5 8 ± 14 0

Cosmarium sp. 0 0 0 4±7 0 15 ± 27 0 3±5 0

Dictyosphaerium sp. 135 ± 105 642 ± 516 373 ± 541 100 ± 73 32 ± 55 452 ± 460 281 ± 427 178 ± 268 203 ± 158

Fragilaria capucina Desmazieres 87 ± 333 16 ± 16 28 ± 24 84 ± 75 0 8 ± 13 39 ± 36 23 ±40 8 ± 14

Fragilaria construens (Ehr.) Grun. 118±124 0 16 ± 18 20 ± 35 0 8 ± 15 0 12 ± 20 3±5

Melosira varians Agardh 4±8 5±9 0 0 0 0 0 0 0

Monoraphidium sp. 0 3±5 0 0 0 0 0 0 0

Navicula criptocephala Klitz. 8 ± 16 33 ± 16 12 ± 21 24±2 12 ± 6 54 ± 74 39 ± 67 0 12 ± 12

Nitzschia fonticola Grun. 79 ± 60 117 ± 140 22 ± 19 16 ± 18 4±5 70 ± 46 78 ± 67 0 13 ± 12

Nitzchia linearis Smith 153±81 304 ± 350 432 ± 55 360 ± 427 25 ± 20 240 ± 336 764 ± 772 81 ± 141 40 ± 38

Scenedesmus sp. 7 ± 14 0 0 0 0 109 ± 117 0 11 ± 18 16 ± 28

Stigeoclonium tenue (Ag.) Klitz (basal cells) 220 ± 106 93 ± 161 103 ± 58 48 ± 55 23 ± 24 151 ± 506 109 ± 133 452 ± 274 184 ± 76

Stigeoclonium tenue (filaments) 59 ± 102 81±141 10 ± 18 256 ± 285 23 ± 27 1,875 ± 1,269 211 ± 253 746 ± 923 208 ± 145

Synedra ulna (Nitzsch.) Ehr. 21 ± 6 125 ± 135 222 ± 96 108 ± 32 12 ± 6 1,415 ± 426 835 ± 49 571 ± 711 221±155

Total abundance 2,771 ± 1,809 2,555 ± 2,689 2,400 ± 42 1,584 ± 219 478 ± 178 7,451 ± 3,521 5,218 ± 725 3,289 ± 1,800 1,732 ± 929

- 9 -
Table 3. ANOVA probability (p) values (from Table 3 ofstudy).

Factor

Taxa/growth form Atrazine (A) Snail (S) Time (T) AxT SxT AxS AxSxT

Achnanthes lanceolata NSJ NS 0.002 NS NS 0.03 0.01

Achnanthes minutissima NS 0.002 0.008 NS NS NS 0.04

Amphora sp. NS 0.02 NS NS NS NS 0.04

Cocconeis placentula 0.04 0.01 NS NS 0.04 0.001 NS

Nitzschiajonticola NS NS NS NS NS NS 0.002

Stigeoclonium tenue (cells) 0.03 NS 0.003 NS 0.004 NS NS

Stigeoclonium tenue (filaments) 0.02 NS 0.003 0.03 NS NS NS

Filamentous 0.02 NS 0.003 0.03 NS NS NS

Stalked NS 0.01 0.0001 NS 0.002 NS NS

Prostrate NS 0.008 0.0001 NS 0.0022 0.0001 0.0003

Crustose NS 0.02 NS NS NS NS NS

1 Not significant.

- 10 -
Data Summary Table: Periphyton and gastropods in indoor,jlow-through microcosms;

periphyton data represent the "atrazine on£v" conditions, in the absence ofgrazing pressure.

Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pg/L) Occurred from Day Oto when?

When Effect

Occurred

Magnitude of effect

estimated from

Periphyton 14 11 giL; Figure 1.

chlorophyll a 15% decrease measured 14.18 ± 12 Not calculable No Effects not

(llg/cm2) 5.93 11giL statistically

significant

(p=0.86).

Periphyton bio- 141lg/L; Study Magnitude of effect

-40% decrease measured 14.18 ± 18 Not calculable terminated at estimated from volume 5.93 Ilg/L day 18. Figure 1. SSNR1

•

No, but Magnitude of effect

Periphyton control estimated from

14 Ilg/L; decreased to Figure 2. photosynthetic 15% decrease measured 14.18 ± 12 Not calculable level of Effects not activity 5.93 1lg/L atrazine statistically (Ilg C/cm2

/hr) treated at day significant

18. (p=0.38).

No, control

Periphyton and atrazine

photosynthetic 141lg/L; treated Magnitude of effect

efficiency -40% decrease measured 14.18 ± 6 Not calculable decreased to estimated from

(Ilg CIllg 5.93 1lg/L same level at Figure 2. SSNR.

ch10rophy1l/hr) days 12 and

18.

Magnitude of effect

141lg/L; Study estimated from

Filamentous ~90% decrease measured 14.18 ± 18 Not calculable terminated at Figure 3.

periphyton 5.93 Ilg/L day 18. Effects statistically

significant (p=0.02,

Table 3).

Magnitude of effect

estimated from

Stalked 14 11giL; Study Figure 3.

periphyton -5% decrease measured 14.18 ± 18 Not calculable terminated at Effects not

5.93 11giL day 18. statistically

significant (p>0.05,

Table 3).

- 11 -
Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pg/L) Occurred from Day Oto when?

When Effect

Occurred

Magnitude of effect

estimated from

Prostrate 14 flg/L; Study Figure 3.

periphyton 10% decrease measured 14.18± 18 Not calculable terminated at Effects not

5.93 flg/L day 18. statistically

significant (p>0.05,

Table 3).

Magnitude of effect

estimated from

Crustose 14 flg/L; Study Figure 3.

periphyton ~40% decrease measured 14.18 ± 18 Not calculable terminated at Effects not

5.93 flg/L day 18. statistically

significant (p>0.05,

Table 3).

Magnitude of

effects calculated

from results in

Achnanthes 14 flg/L; Yes, day 18, Table 2 ofstudy

lanceolata 90% decrease measured 14.18 ± 3 Not calculable (+11% (attached

Breb. 5.93 flg/L stimulation). Worksheet).

(abundance) Effects not

statistically

significant (p>O.05,

Table 3).

Magnitude of effect

calculated from

Achnanthes results in Table 2 of

minutissima 14 flg/L; Study study (attached

Klitz. 91 % decrease measured 14.18 ± 18 Not calculable terminated at Worksheet).

(abundance) 5.93 flglL day 18. Effects not

statistically

significant (p>0.05,

Table 3).

Magnitude of

effects calculated

from results in

14 flg/L; Magnitude of Table 2 ofstudy

Amphora sp. 85% decrease measured 14.18 ± 3 Not calculable effect 26% (attached

(abundance) 5.93 flg/L decrease at Worksheet).

day 18. Effects not

statistically

significant (p>O.05,

Table 3).

- 12 -
Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pg/L) Occurred from Day Oto when?

When Effect

Occurred

Magnitude of

effects calculated

from results in

Cocconeis 14 flg/L; Yes, day 18, Table 2 ofstudy

placentula Ehr. 19% decrease measured 14.18 ± 3 Not calculable (+79% (attached

(abundance) 5.93 flg/L stimulation). Worksheet).

Effects statistically

significant (p=0.04,

Table 3).

Magnitude of effect

Cosmarium sp.

14 flg/L; Study calculated from

100% decrease measured 14.18 ± 18 Not calculable terminated at results in Table 2 of (abundance) 5.93 flg/L day 18. study (attached

Worksheet). SSNR.

Magnitude of

14 flg/L; Magnitude of effects calculated

Dictyosphaerium 42% decrease measured 14.18 ± 3 Not calculable effect 38% from results in

sp. (abundance) 5.93 flg/L decrease at Table 2 ofstudy

day 18. (attached

Worksheet). SSNR.

Fragilaria Magnitude of effect

capucina +388% 14 flg!L; Study calculated from

measured 14.18 ± 18 Not calculable terminated at results in Table 2 of Desmazieres stimulation 5.93 flg/L day 18. study (attached (abundance) Worksheet). SSNR.

Fragilaria Magnitude of effect

contruens (Ehr.) 14 flg/L; Study calculated from

100% decrease measured 14.18 ± 18 Not calculable terminated at results in Table 2 of Grun. 5.93 flg/L day 18. study (attached (abundance) Worksheet). SSNR.

Magnitude of effect

Melosira varians 14 flg/L; calculated from

Agardh 100% decrease measured 14.18 ± 3 Not calculable No results in Table 2 of

(abundance) 5.93 flg/L study (attached

Worksheet). SSNR.

Magnitude of effect

Monoraphidium 14 flg/L; calculated from

100% decrease measured 14.18 ± 3 Not calculable No results in Table 2 of

sp. (abundance) 5.93 flg/L study (attached

Worksheet). SSNR.

- 13 -
Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pg/L) Occurred from Day Oto when?

When Effect

Occurred

Magnitude of

Navicula 14 IlglL; Magnitude of effects calculated

criptocephala 64% decrease measured 14.18 ± 3 Not calculable effect 28% at from results in

Klitz. 5.93 Ilg/L day 18. Table 2 ofstudy

(abundance) (attached

Worksheet). SSNR.

Magnitude of

effects calculated

from results in

Nitzschia 14 IlglL; Yes, day 18, Table 2 ofstudy

fonticola Gron. 81% decrease measured 14.18 ± 3 Not calculable (+11% (attached

(abundance) 5.93 IlgiL stimulation). Worksheet).

Effects not

statistically

significant (p>0.05,

Table 3).

Magnitude of

Nitzschia 14 Ilg/L; Study effects calculated

+218% from results in linearis Smith stimulation measured 14.18 ± 18 Not calculable terminated at Table 2 ofstudy (abundance) 5.93 IlgiL day 18. (attached

Worksheet). SSNR.

Magnitude of effect

Scenedesmus sp.

14 IlglL; Study calculated from

100% decrease measured 14.18± 18 Not calculable terminated at results in Table 2 of (abundance) 5.93 IlglL day 18. study (attached

Worksheet). SSNR.

Magnitude of effect

Stigeoclonium calculated from

results in Table 2 of

tenue (Ag.) 14 IlglL; Study study (attached Klitz. (basal 28% decrease measured 14.18 ± 18 Not calculable terminated at

cells, 5.93 IlgiL day 18. Worksheet).

abundance) Effects statistically

significant (p=0.03,

Table 3).

Magnitude of

effects calculated

Stigeoclonium 14 Ilg/L; No, magnitude from results in

tenue (filaments, 88% decrease measured 14.18 ± 3 Not calculable of effect 89% Table 2 ofstudy

abundance) 5.93 Ilg/L decrease at (attached

day 18. Worksheet).

Effects statistically

significant (p=0.02,

- 14 -
Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine DidRecovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pg/L) Occurred from Day Oto when?

When Effect

Occurred

Table 3).

Magnitude of effect

Synedra ulna 141lg/L; Study calculated from

(Nitzsch.) Ehr. 41 % decrease measured 14.18 ± 18 Not calculable terminated at results in Table 2 of

(abundance) 5.93 IlgIL day 18. study (attached

Worksheet). SSNR.

Magnitude of effect

Total periphyton 141lg/L; Study calculated from

30% decrease measured 14.18 ± 18 Not calculable terminated at results in Table 2 of abundance 5.93 Ilg/L day 18. study (attached

Worksheet). SSNR.

There were no

significant

Snail (Physella differences in

acuta) biomass, biomass (p=O.9),

mortality, 14Ilg/L; Study mortality (p=0.09),

number of egg No effect measured 14.18 ± 18 Not calculable terminated at number of egg

masses, number 5.93 IlgIL day 18. masses (p=O.73) or

of eggs per egg number of eggs per

mass egg mass (p=0.66),

as compared to

controls.

1 SSNR = StatIstIcal sIgmficance, as compared to controls, was not reported.

- 15 -
Reviewer Comments:

Although not reported as part of the study, the purity of the atrazine test compound was reported

via email (see Attachment 1) to be greater than 90%. The source water for the flow-through

microcosms was a well located 50 m from the test facility. Atrazine was not found in water from

untreated control microcosms within the detection limits ofthe test (>0.04 J.lg/L).. No other tests

for chemical contaminants were described. Prior exposure of organisms to atrazine or other

chemicals was not established or reported. Microcosm water was not monitored for turbidity,

alkalinity, or particulate and dissolved organic carbon. Oxygen saturation was reportedly

monitored; however, the data were not presented.

The concentration of acetone used as solvent (0.5 mLlL) was 5 times higher than the maximum

recommended solvent level of 0.1 mLiL in flow-through systems (see OPPTS 850.1000

guidelines for Special Considerations for Conducting Aquatic Laboratory Studies). In addition,

the lack of a solvent control confounds the ability to differentiate treatment-related effects from

those that might due to the high solvent concentrations..

The study was designed to test organismal response in the presence or absence of atrazine and

thus was limited to one test concentration (14 J.lg atrazine/L).

The microcosms were maintained on a 8:16 hour light:dark cycle photoperiod, whereas 12:12

hour and 16:8 hour lightdark cycles are considered more typical.

Description of Use in Document: Invalid

Rationale for Invalid Study Classifcation:

The study is classified as invalid because the concentration of acetone solvent is 5 times higher

than the maximum concentration recommended for flow-through systems. According to the

established criteria for acceptance and review of atrazine aquatic micro/mesocosm open

literature studies, a study should be classified as invalid ifthe amount ofsolvent in a flowthrough study exceeds 0.1 mLiL. In addition, the lack of a solvent control confounds the ability

to discriminate treatment-related effects and adds further uncertainty regarding the interpretation

ofresults relative to a negative control with no added solvent.

Limitations of Study: There were numerous reporting and measuring deficiencies. Purity ofthe

test compound was not reported. As noted above, the most serious flaw ofthe study is the

amount ofsolvent in the treatment groups (5 times above the recommended maximum

concentration for flow-through systems) with no associated solvent control. No effort was made

to document whether chemicals other than atrazine were present in the treatment and/or control

microcosms prior to study initiation. It was not established whether the natural periphyton and

snails collected for the study had prior exposure to atrazine. Water quality monitoring was

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limited: microcosm water was not tested for turbidity, alkalinity, or organic carbon; dissolved

oxygen was monitored, but not reported.

Primary Reviewer: Lynne Binari, M.S., Subcontractor to Cambridge Environmental Inc.

Secondary Reviewer: Catherine Aubee, Biologist, Environmental Fate and Effects Division

U.S. Environmental Protection Agency G')t--. I; ,Jv,'YI,,,L ,JOI/

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, U.S. EPA

~~ J;!l'3(t(

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Attachment 1: Correspondence with Lead Author (Dr. Isabel Munoz)

.Jo::Y. _

. -"

History:

Re: atrazine and aquatic grazers

Isabel Munoz to: Catherine Aubea

This message has been replied to.

1012012009 11:20 AM

Dear ('::at.herine,

The a'C.r:azine used Has supplied by l1akhteshim S.A. a manuf3cture.r: er.treprise.

Puridity was higher 0'0% but I do ne.t remember exactly the percentage.

We di~~olved the atrazine in water by sonication ;10-15 min) in order to a\~·Jid tht:': effects of the ac·~,t,:)ne. \111'2 solve 3mall q1Ja~'ltiti~.s <Jf pcn..,Jer in 3.

IJOO rnl o.f Hi.lli Q wat~3l:. t';h\~n da~: Bolved we add a littlt:.~ nlO::'C~ and ':=:0 011. It.

was really hard but we have not Ihore channels to test the effect (If acetone

alc.. ne.

I hope I helped you . .~in("';e}·~ly,

1"")-,,,1 Ht;:";""

iriqinal M€ssag~ -----

FU.'m: <Aubee. Cather inel~epamai1. epa. qov>

To: <munoz l<pcrtho3. t·io. 'JI:.. os>

Sent: Wednesday, October 14, 2009 2:23 PM

Sl.lbject: atrazine,and aquatic grazerE

> [lO;?,3.r IsaJ:>el,

>

> I dlO \'l.riting In Le9a.rd to the study de;3Cl:ibed :-n iiO\J.2:.. 2001 a.rticle

> "Effects of atLaziu@ 0]1 peLiphyton u)ideL gIaziIlg pres~ure« (Aquatic

> T,o;-;.icolo:;1Y). Can yc·u prcvide the .souJ:ce and purity" cf the pOPl"Joe,l;

;. 3tr3zir,e used in the e:~periment7 r.lso, were the atrazine-free

> tre:3.tment.s pr~pared \'li th cr ~"i th,;ju't. aceto~ie?

> t-1any t.hank.'3,

>

'> Catherin.e Aubee

> Biologist - OPpiEFED

>

> Environmental Prctection Agency

> ()ne Pot.ornac Yard

2777 S. Crystal Drive

Arlington, VA 22202

> Mail Code: 7507-2

> Email: aubee~catherine@epa.'-J()v

'-' I?hone: (·'03) 347-3029

Fa:..: ('103) :;d)~;-t)204

>

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