### Open Literature Review Summary Chemical Name: Atrazine 

### CAS No: 1912-24-9 

### ECOTOX Record Number and Citation: Juttner, 1., A. 

### Peither, J.P. Lay, AI. Ketrup, and SJ. Ormerod. 1995. An outdoor mescosm study to assess ecotoxicological effects ofatrazine on a natural plankton community. Arch. Environ. Contam. ToxicoJ. 29:435-441. (Vl1j(/O 

### l-ffs- i

### f3,'!tD'S;) Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study were also used to parameterize the Comprehensive Aquatic System Model (CASM) and establish the model-associated LOC for atrazine. 

### Date ofReview: January 15, 2009 

### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals in experimental pond cylindrical compartments (mesocosms). 

### Summary ofStudy Findings: Methods: An experimental pond, 15 m2 

### and 0.8 m deep in a small deciduous forest 

### in southern Bavaria, Germany, was compartmentalizedby seventransparentPVC cylinders (1.6 m high, 1.25 m diameter, ofvolume about 1,000 L), pressed 20 cm into the sedimentto prevent water exchange. Six ofthe enclosures were treated with atrazine on July 31, 1990, with supplemental treatments on August 28 and September 21, to maintain concentrations near nominal. Each enclosure received a different treatment level. Nominal concentrations were 5, 10,25, 75, 200, and 360 

### J.tg/L, while actual mean concentrations were 5, 10, 22, 68, 182, and 318 

### J.tg/L overthe two month study period (Figure 1). Water samples were collected weekly for atrazine analysis, pre-concentrated from volumes of5 to 150 mL, extracted into 50:50 (v/v) hexane:acetone with C18 SPE columns, evaporated, and redissolved in 1 mL methanol, then analyzed by HPLC (HP 1090, methanol/H20 80120 

### as mobile phase, 5 J.tm Hypersil ODS column 250 mm x 

### 4.6 mm 1.D. [Shandon], diode-array detection at 230 nm, detection limit 0.5 

### J.tg/mL).



### 1 1 ••

# 11••111 2087854Temperature, dissolved oxygen, pH, and conductivity were measured twice a week between 11 :00 and 13:00 using portable meters (WTW). Simultaneously, plankton samples were taken with a specially constructed sampler consisting ofa plexiglass cylinder (70 cm long, 7 cm diameter, volume 2.5 L), with four samples taken at different points in the enclosure and bulked before removal ofa 100 mL subsample for phytoplankton enumeration. The subsamples were preserved in acid Lugol's and counted by the Utermoehl method. Forzooplankton sampling, the 10 L sample was passed through a 36 flm net and the filtered fraction preserved in sugar formalin solution. Adult females, males, the number ofeggs per female (egg ratio), andjuveniles were counted for the major cladoceran

### Daphnia longispina. Forcyclopoids, adults and copepodites were combined, and nauplii were counted separately. Rotifers, which occurred in high numbers, were assessed by removing four or five subsamples of 1 mL from the total filtered zooplankton sample after resuspending it into a 100 mL volume.
No statistics were perfonned (there was no replication).



### Results: Atrazine concentrations were maintained relatively constant (Figure 1) and were not far from nominal, except for the two highest doses. Follow-up measurements revealed that 50-67% ofthe atrazine from Septemberremained in the enclosures in April 1991, the next spring. Oxygen concentration, pH, and conductivity changed over time, with some relation to atrazine concentration, especially in the oxygen level (Figure 2). pH decreased somewhat with atrazine treatment level, with the two highest doses showing the strongest effect; oxygen declined noticeably at atrazine concentrations exceeding 22 flg/L, with declines in the two highest atrazine treatments (182 and 318 Jlg/L) to less than 30% of pretreatment levels within 6 days ofthe first application. According to the study text, conductivity decreased mostly in the control and 5 Jlg/L treatment enclosures, had less decline in the 10-68 Jlg/L treatments, and no decline at 182 and 318 Jlg/L; from Figure 2, however, it appears that conductivity was highest in the control and 5 Jlg/L treatment enclosures). Although the authors note thatperiphyton was apotentially significant contributorto photosynthesis in the mesocosms, due to the enclosure surfaces providing much ofthe substrate, periphyton were not measured in the experiment. The authors cite unpublished data from a bottle study showing lowest effect concentrations of50 Jlg/L for primary production and 25 Jlg/L for respiration for periphyton on artificial substrata, which was similar to the mesocosm thresholds for phytoplankton. The authors also note that

2

### periphyton cover "was less in the higherdosage enclosures, but this was not quantified further".
Effects on phytoplankton and zooplankton were generally detected only at 182 or 318 

### Ilg/L (Figure 3

- Figure 8, Table 1), and the non-lineartrends and fluctuations overtime due to seasonal changes interfered with statistical (time-series) analyses.
No atrazine effects on phytoplankton cell density were found up to 182 

### Ilg/L, but cell density at 318 

### Ilg/L was lowerthan any othertreatments throughoutthe entire test perio (Figure 3). The Shannon-Weinerdiversity index, H', was lower at 182 and 318 

### Ilg/L than in othertreatments because ofsmallernumbers ofspecies and more variability (inequitability) in theirrelative abundance overthe second halfofthe testperiod (no figures ordata are provided). Diatoms were the least sensitive to atrazine and dominated the phytoplankton community at 182 

### Jlg/L between days 35 and 53, and at 318 

### Jlg/L throughoutthe test period. The authors cite single-species toxicity tests showing that several diatoms (Bacillariophyceae) can tolerate higher atrazine levels thattaxafrom Chlorophyceae, Cyanophyceae, and Chrysophyceae. Also, the relative abundance of 

### Trachelomonas sp. (Euglenophyceae) was highest at 182 and 318 

### Ilg/L. The Cryptophyceae 

### Cryptomonas marsonii and C. 

### erosalovata were not present in the early summer, but appeared as expected in the autumn in all enclosures except 182 and 318 

### Ilg/L (Figure 4). 

### Mallomonas sp. (Chrysophyceae) peaks occurred in the control and lower treatment level enclosures before day 25, but did not occur at 182 and 318 

### Jlg/L (Figure 5). Finally, 

### Planktosphaeria sp. (Chlorophyceae) had much lowercell numbers after day 30 at 182 and 318 

### Jlg/L than at lower atrazine concentrations (no figures ordata provided). 

### Daphnia longispina total numbers declined in all enclosures after atrazine application, and egg ratios also declined from days 3-21, increasing afterday 21 to drive increases in numbers of juvenile and adult daphnids. At 318 

### Jlg/L, the peakegg ratio was lowerthan in other enclosures and also more delayed in its arrival (Figure 6), which the authornotes may reflect indirect effects offood availability. Males appeared in all enclosures after day 25, but no ephippia occurred in the 318 JlglL enclosures despite appearing elsewhere (data not provided). Copepod nauplii increased in control and low dose enclosures up to 

### 22 Jlg/L atrazine after day 18, with only a slight increase at 68 

### Jlg/L and no increase at 182 and 318 

### Jlg/L (Figure 7). Rotifers declined in abundance in all enclosures within the first two weeks ofatrazine application, which the authors state may be due to seasonal effects. The initial decrease in 

### Synchaeta sp. numbers was more pronounced at 182 and 

### 318 Jlg/L than at otherconcentrations, and subsequent densities remained lower (Table 

### I). Polyarthra sp. declined in abundance earlier at 318 

### Jlg/L than at otherconcentrations, and was nearly absent after day 18 (Figure 8).

### "'OOO~l

lOO**\~' -_.~---------;**  

# I**-+-~** S P' d/1 

### ! -r-" 10 fJ9il 

## I I**~--+-** 25 \-lGVi i i --:+- 75 pgi', 

## I 

# I 2DOWJ.i1**-~"** 3GO**~lg/I** 

### l_,_,_.__.__. __._,--'

7 1<1 21**2~.** 35 42 49 5:3 GO

Figure 1. Atrazine concentrations in 1,000 L enclosures during the test period ofthe mesocosm experiment. (study figure 1)

4pH



### ojJg!1



# -- 5 jJgi'



# -- 10

--+- 22 IJgil 68jJg/1 -4- 182 jJgil 3181Jg/i 

### o 7 10141821252832353942464953565963 (A)



### Be

20 60

88 94 ('>0 ( B) :::.-'.

# --__;:----._---**-~** P'

# I
I 1:15 

# 1 

# I 

# :'1 I 

# O'l**n~'·~-.....,..-...,__,..._._..,.....-,___r_y__,c_,-,·-~--____'** 75', ·-",..-,......,---,.1""""1-r,-""1-',-,- , I 

### o 7 10 141821252832353942464953565963 daiS**~'Q** ( C ) 110, 106
.

o 710 141821252832.353942464953565963 days Figure 2. Percentage change in oxygen saturation (A), pH (B), and conductivity (C) in 1,000 L enclosures during the test period ofthe mesocosm experiment. Values are expressed as percentages ofthe values measured on day O. (study figure 2)18,2 pgii --11-- 0 1.lg/1



### 39**4~** 

### 46 49 53 56 fr !l,r'jl 

# I'

'.W

- ,I 

## I \ 

# J\ 

# \

10 '4 18 21 25 28 

# /\

/ I 

## I \ 

# \ I 

# !

# f----,='--~..._,___,__.__~--r-----:-.J 300 - 50J 

### -1 

# i

day.;; Figure 3. Phytoplankton celJ density in 1,000 L enclosures between day 10 and 56 after the initial application ofatrazine. (study figure 3)

CryplO,T.On3S marsoni

.. + erusa/ova:a (celift!1 x 10:::0) 

### 180,r-------------------, 160
. 

# 140j. 120 100



### 80 60- 40- 20**o-+~,.. ~1!!!~~~--=~** 

### ......**....:::~~;;:I~~~~::::;..-n---;-i.~** 10 1J 18 21 25 28 32 35 39 42 46 49 53 56 _._IJ- 0 fJ9/1 

# ----.- 5 p;Jii 

# -'- '0 1J9/i -+.- 22**jJ~/**

_. 68 jJg./l --4- 182 1-19/1**~".-** 318 p-g/I L

C;JVS Figure4. Population density ofCryptomonas marsonii+ Cryptomonas erosa/ovata in 1,000 L enclosures between day 10 and 56 afterthe initial application ofatrazine. (study figure 4)



### 6**Ma/,icmor.f1{~** 

# sr. (celis!'l xWOO} 1BO.-----------------------. 160 140 120 100



### 80 60 40 

### 20 

### 10 14 lU 21 25 28 32 35 39 42 46 49 53 56 C".;.'"1ys _a_- 0 

### pg/i 

### -+- 5 pg'J**--.-.~** 1Q wg/I -+- 

### 22 iJ9/1 

### 68 IJgil



# -- 182 lJg/i 318 pg/l

Figure 5. Population density ofMallomonas sp. in 1,000 L enclosures between day 10 and 56 after the initial application ofatrazine. (study figure 5)

DaphniaJcngispina, egg ratio



### 1.4 1,2
0,8
0,4
{) 3 7 1014 1821 25283235394246495356 dai'S -II- 

### o1)9/1 -+- 5 pg/I 10**~,g/l** -+- 22 IJgil -&- 68 119/1

-.. 

### 182**~g/I** -.,,;-- 318 I-lg/l

Figure 6. Egg ratio ofDaphnia longispina in 1,000 L enclosures between day 10 and 56 after the initial application ofatrazine. (study figure 6)**COP0C(;(j~i** naLplii {lnd/1) -It- 

## opgil



# ---- 5 pg/i 

## -..-, 10 --+-. 22
--+:-:-- 

### 68 pg/l**-.:I>.~** 182 U**~;!/l ~--**  ">;8**~Jq/J** i ") i OW { 

## , \

I 

## I**.~** 

# .. :3 7 10 14. 1821 25**2~** 32 353942 ·.6 495:3 56 40 

### 80 160 ...,...-------------...--.-.....**~-----.~-,** 160

Figure 7. Copepoda nauplii in 1,000 L enclosures during the test period ofthe mesocosm experiment. (study figure 7)

Poiy,'ilthra

### Sp. {lndl1l 2000-,----------------------- 1800- 1600
.. 1400 1200 1000-- 800 600
.. 

### 400

# 2°~.L.,....._~;~t;~~f!~~~;~ijt~ 22 pg/!

•82 fJg/1 318 ug/I



## o 3 7 '\0 14 18 21 25 2.8 32 35 39 42 ·:6 49 53 56 d,;'yS Figure 8. Polyarthra sp. in 1,000 L enclosures during the test period ofthe mesocosm experiment. (study figure 8)
8Table 1. Effects ofconcentrations ofatrazine on phytoplankton and zooplankton species in pond mesocosms. (study table 1)**---------------~~----** Effectconcentration Diatoms Cryptomonas marsonii Cryptomonas erosa/ovata Cryptomonas rostratiformis Planktosphaeria sp. Trachelomonas sp. Mal/omonas sp. Daphnia longispina egg ratio numberof juveniles number ofadults Copepoda nauplii Rotifers Synchaeta sp. Polyarthra sp. most numerous algae seasonal blooms prevented seasonal blooms prevented absent seasonal blooms prevented relative abundance highest seasonal blooms prevented delayed peak; smaller delayed peak delayed peak low numbers seasonal decline earlier; low numbers low numbers 182,318 182,318 182,3/8 318 182,318 182,318 182,318 318 318 318 68, 182,318 182,318 318


### Data Summary Table: Magnitude Nominal Day of Time Mean Did Comments of

Effect Atrazine Study Weighted Measured Recovery relative to Concentration Effect Average Concentratio Occur- if controls (Ilg/ L

) Occurred Atrazine nforStudy so, when Concentration Duration (see from Day 0to comment)

When Effect ? 

## I

# ~~ flg/L Occurred 

## I No adverse NA 8 /lg/L 5 N/A 1 effect

No adverse 10 effect 10 /lg/L NA ] 1/lg/L N/A (increase Decline in occurred 

# :=-1 Magnitudes No adverse 22 rotifers effect estimated (Polyarthra sp.) (increase 25 flg/L NA 22 flg/L N/A from Fig. 8. 

## I 68 Yes

- Day 

## I 18 I NA

## ~=l Decrease in dissolved oxygen (% of pretreatment level)? Weighted Average Atrazine Concentration from Day 0 to Effect byapprox.

63**~lg/L** 30%

Magnitudes

### ,-=-~----t-----+_

# ...__..**--i--:""'::::'-_-~_.:-==-----~** ..

### ----L21~ 

# ..__.J estimated from Fig. 2 (C) Increase (% of pretreatment level)

Decrease in Mallomonas sp. (no seasonal bloom)

Decrease in Cryptomonas spp. (no

10**r--=-=---·.·.--.--::-----:---,----~·:----.----·...,-----··-.._:__::__···---:__:~c---r·__=.-.----** Effect Magnitude Nominal Day of Time Mean Did rCiOlnm(mts of Effect Atrazine Study Weighted Measured Recovery relative to Concentration Effect Average Concentratio Occur

- 

### if controls (p.g/L) Occurred Atrazine nforStudy so, when Concentration Duration (see from Day 0to comment)

If'henEffect ? Occurred 100% 200 !!g/L Day 32 194 llg/L 182 No f--. r:l--c:-:0::-OtX-=.o

__+.}=:-16::...:...L0f..l9g!
..:L.:::

..._--t-=..:;;;LDay-=.3.:::c.2**---+-:~_L_--t-3::-18** ........., N_
... o

__----oI

_ NR 5 [lg/L NA NA 5 NA i NR 10 J..lg/L i NA NA 10 NA i

### r~ Day 18

10 5
1 

## 10 i I 

# I /lg/L

Day Day 35 Day 35

75/lgfL 

## \10 IlgfL I

10% ,200 /lgfL +10% (increase) +8% (increase) 22

### i~ Day Magnitudes f------t------.----I-----+-------f-------t-----.---i estimated 68

### i~ Day from Fig. 6. 182 Yi Tt.,u 39

- L/UJ Increase in diatoms and Trachelomonas sp. relative abundances 

### *

Decrease in Daphnia**longi~pina** reproduction 

### *

50% 360 /lg/L 11 /lg!L 318

### i~ Day

Decrease in copepod nauplii 

### * NR 5..Mc.=fL:.........._-+-:N:..;.:A:...:.-._+.:.N,:.::-I;A=-- -+--=S -+.:..N.:.:A:..........---j NR 10 I-igfL NA NA 10 NA Decrease in**f------~---'---'-'----+** -----t----'------I------t- 

## I

. NR 25 lJ.g/L NA I NA 22 NA Planktosphaeri**~.=---::.-...----+..c...:...=<i'--:=-----+'-~--+--..:..:..----++------+...** :"""""--1 I NR 75 Ilg/L NA NA 68 NA 3 a sp. (no f--------I---'---'-'----I-----+ I. Textp. 4 8. , "much 182**~easonaI** bloom) 

## I lower" I 200/lg/L Day 30 194 /lg/L No 

## I 

## i

# ~::e~~' i 360 Ilg/L Day 30 298 llg/L 3 I8 I No 

# I f---------t---'--'------+-------+----+-----+------!-------I-----.- i 

## L

+300% 

# I 5 IL D 5 Yes

- Day {i!1..crease)
.

### .-l._
.._Il_g, -+_... ay 42 i +300% 

# I 10 1!L Day 10 Yes

- Day

(increase) : /lgl I 39 Magnitudes +300% 22 

## I y, Day estimated (increase) _2_S_11_g
..I.L

__--+_D_a_y_2_5_+-2
..1._Il_g.

__ /L

__---+. --+._4 _2_
.._-+ from Fig. 7. 750/<.::..0

__**--+_7.~.::../L**__-+_D_aL.Y3:._S_l-6_9_
.......... I-lg/.::..IL

__-+--6 c.S:.--. +--N
..... o_._--+ 50% 200 J.l-g/.I: Day 25 194 ug/L 182 No '-----_. ----Jl.....5._O_0/<..::.Q

__

### --'-1
.... 3_6_0-'-f..l""'g'/_L

__-'--D_a"'--Y2_5----Jl.....2_8_6-'-u-'-'--Lg!L

__.L-.3_18_.

__**--'--_N_o~.**__-'- i
.. _ Note: NR=Not Reported, NA=Not Applicable 

### Time weighted averages (TWAs) were computed from the atrazine concentration measurements presented in Figure 1. 

### Because ofthe log scale on the graph, errors in the visual estimations are likely. Daily chemographs (day 0-35) were constructed assuming lineartransitions between measurementpoints, and time-weighted averages for days 0-35
11

### were constructed as cumulative averages ofthe estimated daily concentrations. See linked Excel spreadsheet, below, for the resulting TWAs. 

### (Othercalculations and comparisons are within the spreadsheet.)
TWAs effect days Day 5 ug/L 10 ug/L 25 ug/L 75 ug/L 200 ug/L 360 ug/L 0 10 13 22 66 213 307 1 10 12 22 65 212 313 2 10 12 22 65 210 320 3 9 12 22 65 208 327 4 9 12 22 64 207 333 5 8 11 22 64 205 340 6 8 11 22 63 203 347 7 8 11 22 63 202 353 8 7 11 22 63 201 356 9 7 10 21 62 200 355 10 7 10 21 62 199 353 11 7 10 21 62 199 349 12 7 10 21 62 199 343 13 6 10 21 62 199 337 14 6 10 21 62 199 330 15 6 10 21 61 199 323 16 6 10 21 61 199 317 17 6 10 21 61 198 311 18 6 10 21 60 198 306 19 6 10 21 60 197 301 20 6 10 21 60 196 296 21 6 10 21 59 196 291 22 6 10 21 59 195 288 23 6 10 21 59 195 286 24 6 10 21 60 194 285 25 5 10 21 61 194 286 26 5 10 21 61 194 287 27 5 10 21 63 194 290 28 5 10 21 64 194 293 29 5 10 22 65 194 296 30 5 10 22 66 194 298 31 5 10 22 67 194 301 32 5 10 22 67 194 304 33 5 10 22 68 194 306 34 5 10 22 68 194 308 35 5 10 22 69 194 311

12

### Reviewer Comments: This study examined a wide range ofendpoints and atrazine concentrations. However, fundamental problems with the experiment design, as well as problems in the presentation ofthe results, limit the study's utility. The experimental design is limited by the lack ofreplication as well as the use of relatively small (1.25 m diameter) cylindrical enclosures to create the mesocosms. The lack ofreplication means that no statistics can be computed to evaluate effects of variability within a treatment level. The authors state in their conclusion that "the replication oftreatments, while potentially desirable in future mesocosm studies, might not circumvent
... statistical problems [arising from non-linear seasonal changes] since population fluctuations among plankton will not necessarily be synchronous, nor similar in magnitude across all experimental units." However, because ofsuch complexities, it is preferredthat mesocosm experiments include as much replication as feasible, because otherwise it is impossible to know to what extentthe results can be generalized, even to almost exactly similar aquatic environments. The authors do, however, highlightthe importance ofthe timing ofthe atrazine exposurerelative to the seasonal blooms and declines in the various plankton communities. The enclosure effects in these relatively small cylindrical enclosures were probably substantial over the 60-day study. Primarily, the presence ofsubstrate forperiphyton growth was referenced by the authors, and they notedthatthe highest atrazine treatments had "less" growth than the others, thereby implying that the growth on all enclosures was quite noticeable. Possible effects ofthe enclosures, including indirect effects from the periphyton growth, include lackofnutrient exchange, decreased light availability, and edge effects on arange ofvariables such as temperature and zooplankton density. Finally, the study results were very briefly and incompletely presented considering the large numberofendpoints examined (see Table 1). Not all the endpoints in Table 1 had data or figures provided for them. At one point, the authors mention changes in the Shannon-Weaverdiversity index, H', but no data are provided; also, no data areprovided on Trachelomonas sp. ordiatoms, which apparently increased in the highertreatment enclosures. The Data Summary Table could not be completely populated due to these deficiencies. Also, there was an apparent discrepancy in the discussion ofconductivity results; the graph shows conductivity growing in the control and lowesttreatment enclosures, butthe text describes these as showing the largestdecrease. It 

### was assumed that the figure was correct and the text was incorrect. Description ofUse in Document (QUAL, QUAN, INV): INV 

### ]3

### Rationale for Use: The study had no replication and its observed treatment effects could not be generalized. Also, the presence ofatrazine was not evaluated in controls, use ofa solvent was not reported and chemical purity was not reported. 

### Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc 

### Secondary Reviewer: Brian Anderson, U.S. EPA

14