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 of atrazine on a natural plankton

community. Arch. Environ. Contam. ToxicoJ. 29:435-441. (Vl1j(/O l-ffs-if3,'!tD'S;)

Purpose of Review:

Determine potential effects of atrazine 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 of Review:

January 15, 2009

Purpose of Study:

This study examines the potential effects of atrazine to aquatic plants and animals in

experimental pond cylindrical compartments (mesocosms).

Summary of Study Findings:

Methods:

An experimental pond, 15 m

2 and 0.8 m deep in a small deciduous forest in southern

Bavaria, Germany, was compartmentalized by seven transparent PVC cylinders (1.6 m

high, 1.25 m diameter, of volume about 1,000 L), pressed 20 cm into the sediment to

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 over the two month study period

(Figure 1). Water samples were collected weekly for atrazine analysis, pre-concentrated

from volumes of 5 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

2087854
Temperature, 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 of a 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 of a 100 mL subsample for

phytoplankton enumeration. The subsamples were preserved in acid Lugol's and counted

by the Utermoehl method. For zooplankton 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 of eggs per female (egg ratio), and juveniles were counted for

the major cladoceran Daphnia longispina. For cyclopoids, 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 September remained 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 that periphyton was a potentially significant contributor to

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 of 50 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 higher dosage 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-linear trends and fluctuations over time

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 lower than any other treatments throughout the entire test perio

(Figure 3). The Shannon-Weiner diversity index, H', was lower at 182 and 318 Ilg/L

than in other treatments because ofsmaller numbers ofspecies and more variability

(inequitability) in their relative abundance over the second half ofthe test period (no

figures or data 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

throughout the test period. The authors cite single-species toxicity tests showing that

several diatoms (Bacillariophyceae) can tolerate higher atrazine levels that taxa from

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 lower cell numbers

after day 30 at 182 and 318 Jlg/L than at lower atrazine concentrations (no figures or data

provided).

Daphnia longispina total numbers declined in all enclosures after atrazine application,

and egg ratios also declined from days 3-21, increasing after day 21 to drive increases in

numbers ofjuvenile and adult daphnids. At 318 Jlg/L, the peak egg ratio was lower than

in other enclosures and also more delayed in its arrival (Figure 6), which the author notes

may reflect indirect effects of food 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 of atrazine 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 other concentrations, and subsequent densities remained lower (Table

I). Polyarthra sp. declined in abundance earlier at 318 Jlg/L than at other concentrations,

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 of the mesocosm

experiment. (study figure 1)

4
pH

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

of the 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 of atrazine. (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

Figure 4. Population density of Cryptomonas marsonii + Cryptomonas erosa/ovata in 1,000 L

enclosures between day 10 and 56 after the initial application of atrazine. (study figure 4)

6
Ma/,icmor.f1{~ sr. (celis!'l x WOO}

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 of Mallomonas sp. in 1,000 L enclosures between day 10 and 56 after

the initial application of atrazine. (study figure 5)

Daphnia Jcngispina, 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 of Daphnia longispina in 1,000 L enclosures between day 10 and 56 after the

initial application of atrazine. (study figure 6)
COP0C(;(j~i naLplii {lnd/1)

-It- o pgil

---- 5 pg/i

-..-, 10

--+-. 22

--+:-:-- 68 pg/l

-.:I>.~ 182 U~;!/l

~-- ">;8 i ") i OW ~Jq/J { , \

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 of the 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 of the mesocosm experiment.

(study figure 8)

8
Table 1. Effects of concentrations of atrazine on phytoplankton and zooplankton species in pond

mesocosms. (study table 1)

---------------~~----

Effect concentration

Diatoms

Cryptomonas marsonii

Cryptomonas erosa/ovata

Cryptomonas rostratiformis

Planktosphaeria sp.

Trachelomonas sp.

Mal/omonas sp.

Daphnia longispina

egg ratio

number ofjuveniles

number of adults

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

ofEffect 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 0 to 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

No :=-1 Magnitudes 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.

30% 63 ~lg/L

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

ofEffect Atrazine Study Weighted Measured Recovery

relative to Concentration Effect Average Concentratio Occur - if

controls (p.g/L) Occurred Atrazine nfor Study so, when

Concentration Duration (see

from Day 0 to comment)

If'hen Effect ?

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. Text p. 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__-+--6c.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

linear transitions between measurement points, 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. (Other calculations 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 of endpoints 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 of replication 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 of treatments, 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 of such complexities, it is

preferred that mesocosm experiments include as much replication as feasible, because

otherwise it is impossible to know to what extent the results can be generalized, even to

almost exactly similar aquatic environments. The authors do, however, highlight the

importance ofthe timing of the atrazine exposure relative 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 for periphyton

growth was referenced by the authors, and they noted that the 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 lack of nutrient exchange, decreased light availability, and

edge effects on a range of variables such as temperature and zooplankton density.

Finally, the study results were very briefly and incompletely presented considering the

large number of endpoints 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-Weaver diversity index, H', but no data are provided; also, no data are provided

on Trachelomonas sp. or diatoms, which apparently increased in the higher treatment

enclosures. The Data Summary Table could not be completely populated due to these

deficiencies. Also, there was an apparent discrepancy in the discussion of conductivity

results; the graph shows conductivity growing in the control and lowest treatment

enclosures, but the text describes these as showing the largest decrease. It was assumed

that the figure was correct and the text was incorrect.

Description of Use 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 of atrazine was not evaluated in controls, use of a

solvent was not reported and chemical purity was not reported.

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer: Brian Anderson, U.S. EPA

14