Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Brockway, D.L., P.D. Smith, and F.E. Stancil. IYM. Fate and effects of atrazine on

small aquatic microcosms. Bull. Environ. Contam. Toxicol. 32:345-353. (MRID

47543602).

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:

December 1l, 2008

Purpose of Study:

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

plankton plus AufWuchs) in both static and continuous-flow microcosms.

Summary of Study Findings:

Methods: Two sets of experiments were conducted: one in continuous-flow aquaria, and

one in static beakers. Atrazine of98.2% purity was obtained from the Reference

Standards Repository, USEPA, Research Triangle Park, North Carolina. Analyses used

high performance liquid chromatography, a 2% water-methanol mobile phase, a

Whatman Partisil ODS-2 column, and a variable wavelength UV-VIS detector at 222 nm.

Continuous-flow experiments

Continuous-flow experiments occurred in 12 seamless glass aquaria, 20 em x 30 em x 18

em, in a temperature-controlled room at 22°C. Light sources were daylight-balanced

high intensity fluorescent lamps with intensity 6500±500 lux, on a 12 hours on, 12 hours

off photoperiod. Microcosms were initialized with a 0.5-cm layer of pond sediments, 7 L

of pond water, and "an inoculum" (size/concentration not described) of net plankton plus

AufWuchs from a mesotrophic, soft water, 2 ha pond (location, land use, etc. not

described). Microcosms also received weekly additional inocula for the duration ofthe

1811.11111

2087801
49-week experiment. Freeman's standard reference water was added through a glass and

stainless steel system at a rate of 0.6 mL/min, giving an 8.1 day retention time in the

microcosms. Microcosms were continuously mixed by chain-driven steel stirrers at 50

rpm.

Twelve microcosms were organized into 4 sets of 3 containers, each set on a separate

nutrient feed system. After allowing 39 weeks for microcosms to develop, atrazine ,,' C'A/ '_ 1\ At"' t"' l .... A __ ~{T . " 1~ __1 .• __ 4.-L ..Ln~ __ L ~_l

COfl(.:eruraUOIlS 01 v COIlLIV1), V . .J, .J, eUlU .JV Ilg1 L Wt::lt:: ellJVIIC;U l/LU lilt:: IIUll lC;[1l 1>UVVlY

systems for the four respective sets. The microcosms were not spiked, so the

concentration increased gradually. For acclimation tests, concentrated spikes were added

to each microcosm directly. For resilience tests, 50% ofthe nutrient medium was

removed and the tank refilled with uncontaminated nutrient medium on two successive

days, and the normal nutrient input was switched to uncontaminated medium.

Treatments were as follows:

Treatment 1: 5 ug/L

Treatment 2: 50 ug/L (Day 0 to 55) + 0 ug/L (removal of atrazine from the system at Day

56) + 50 ug/L (Day 63)

Treatment 3: 0.5 ug/L (Days 0 to 28) + 50 ug/L (Day 29) + 100 ug/L (Day 35) + 0 ug/L

(removal of atrazine from the system at Day 50) + 100 ug/L (Day 63)

Six parameters were monitored in the continuous-flow experiments. Dissolved oxygen

(DO) and pH were measured before and after the 12-hour light period, using an

Orbisphere DO meter and an Extech Model 609 digital pH meter. Total dissolved

phosphorus, nitrate, nitrite, and ammonia concentrations were determined weekly, on the

same day and at the same time (1 hour after lights-on), using a Technicon Auto Analyzer

II (following USEPA, 1979, Methods for chemical analysis ofwater and wastes).

Static Experiments

Static experiments used l-L glass beakers containing 600 mL ofthe nutrient medium.

The beakers were inoculated with 2-g aliquots (wet weight) of mixed biota from I-yearold laboratory flow systems established for stock purposes. The inoculum was

dominated by Spirogyra sp., and also included Oedogonium, Microcystitis, Apthanothece,

Scenedesmus, other algal species, flagellate protozoans, and nematodes. Temperature,

lighting, and DO and pH monitoring proceeded as in the continuous-flow experiments.

Four replicates each at atrazine treatment levels of 0 (control), 50, 500, and 5,000 )lg/L

were tested for 12 days in the static experiments, with one set ofreplicates sacrificed on

days 1 and 7. A I-replicate abiotic control was also included. A second experiment used

3 replicates at each atrazine treatment levels of 0 (control), 0.5, 5, and 100 )lg/L and

lasted for 7 days.

2
Results:

Measured atrazine concentrations were close to nominal and stable in the continuousflow systems (Figure 1), with the static tests showing slightly more variability (results not

described).

In the static experiments, net DO production during the day was similar to controls in the

atrazine treatment levels of 0.5 and 5 Ilg/L, decreased by 25-30% in the 50 Ilg/L

treatments, decreased 40-50% in the 100 Ilg/L treatments, decreased 90% in the 500 Ilg/L

treatments, and went to zero or negative production in the 5,000 Ilg/L treatments (see

Figure 2). Within-treatment variations were very low (coefficients of variation or CYs

were typically below 7%). (No significance testing results were provided.) Biotic

community composition was similar in all treatments, and appeared healthy at and below

atrazine treatments of 500 Ilg/L, although after 12 days at 5,000 Ilg/L the algae were

totally bleached (viable rotifers and nematodes were still present).

In the continuous-flow experiments, during the pretreatment microcosm development

phase, net DO production during the day increased from about 4 mg/L in week 1 to 11

mg/L during week 14, leveled off until week 24, and then slowly declined until the end of

the pretreatment phase. After atrazine treatment commenced, oxygen production (as %

of control production) fluctuated in response to various treatments, spike, and dilutions

(see Figure 3). The 5 Ilg/L constant treatment appeared to eventually recover (around

day 50 oftreatment) with respect to the net diurnal DO production, whereas the more

complex treatment regimes showed clear drops immediately after atrazine spikes were

added and sudden rises immediately after dilutions occurred, apparently regardless of

previous exposures. Within-treatment CYs were very good, typically below 19%, though

controls varied by ± 10% during a period ofseveral days, so treatment effects were not

judged as qualitatively significant (no statistical significance test results provided).

Ofthe other chemical parameters measured, only dissolved nitrate changed significantly

after atrazine treatment. Nitrate concentrations increased significantly when Treatment 3

was spiked to 100 Ilg/L (Student's t-test, p-value < 0.01). Dilutions were similarly

marked by decreases in nitrate (no statistics provided). See Figure 4. The authors

calculated that the nitrate decrease was what could be expected from the decrease in

nitrate utilization caused by inhibition of photosynthesis, as estimated from DO changes.

Microscopic observations of biota throughout the experiments showed similar

composition through all times and treatment levels. Bluegreen algae dominated, green

algae and diatoms were also abundant, and copepods, ostracods, and annelids were also

present.

3
..t.

.,.e-&" -a- -l!f - .ffi- e -e- ~--aK

%

I

I

5 m JlIi - -J;. "i( - ~- -iT .-~ .....-~ ...-' * }( ......¥ / + .<!> ,~ - 5 ).Lg/L Input

1+ r.J I I - 50 1J.g/L Input

I - - .- - - Calculated 10"(..,L..--.,--r--..,.----.-----...----,-,.-.----,---=:..:::,1,::..:::::.:::..:..;.:::..-

10 20 30 40 50

Time During Treatment Phase (days)

Figure 1. Measured and calculated atrazine concentrations in continuous-flow microcosms.

1 2 3 4 5 6 7

Time (days) Time (days)

500 J..Li/L ..-.. [!] Control 4:l. 5.0 fJ.g/L .~

*5000 p..g/L ,.q C) 0.5 p,g/L*100 J.l.g/L

N 20

.,....j 18

~16

"---14

tt01Z 510

(l) 8

on6

Q 4 -it. ;1:..'_ ....t

~------~ -----.....t.J. 2 .........

U 0 -"--..---.---.--.--..--.--

1 2 3 4 5 6 7 8 9 101112 0

Q

..-.. Q] Control l! C9 50 J.'g/L

~121

~10

~8

on

S 6 ........"

0) 4-

0.0

2

ro

..c: 0

u

o

Q

Figure 2. Net diurnal dissolved oxygen (DO) production in the two static tests.

4
\\

~~/~

tt tt t

/

~~~I

15 20 25 30 35 40 45 50 55 60

Time During Treatment Phase (days)

o 5 10

130

120

110

100

90

-+oJ' 80

Q

w 70

()

60

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50

40 -I • Control t

30 -{ .... Trea.tment 1

CJ Treatment Z t 3 4 S r I

ZO-i (,)Treatment 3 1 Z T I I I I ds 70 75 10-{ I I 1 r o I I I I j

Figure 3. Net diurnal dissolved oxygen production, as percentage of controls, during the continuous-flow experiments. Treatment 1: 5 I!g/L; Treatment

2: 50, then 0, then 50 I!g/L; Treatment 3: 0.5, then 50, then 100, then 0, then 100 I!g/L. Timing of Treatment 3 spikes given by Arrows 1,2, and 5; of

Treatment 3 dilution given by Arrow 3. Timing of Treatment 2 dilution given by Arrow 4 and Treatment 2 spike given by Arrow 5. Each point is the

mean of 3 replicates.

5
CD

I

I

I

I

I

Pretreatment

0.04

2 0.08

tU

l-l

..,.>

• r-l

Z

O.

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Control

t.t. Treatment 1

Treatment 2

...-. 0.20 C} Treatment 3

....,""

........... S 0,16

(l)

tl.Oe 0.12

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Figure 4. Dissolved nitrate concentrations as mg NIL before and after atrazine treatment.

Treatment 1: 5 Ilg/L; Treatment 2: 50, then 0, then 50 /-lg/L; Treatment 3: 0.5, then 50, then 100, then

0, then 100 /-lg/L. Timing of Treatment 3 spikes given by Arrows 1,2, and 5; of Treatment 3 dilution

given by Arrow 3. Timing of Treatment 2 dilution given by Arrow 4 and Treatment 2 spike given by

Arrow 5. Each point is the mean of 3 replicates.

6
Data Summary Table: Note that all endpoints appear in Attachment 2, but with

misleading Test Cone, Results, Plant Group, and Recovery information.

Effect Magnitude Nominal Day of Time Did Recovery Comments

of Effect Atrazine Study Weighted Occur - if so,

relative to Concentration Effect Average when (see

controls (llg/L) Occurred Atrazine comment)?

Concentration

from Day 0 to

When Effect

Occurred

no effect 0.5 J-lg/L NA NA/variable NA

Decrease in Continuous-flow

20% 5 J-lg/L Day 30 3.8 J-lg/L Yes - Day 48 experiments with

net diurnal Partial by Day time-varying dissolved 30% 50 J-lg/L Day 30 33.5 J-lg/L 55 treatments.

oxygen Day] Magnitudes estimated production No (>]5 days 40% ]00 J-lg/L after spike 11.4 J-lg/L after spike)

from Fig. 3.

treatment

no effect 0.5 J-lg/L NA NA/variable NA

no effect 5 J-lg/L NA NA/variable NA Continuous-flow

Increase in 50% 50 J-lg/L Day] 8(?) 23.4 J-lg/L No (>40 days) experiments with

dissolved time-varying

nitrate 65% treatments.

concentration (different Day I(?) No (>15 days Magnitudes estimated

from 100 J-lg/L after spike I ].4 J-lg/L after spike) from Fig. 4.

control at treatment

p=O.Ol)

no effect 0.5 J-lg/L NA 0.5 J-lg/L NA

Decrease in no effect 5 J-lg/L NA 5 J-lg/L NA

net diurnal 25% 50 J-lg/L Day] 50 J-lg/L No (>12 days) Static experiments.

dissolved Magnitudes estimated

oxygen 40% 100 ).1g/L Day I ]00 J-lg/L No (>7 days) from Fig. 2.

production 90% 500 J-lg/L Day] 500 J-lg/L No (>12 days)

>100% 5,000 J-lg/L Day 1 5,000 ).1g/L No (>12 days)

Note: NA=Not Applicable

No data on atrazine concentrations for the static experiments were provided, so for those

experiments, time-weighted average atrazine concentration was assumed to be nominal.

For the continuous-flow experiments, atrazine concentrations (as shown in Figure 1)

slowly increased over the first 20 days ofthe experiment. Additionally, calculation of

time-weighted averages was complicated by the time-varying treatment regimes for

Treatment 2 (50 Ilg/L, then 0 Ilg/L, then 50 Ilg/L) and Treatment 3 (0.5 Ilg/L, then 50

Ilg/L, then 100 Ilg/L, then 0 Ilg/L, then 100 Ilg/L). Chemographs were determined from a

combination of Figure 1, assuming concentrations equal to nominal (assuming nominal

spike or dilution concentrations) after Day 20, and the timing ofspikes and dilutions (i.e.

from Figure 3 arrows), and then running averages were computed. For simplicity, linear

increases between data points on Figure 1 and zero concentrations on Day 0 were

assumed for creation of chemographs, and it was assumed that the nominal 0.5 Ilg/L

7
treatment (Treatment 3) followed the pattern of Treatment 1 for the first 20 days, with

concentrations at l/lOth the level, since these data were not provided in Figure 1. Timeweighted averages are computed for all time elapsed since Day 0 ofthe treatment phase.

The authors found the latter was a better determiner ofthe effects they saw. See the

linked Excel spreadsheet below. (It continues beyond the end ofthe page to cover the

whole 75-day interval.)

8
Treatment 1 Treatment 2 Treatment 3

Day Cone. TWA Cone. TWA Cone. TWA

0 0.0 0.0 0.0 0.0 0.0 0.0

1 0.4 0.2 1.4 0.7 0.0 0.0

2 0.8 0.4 2.8 1.4 0.1 0.0

3 1.1 0.6 4.1 2.1 0.1 0.1

4 1.5 0.8 5.5 2.8 0.2 0.1

5 2.0 1.0 10.3 4.0 0.2 0.1

6 2.5 1.2 15.2 5.6 0.3 0.1

7 3.0 i.4 20.0 7.4 0.3 O.-i

8 3.3 1.6 22.5 9.1 0.3 0.2

9 3.5 1.8 25.0 10.7 0.4 0.2

10 3.8 2.0 27.5 12.2 0.4 0.2

11 4.0 2.1 30.0 13.7 0.4 0.2

12 4.2 2.3 33.3 15.2 0.4 0.2

13 4.3 2.4 36.7 16.7 0.4 0.2

14 4.5 2.6 40.0 18.3 0.5 0.3

15 4.6 2.7 40.8 19.7 0.5 0.3

16 4.8 2.8 41.6 21.0 0.5 0.3

17 4.9 2.9 42.4 22.2 0.5 0.3

18 5.0 3.1 45.0 23.4 0.5 0.3

19 5.0 3.2 46.7 24.5 0.5 0.3

20 5.0 3.2 48.3 25.7 0.5 0.3

21 5.0 3.3 50.0 26.8 0.5 0.3

22 5.0 3.4 50.0 27.8 0.5 0.3

23 5.0 3.5 50.0 28.7 0.5 0.3

24 5.0 3.5 50.0 29.6 0.5 0.4

25 5.0 3.6 50.0 30.3 0.5 0.4

26 5.0 3.6 50.0 31.1 0.5 0.4

27 5.0 3.7 50.0 31.8 0.5 0.4

28 5.0 3.7 50.0 32.4 0.5 0.4

29 5.0 3.8 50.0 33.0 50.0 2.0

30 5.0 3.8 50.0 33.5 50.0 3.6

31 5.0 3.8 50.0 34.0 50.0 5.0

32 5..0 3.9 50.0 34.5 50.0 6.4

33 5.0 3.9 50.0 35.0 50.0 7.7

34 5.0 3.9 50.0 35.4 50.0 8.9

35 5.0 4.0 50.0 35.8 100.0 11.4

36 5.0 4.0 50.0 36.2 100.0 13.8

37 5.0 4.0 50.0 36.6 100.0 16.1

38 5.0 4.1 50.0 36.9 100.0 18.2

39 5.0 4.1 50.0 37.2 100.0 20.3

40 5.0 4.1 50.0 37.5 100.0 22.2

41 5.0 4.1 50.0 37.8 100.0 24.1

42 5.0 4.1 50.0 38.1 100.0 25.8

43 5.0 4.2 50.0 38.4 100.0 27.5

44 5.0 4.2 50.0 38.6 100.0 29.1

45 5.0 4.2 50.0 38.9 100.0 30.7

46 5.0 4.2 50.0 39.1 100.0 32.1

47 5.0 4.2 50.0 39.4 100.0 33.6

48 5.0 4.2 50.0 39.6 100.0 34.9

49 5.0 4.3 50.0 39.8 100.0 36.2

50 5.0 4.3 50.0 40.0 0.0 35.5

51 5.0 4.3 50.0 40.2 0.0 34.8

52 5.0 4.3 50.0 9 40.4 0.0 34.2

53 5.0 4.3 50.0 40.5 0.0 33.5

54 5.0 4.3 50.0 40.7 0.0 32.9
Reviewer Comments:

The continuous-flow study data were difficult to interpret within the framework oftimeweighted averages, days to recovery, etc., due to the time-varying nature oftwo ofthe

three treatment regimes. Additionally, statistical significance tests were not conducted

for the vast majority ofthe variables measured, so despite the reasonably good replication

it is not clear how significant are most ofthe results.

Microcosms, with simplified biota communities, were the subject ofstudy. The source

pond for the continuous-flow biota was not well described. Biota consisted only of drift

(net) plankton and Aufwuchs that could grow from an inoculum. Microcosms were given

a reasonable (nearly 40 week) amount oftime to acclimate to the laboratory and grow to

steady-state, however, they remained simple (consisting of mainly algae with some small

animals). Re-introduction of biota during weekly inoculum additions may have unduly

affected results as well.

Description of Use in Document (QUAL, QUAN, INV): QUAL

Rationale for Use: Study contributes to weight of evidence regarding potential atrazine

effects to aquatic communities.

Limitations of Study: Data Summary Table must be interpreted with care due to timevarying nature of continuous-flow atrazine treatments. Statistical tests were not

performed for most data, and raw data are not provided. Microcosms, though the biota

were natural in origin, were very artificial, and results may not be applicable to more

complex natural ecosystems.

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

Secondary Reviewer: Brian Anderson

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