Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Fairchild, J.F., T.W. LaPoint, and T.R. Schwarz. 1994. Effects of a herbicide and

insecticide mixture in aquatic mesocosms. Arch. Environ. Contam. Toxicol. 27:527-533

(MRID 47543612).

Purpose of Review:

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:

November 24, 2009

Purpose of Study:

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

experimental ponds (mesocosms).

Summary of Study Findings:

Methods: This study examined combined effects of atrazine and a pyrethroid

insecticide, esfenvalerate, on aquatic biota. Methods relating solely to insecticide

treatment are not discussed in detail in this review summary.

Twelve mesocosms were used for the experiment, six as atrazine controls and six treated

with 50 /-lg/L atrazine on May 26 (single application) (year not provided). The

mesocosms were 0.1 ha, earthen-lined, with a mean depth of 1.5 m and volumes between

600 and 850 m

3

, and were located at the National Fisheries Contaminant Research

Center, National Biological Survey, Columbia, Missouri. Atrazine (40.8% liquid field

formulation; Shell Corporation) was applied as an aqueous slurry poured behind a boat.

All mesocosms also received 0.02 mLiL acetone, since this was the insecticide solvent; it

was injected at 0.5 m beneath the water surface using a hand sprayer behind a non-driven

boat over a 30-minute mixing period. 50 /-lg/L was selected as the atrazine treatment

level because it was expected to cause noticeable changes in vegetation, but not to cause

1••11••111

2087820
direct effects on fish and invertebrates, and also because it was seen as a common level in

runoff adjacent to intensively farmed land. The insecticide used, esfenvalerate, is a

synthetic pyrethroid with a short (hours) half-life and low perceived environmental

toxicity. The insecticide treatments were applied on May 26, the day of atrazine

application, and on June 8, 6 weeks after the first treatment, at six concentrations (from 0,

control, to 1.71 Ilg/L nominal). Given that esfenvalerate was added to five ofthe six

atrazine treatments, only one replicate pond was sampled at an atrazine concentration of

50 Ilg/L (with no added esfenvalerate). Each mesocosm thus received a unique

combination of atrazine and esfenvalerate treatments. (A table summarizing the

herbicide and insecticide treatment protocol is provided in the original article but is not

reproduced here.)

Atrazine concentrations in the mesocosms were measured 2 hours after application, and

then at 2, 7, 14, and 28 days following treatment. A single depth-integrated sample was

taken with a I-m glass tube and atrazine was immediately isolated from aIL water

sample using a C18 column (J.T. Baker Co.) with a Baker-Mizer pre-filter (J.T. Baker

Co.), once the water had been adjusted to pH 6.6 using sulfuric acid or KOH. Residues

were eluted from the column using 3 mL isopropanol followed by 3 mL hexane, then

diluted to 5 mL in hexane and analyzed with a gas chromatograph: Varian Model 3700,

EC detection, 15 m x 0.25 mm DB-5 column, J&W Scientific; H2 carrier at 0.56 kg/cm2

with N2 make-up gas at 20 mLimin, and temperature programming from 70-275°C

(increase 5°C/min, final time 5 minutes). Triplicate spikes and blanks were analyzed, and

atrazine recovery was 104% (standard deviation 16%, n=3, 12.5 /lg/L spike). Sample

values were corrected for recovery.

Water quality, nutrients, and primary productivity were measured every two weeks.

Temperature, pH, conductivity, dissolved oxygen, and oxidation-reduction potential were

measured in situ at depths of 0, 0.5, and 1 m using a Hydrolab Surveyor II water quality

monitor. Primary productivity and nutrients were analyzed from depth-integrated water

samples taken with a I-m PVC tube, with duplicate measures performed for dissolved

organic carbon, total nitrogen, total phosphorus, pH, alkalinity, and algal biomass as

chlorophyll a and particulate organic carbon (POC). (A 1987 article headed by the lead

author is cited for further methodological details.) Additionally, whole-system

measurements of gross photosynthesis, community respiration, and net primary

production were determined using the diurnal oxygen method (a 1992 article headed by

the lead author is cited for further methodological details).

Macrophyte biomass and species composition were determined from monthly samples,

four samples per mesocosm. For sampling, a stovepipe sampler (0. I m2 area) was used,

and macrophytes were removed with a rake and washed to remove sediments. Percent

species composition was visually estimated to the nearest 10%, and the macrophytes

were dried at 105°C for 24 hours and weighed to the nearest 0.1 g for dry weight

biomass. Naja sp. and Chara sp. dominated the macrophyte community.

2
Zooplankton were sampled each week with a battery-powered metered pump. Two

replicate 114-L, depth-integrated composites (38 L each from depths of 0, 0.5, and 1 m)

were taken and sieved through an 80 11m Wisconsin net, and the zooplankton stored in

99: 1 ethanol:glycerin for future identification to order or sub-order (identification method

details are not provided).

The mesocosms were stocked with bluegill (Lepomis macrochirus) on May 15. The fish

mean length was 4.18 cm, mean weight was 1.39 g, and they were stocked to achieve a

density in the pond of 0.275 g/m3

, corresponding to at least 100 fish per pond. Adults

and recruits were removed, counted, and individually weighed and measured in early

October (no exact date provided; the 1992 article is again cited for details). Condition

was calculated as w/13

x lOO, where w=weight in g and l=length in cm. Additionally, a

two-week toxicity study was conducted in situ, wherein 60 bluegill from the original

population were placed in 2 polyethylene mesh cages (30 per cage) at each mesocosm

surface for 2 weeks, and observed daily for loss of equilibrium and mortality (dates not

provided).

Since the 1992 article headed by the lead author had showed no direct effects of

esfenvalerate on algae or macrophytes, data were combined across insecticide treatments

to examine effects of atrazine on plants, and tested using non-parametric one-way

analysis of variance with the Wilcoxon Rank-Sum test on each date. P-values less than

0.05 were considered significant. Combined effects on fish and invertebrates were not

statistically analyzed.

Results: This study examined combined effects of atrazine and an insecticide,

esfenvalerate, on aquatic biota. Since the plants were apparently unaffected by the

insecticide, atrazine treatments were pooled over the insecticide treatments for

examination of plant endpoints. Results for animal endpoints, which were broken down

by insecticide treatment regime, will not be reproduced in full detail, but the results for

control levels of insecticide will be summarized.

Average measured atrazine concentrations in the 6 treatment mesocosms were equal to

nominal (50 I1g/L) 2 days after application with a standard deviation of 13 I1g/L, but

declined to 20 I1g/L after 8 weeks. No further data are provided, but the calculated halflife of atrazine in the mesocosms was 41 days (r = -0.87 with a negative exponential

model, p:::; 0.001). Water quality results (ranges over all mesocosms) were:

pH: 7.3-9.1

alkalinity as CaC03: 150-230 mg/L

conductivity: 339-510 I-tS/cm

dissolved organic carbon: 5-9 mg/L

total nitrogen: 400-842 I-tg/L

total phosphorus: 20-52 I-tg/L

3
Changes in mesocosm water quality with atrazine treatment are not discussed in the

article.

As shown in Figure 1, atrazine slightly increased algal biomass one week after

application, but then decreased relative to controls. These differences were occasionally,

though not consistently, statistically significant (p :::; 0.05): POC was significantly

reduced in treatments on 3 ofthe 9 sampling days, showing recovery in between each

reduction, and chlorophyll a was significantly reduced on only I ofthe 9 sampling days.

The two algal biomass measures, chlorophyll and POC, were highly correlated (r = 0.764,

p:::; 0.001).

Treatment with atrazine altered the composition ofthe macrophyte community, but not

the overall biomass. Prior to atrazine addition, Naja sp. comprised about 20% ofthe

macrophytes, and that share grew to 60% ofthe total macrophytes by August in the

herbicide controls. In the atrazine treatments, in contrast, Naja sp. was nearly eliminated.

Macrophyte biomass, although changing with time in all mesocosms, did not

significantly differ between controls and atrazine treatments, except for one date in early

July when the treatment mesocosms had significantly more macrophyte biomass than

controls (p:::; 0.05). See Figure 2 for plots ofthe macrophyte data. Additionally, wholesystem measures of productivity did not show significant changes due to atrazine

treatment (see Figure 3). Gross photosynthesis and community respiration were highly

correlated, resulting in an average P/R ratio around 1, even when both measures showed

significant (p :::; 0.05) decreases in treatment mesocosms 1 week after atrazine application

and again in late August. In both cases, these measures quickly recovered to again be

similar in treatment and control mesocosms.

Atrazine was not found to significantly or consistently modify the response of

zooplankton or fish to esfenvalerate. Raw data tables and figures of zooplankton and fish

responses are provided in the article but are not reproduced in full here. The data at

control levels of esfenvalerate (mean followed by standard deviation in parentheses, two

replicates from each mesocosm, C=control and T=atrazine treatment) are as follows:

# of crustacean zooplankton per liter, 2 replicate water samples: (study table 2-3)

May 21 (pre-treatment) C: 74 (74), T: I (I)

May 28 C: 265 (149), T: 60 (13)

J1.j.ne 4 C: 66 (4), T: 79 (14)

July 2 C: 70 (3), T: 10 (3)

July 10 C: 9 (2), T: 7 (7)

July 16 C:4(2),T:3(1)

% mortality of bluegill after 48 hours, 2 replicate cages (30 fish per cage): (study table 4)

May 26 C: 2 (2), T: 0

July 8 C: 2 (2), T: 0

As estimated from the study's Figure 4 (not reproduced here), the average bluegill

recruitment (#/adult) was 39 in atrazine controls, and 109 in atrazine treatments

4
(insecticide controls only), while the recruit condition (mean of 100 fish) was roughly

equal between atrazine controls and treatments (insecticide controls only).

June JUly Au·gL.sl Sept

0

12,5CO

10,000

::J-.en

2- 7,50::1

(J

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2,5GO

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May

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en

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0 120 T

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Date

Figure 1. Chlorophyll a and particulate organic carbon (POC) of phytoplankton in mesocosms with

and without atrazine. Each point represents the mean (± standard deviation) of n=6 mesocosms.

Arrows denote treatment dates (atrazine was only applied on the first treatment date). Asterisks

denote significant differences between herbicide treatments at p :s 0.05.

5
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D 125 .1 ' as

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0

MaV Juno

Date

Figure 2. Species composition and total biomass of macrophytes in mesocosms with and without

atrazine. Each point represents the mean (± standard deviation) of n=6 mesocosms. Arrows denote

treatment dates (atrazine was only applied on the first treatment date). Asterisks denote significant

differences between herbicide treatments at p 0.05.

6
Aug

.......- C'/.o3:~ AL-azlnll

..... 50 ,IJ!JI1.Io~nt.zI-9

June July

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Date

Figure 3. Total system metabolism (gross photosynthesis, community respiration, and P/R ratio) in

mesocosms with and without atrazine. Each point represents the mean (± standard deviation) of n=6

mesocosms. Arrows denote treatment dates (atrazine was only applied on the first treatment date).

Asterisks denote significant differences between herbicide treatments at p :5 0.05.

7
Data Summary Table:

Effect Magnitude of Nominal Day of Time Did Comments

Effect Atrazine Study Weighted Recovery

relative to Concentratio b1fecl Average Occur- if

controls n (fig/I) Occurred Atrazine so, when

Concentratio (.~ee

nfrom Day () comment)

to When ?

Effect

Occurred

Increase in % no (>105 Magnitude

Chara sp. (vs. 240% 50 J.lg/L ~80 23.2 J.lg/L days) estimated from

Naja sP.) Fig. 2.

Total biomass From text p. of No effect 50 J.lg/L NA NA NA

macrophytes 529 and Fig. 2

No From Fig. 1.

consistent The few days

effect with (p<0.05)

(initial 100% statistically

Algal biomass increase, 50 J.lg/L NA NA NA significant

effects all followed by showed occasional

recovery by transient

reductions) the next

sampling day.

Recovery

System noted on next

metabolism sampling day.

(gross 25% 50 J.lg/L 2 51.2 J.lg/L Yes (Day P/R remained

photosynthesis decrease 9) around 1.

, community Magnitudes

respiration) estimated from

Fig. 3.

Zooplankton No effect 50 J.lg/L NA NA NA From text p.

530

Fish No effect 50 J.lg/L NA NA NA From text p.

530

Note: NR=Not Reported, NA=Not ApplIcable,

The time weighted averages are calculated using the given information in the study, that

the half-life of atrazine in the mesocosms was 41 days, and that concentration on Day 2

was 50 flg/L. Unfortunately, the modeled chemograph does not match the other

information given, that the concentration after 8 weeks was 20 flg/L (the model predicts a

concentration of about 13 flg/L at Day 56), but it was used anyway because it provided a

complete chemograph. Daily modeled concentrations were cumulatively averaged over

all 80 days to derive time weighted averages. See calculations below and in the linked

Excel spreadsheet.

Day Data

Calc.

cone. TWA

8
0 52.5 52.5 1 51.2 51.9 2 50 50.0 51.2 3 48.8 50.6 4 47.6 50.0 5 46.5 49.4 6 45.4 48.9 7 44.3 48.3 8 43.2 47.7 9 42.2 47.2

10 41.1 46.6

11 40.1 46.1

12 39.2 45.5

13 38.2 45.0

14 37.3 44.5

15 36.4 44.0

16 35.5 43.5

17 34.7 43.0

18 33.8 42.5

19 33.0 42.1

20 32.2 41.6

21 31.5 41.1

22 30.7 40.7

23 30.0 40.2

24 29.2 39.8

25 28.5 39.4

26 27.8 38.9

27 27.2 38.5

28 26.5 38.1

29 25.9 37.7

30 25.3 37.3

31 24.6 36.9

32 24.1 36.5

33 23.5 36.1

34 22.9 35.7

35 22.4 35.4

36 21.8 35.0

37 21.3 34.6

38 20.8 34.3

39 20.3 33.9

40 19.8 33.6

41 19.3 33.3

42 18.8 32.9

43 18.4 32.6

44 18.0 32.3

45 17.5 31.9

46 17.1 31.6

47 16.7 31.3

48 16.3 31.0

49 15.9 30.7

9
50 15.5 30.4

51 15.1 30.1

52 14.8 29.8

53 14.4 29.5

54 14.1 29.3

55 13.7 29.0

56 20 13.4 28.7

57 13.1 28.4

58 12.8 28.2

59 12.5 27.9

60 12.2 27.7

61 11.9 27.4

62 11.6 27.1

63 11.3 26.9

64 11.0 26.7

65 10.8 26.4

66 10.5 26.2

67 10.2 25.9

68 10.0 25.7

69 9.8 25.5

70 9.5 25.3

71 9.3 25.0

72 9.1 24.8

73 8.8 24.6

74 8.6 24.4

75 8.4 24.2

76 8.2 24.0

77 8.0 23.8

78 7.8 23.6

79 7.6 23.4

80 7.5 23.2

Reviewer Comments:

For the purposes ofthis literature review, the macr.ophyte observations are the most

useful part ofthis study. However, although the authors assert that the insecticide,

esfenvalerate, has no noticeable impact on the macrophyte community, only one replicate

was tested at the atrazine treatment level of 50 /lg/L. Therefore, there is insufficient

replication of ponds treated with only atrazine relative to the control, and this study is

classified as invalid..

The replacement ofNaja by Chara has been noted by deNoyelles and colleagues in the

Kansas studies; however, this study provides evidence ofthis change as a result of a

single application of 50 /lg/L atrazine. Likewise, the theory that total macrophyte

biomass is not sensitive to atrazine at these levels, as a result ofthe community

composition alteration to more tolerant species, is supported by the results from the one

tested replicate.

10
Although the study author state that there are no insecticide impacts on plants, effects of

the insecticide esfenvalerate on grazer populations could have feedbacks on the plant

community.

The whole-system measures (gross photosynthesis, community respiration) were not well

explained (the methodology is apparently described in other, related articles), and did not

show strong effects from the atrazine. Without knowing the methods used, it is difficult

to interpret these results. Taken together with the algal biomass measures, however, the

results from the one replicate show that 50 IJg/L atrazine does not destroy the algal

community. This observation reflects a similar dynamic in the macrophyte community,

that although community composition may adjust, the community as a whole is able to

adapt to a single 50 IJg/L application of atrazine. Change in algal community structure

was not examined, unfortunately, and may have been significant.

The results for zooplankton and fish are of little note. These endpoints were likely to be

affected by the insecticide treatment; therefore, only the results for control levels of

insecticide were reviewed. There was no mesocosm-scale replication, however, given

that the scope ofthe study was constrained to only these mesocosms. Therefore, the

animal endpoints were derived based on only a single atrazine control mesocosm and a

single atrazine treatment mesocosm. No effects were found; however, this may have

been due to lack ofreplication.

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

Limitations of Study: Although this study provides evidence of a shift in macrophyte

community structure as a result of a single application of 50 IJg/L atrazine, only one

replicate pond was tested at 50 IJg/L atrazine (with no added esfenvalerate). Based on the

quality criteria established for review of atrazine cosm studies, this study is classified as

invalid based on insufficient replication.

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

Secondary Reviewer: Anita Pease, M.S., Senior Biologist, Environmental Fate and

Effects Division, U.S. Environmental Protection Agency.

b((?;( \l

11