### 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 ofaherbicide and insecticide mixture in aquatic mesocosms. Arch. Environ. Contam. Toxicol. 27:527-533 (MRID 47543612).

Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidenceregarding effects ofatrazine on aquatic plant communities and associated determination ofaLevel ofConcern (LOC).

Date ofReview: November24, 2009

Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals in experimental ponds (mesocosms).
Summary ofStudy Findings:

Methods: This study examined combined effects ofatrazine and apyrethroid insecticide, esfenvalerate, on aquatic biota. Methods relating solely to insecticide treatment are not discussed in detail in this review summary.

Twelve mesocosms were used forthe experiment, six as atrazine controls and sixtreated with 50 /-lg/L 

### atrazine on May 26 (single application) (yearnotprovided). The mesocosms were 0.1 ha, earthen-lined, with amean depth of1.5 m and volumes between 600 and 850 m3

, 

### and were located atthe National Fisheries ContaminantResearch Center, National Biological Survey, Columbia, Missouri. Atrazine (40.8% liquid field formulation; Shell Corporation) was applied as an aqueous slurry pouredbehind aboat. All mesocosms also received 0.02 mLiL acetone, since this was the insecticide solvent; it was injected at 0.5 mbeneath the water surface using ahand sprayerbehind anon-driven boat overa 30-minute mixing period. 50 /-lg/L 

### was selected as the atrazinetreatment level because it was expectedto cause noticeable changes in vegetation, butnot to cause

1••

# 11••111 2087820direct effects on fish and invertebrates, and also because itwas seen as a common level in runoffadjacentto intensively farmed land. The insecticide used, esfenvalerate, is a synthetic pyrethroid with a short (hours) half-life and low perceived environmental toxicity. The insecticidetreatments were applied on May 26, the day ofatrazine application, and on June 8, 6 weeks afterthe 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 aunique combination ofatrazine and esfenvalerate treatments. (A table summarizing the herbicide and insecticide treatment protocol is provided in the original article butis not reproduced here.)

Atrazine concentrations in the mesocosms were measured 2hours 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. BakerCo.) with aBaker-Mizerpre-filter (J.T. Baker Co.), once the waterhadbeen adjustedto pH 6.6 using sulfuric acid orKOH. 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 N2make-up gas at 20 mLimin, and temperature programming from 70-275°C (increase 5°C/min, final time 5 minutes). Triplicate spikes andblanks 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, andprimary productivity were measured every two weeks. Temperature, pH, conductivity, dissolved oxygen, and oxidation-reduction potential were measured in situ at depths of0, 0.5, and 1 m using a Hydrolab SurveyorII water quality monitor. Primary productivity andnutrients 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 andparticulate organic carbon (POC). (A 1987 article headed by the lead authoris cited for further methodological details.) Additionally, whole-system measurements ofgross photosynthesis, community respiration, and net primary production were determinedusing the diurnal oxygen method (a 1992 article headedby the lead author is cited for further methodological details).
Macrophytebiomass and species composition were determined from monthly samples, four samples permesocosm. For sampling, a stovepipe sampler (0.I m2 area) was used, and macrophytes were removed with arake 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. dominatedthe macrophyte community.

2Zooplankton were sampled each weekwith abattery-powered meteredpump. Two replicate 114-L, depth-integrated composites (38 L each from depths of0, 0.5, and 1 m) were taken and sievedthrough an 80 11m Wisconsin net, andthe zooplankton stored in 99: 1 ethanol:glycerin for future identificationto orderor sub-order (identification method details are notprovided).

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, andthey were stocked to achieve a density in the pond of0.275 g/m3

, corresponding to at least 100 fish perpond. 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 2polyethylene mesh cages (30 percage) at each mesocosm surface for 2 weeks, and observed daily for loss ofequilibrium and mortality (dates not provided).

Since the 1992 article headed by the lead authorhad showedno direct effects of esfenvalerate on algae ormacrophytes, datawere combined across insecticidetreatments to examine effects ofatrazine onplants, and testedusing non-parametric one-way analysis ofvariance withthe 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 ofatrazine and an insecticide, esfenvalerate, on aquatic biota. Sincethe plants were apparently unaffected by the insecticide, atrazine treatments were pooled overthe insecticide treatments for examination ofplant endpoints. Results for animal endpoints, which were broken down by insecticide treatmentregime, will notbe reproduced in full detail, butthe results for control levels ofinsecticide will be summarized.

Average measured atrazine concentrations in the 6 treatmentmesocosms were equal to nominal (50 I1g/L) 2 days after application with a standard deviation of13 I1g/L, but declinedto 20 I1g/L after 8 weeks. No further data are provided, butthe calculated halflife ofatrazine in the mesocosms was 41 days (r= -0.87 with anegative exponential model, p:::; 0.001). Waterquality results (ranges overall 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

3Changes 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).

Treatmentwith atrazine alteredthe composition ofthe macrophyte community, butnot 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 differbetween 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 forplots ofthe macrophyte data. Additionally, wholesystem measures ofproductivity 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 similarin treatment and control mesocosms.

Atrazine was not found to significantly orconsistently modify the response of zooplankton or fish to esfenvalerate. Raw data tables and figures ofzooplankton and fish responses are provided in the article but are notreproduced in full here. The data at control levels ofesfenvalerate (mean followed by standard deviation in parentheses, two replicates from each mesocosm, C=control and T=atrazine treatment) are as follows: # ofcrustacean 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 ofbluegill after48 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 (notreproduced 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 of100 fish) was roughly equal between atrazine controls and treatments (insecticide controls only).

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Figure 1. Chlorophylla and particulate organic carbon (POC) ofphytoplankton in mesocosms with and without atrazine. Each point represents the mean (± standard deviation) ofn=6 mesocosms. Arrows denote treatment dates (atrazine was only applied on the first treatmentdate). Asterisks denote significant differences between herbicide treatments atp

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Figure 2. Species composition and total biomass ofmacrophytes in mesocosms with and without atrazine. Each point represents the mean (± standard deviation) ofn=6 mesocosms. Arrows denote treatment dates (atrazine was only applied on the first treatmentdate). Asterisks denote significant differences between herbicide treatments at p 0.05.

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Figure 3. Total system metabolism (gross photosynthesis, community respiration, and P/Rratio) in mesocosms with and without atrazine. Each point represents the mean (± standard deviation) ofn=6 mesocosms. Arrows denote treatment dates (atrazine was only applied on the first treatment date). Asterisks denote significant differences between herbicide treatments atp :5 0.05.

7Data 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 textp. 530 Fish No effect 50 J.lg/L NA NA NA From text p. 530 Note: NR=NotReported, NA=NotApplIcable,

The time weighted averages are calculatedusing the given information in the study, that the half-life ofatrazine in the mesocosms was 41 days, andthat concentration on Day 2
was 50 flg/L. Unfortunately, the modeled chemograph does notmatch the other information given, thatthe concentration after 8 weeks was 20 flg/L (the model predicts a concentration ofabout 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

80 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

950 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: Forthe purposes ofthis literature review, the macr.ophyte observations are the most useful part ofthis study. However, although the authors assertthatthe insecticide, esfenvalerate, has no noticeable impact onthe macrophyte community, only one replicate was tested atthe atrazine treatment level of50 /lg/L. Therefore, there is insufficient replication ofponds 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 aresult ofa single application of50 /lg/L atrazine. Likewise, the theory thattotal macrophyte biomass is not sensitive to atrazine atthese levels, as aresult ofthe community composition alteration to more tolerant species, is supported by the results from the one tested replicate.

10Although the study author state thatthere are no insecticide impacts on plants, effects of the insecticide esfenvalerate on grazerpopulations couldhave 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. Withoutknowing the methods used, it is difficult to interpretthese results. Taken togetherwith 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 similardynamic inthemacrophyte community, that although community composition may adjust, the community as awhole is able to adaptto a single 50 IJg/L application ofatrazine. Change in algal community structure was not examined, unfortunately, and may have been significant.

The results for zooplankton and fish are oflittle note. These endpoints were likely to be affectedby the insecticide treatment; therefore, onlythe results for control levels of insecticide were reviewed. There was no mesocosm-scale replication, however, given thatthe scope ofthe study was constrained to only these mesocosms. Therefore, the animal endpoints were derivedbased 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 ofUse in Document (QUAL, QUAN, INV): Invalid

Limitations ofStudy: Althoughthis study provides evidence ofa shift in macrophyte community structure as aresultofasingle application of50 IJg/L atrazine, only one replicate pondwas tested at 50 IJg/L atrazine (with no added esfenvalerate). Basedon the quality criteriaestablished forreview ofatrazine cosm studies, this study is classifiedas invalid based on insufficientreplication.


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


### Secondary Reviewer: AnitaPease, M.S., SeniorBiologist, Environmental Fate and EffectsDivision, U.S. Environmental Protection Agency. 

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