Open Literature Review Summary
Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation: Kettle, W.D., F. deNoyelles, B.D. Heacock, and A.M. Kadoum. 1987. Diet and reproductive success ofbluegill recovered from experimental ponds treated with atrazine. Bull. Environ. Contam. Toxicol. 38:47-52 (MRID 47543506).
Purpose ofReview: Determine the potentialeffects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plant communities and associated determination ofaLevel ofConcern (LOC).

Date ofReview: November 24, 2009
Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals in experimental ponds (mesocosms). Specifically, the studyreports the effects ofatraiine on macrophyte andbluegill sunfish populations and considers direct and indirect responses to the herbicide.
Summary ofStudy Findings:

Methods: Methods are also described in deNoyelles et al. 1989 (described as "the 136- day study" in the review), and more extensively in deNoyelles and Kettle 1980.

In July 1979, sixponds (0.045 ha each) were drained and then refilled with waterfrom an adjacentreservoirto a depth of2.1 m (no further details about dates ortiming ofdraining and refilling, orwater quality in reservoir). Three species offish were added to each pond, in the following quantities: bluegill sunfish 

### (Lepomis macrochirus), 50 fish of average total length (TL) 85 mm; channel catfish 

### (Ictaluruspunctatus), 20 fish ofaverage TL 116 mm; and gizzard shad 

### (Dorosoma cepedianum), 7 fish ofaverage TL 185 mm. All fish were obtained from local ponds and reservoirs.

I 

# 11.1.11111 2087853On July 24, atrazine in its commercially availableherbicide formulation (CO-OP liquid, EPA registration number 1990-381,40.8% active ingredient) was added (no details on application method orthe extent ofcomplete dissolution were provided) to four ofthe six ponds to create two control ponds, two ponds at 20 flg/L, and two ponds at 500 flg/L (nominal concentrations ofatrazine active ingredient). Atrazine levels were monitored throughoutthe study (complete data notprovided; deNoyelles 1982 is referenced) and 136 days after addition, 70% ofthe original concentration remained.
Macrophytes were monitored by direct visual estimates, qualitative rake hauls, and aerial analysis ofphotographs ofindividual ponds. Individual plants were collected from rake hauls for species identification.

After 136 days had passed since atrazine addition, the ponds were drained overnightto a depth of 1 m and immediately seinedto recover all fish, including young that had been produced. Fish were kiIled and preserved in 10% formaline. Stomachs ofadult bluegill recaptured atthe end ofthe study were removed and the contents analyzed for relative differences in diet at that time.

Statistical significance ofdifferences in young produced in treatment vs. control ponds were analyzed using one way ANaYA (no description ofparticular methods was provided). Other statistical tests ofsignificance used the Kruskal-Wallis test with group separation through non-parametric multiple comparison.

Results:
Bluegill was the only species to reproduce during the study, and reproduced significantly less in the treatment ponds than in the control ponds (p-value < 0.01 by one-way ANaYA). See Table 1. Among the original stocked fish, there was no significant difference in mortality over differentpond treatments (p-value > 0.05 by one-way ANaYA). About 80% ofthe bluegiIl survived compared to more than 95% ofthe gizzard shad and catfish.

Stomach contentresults showed significant differences in dietbetween fish in control and treatmentponds; see Table 2. Bluegill from control ponds had significantly more prey items in their stomach (p-value < 0.001), averaging 25.6 food items/stomach in the control comparedto averages of3.8 and 5.7 items/stomach from 20 flg/L and 500 Ilg/L treatmentponds, respectively.

The food items in bluegill stomachs were from a significantly greaternumber of taxonomical groups in the control ponds compared to the treatment ponds (p-value < 0.001). Analysis ofbluegill stomach contents by insect order (see Table 3) found a

2significantly greater number ofEphemeropterans (p-value < 0.001) and Coleopterans (pvalue < 0.01) in control pond fish than in both levels oftreatment pond fish, while Odanata and Dipterawere regularly found in control pond fish but very rare or absent in treatment pond fish (no significance analyses reported). 86% offish from control ponds had Ephemeroptera intheir stomachs and 42% had Odonata, whereas in 20 /lg/L and 500 /lg/L treatment ponds the percentages were 50% and 38% for Ephemeropteraand near zero at both treatment levels for Odonata.

The macrophyte coverwas noticeably reduced in treatmentponds (no statistical analyses ofmacrophyte observations were provided). Visual estimates found roughly a 60% decline atthe 20 /lg/L treatment level and about a 90% decline at the 500

### /lglL treatment level relative to controls, two months after atrazine addition. Rake hauls verifiedthese relative differences. 10 months after atrazine addition the ponds were drained completely, inMay (when macrophytes are normally well established in ponds subjectto the Kansas climate), and macrophyte cover changes were quantified from aerial photographs. At this time and with this estimation method, the 20 /lg/L treatment ponds had a 90% reduction relative to controls while the 500 /lg/L treatment ponds had a>95% reduction. Changes in macrophyte community composition were also noted: control ponds contained mostly Potamogetonpusillus, P. nodosus, andNajas guadalupensis with small amounts ofChara globularis, whereas treated ponds contained mostly C globularis.

Because bluegill are not susceptible to direct atrazine toxicity, the changes in reproductive success and diet in the bluegill fish were attributed by the authors to the change in macrophyte refugiaproviding substrate and food for the insectprey ofthe fish, with some specific possible mechanisms described.

Table L Reproduction ofadult bluegill stocked in experimental ponds (two ponds at each treatment level). Values are individual pond measurements. Treatments are different from controls at p<O.OL From Kettle Table 1.**r=-~-=---------r--------,-----------,** #young/pond 20pg/L llB

#food itemsljish stomach Table 2. Analysis ofstomach contents ofadult bluegill from experimental ponds (two ponds at each treatment level). Values are individual pond means ± standard error (n=25). Excludes consideration ofcrustacean zooplankton. Treatments are different from controls at p<O.OOl From Kettle Table 2. 

## o

pg/L 20pg/L 

### I 500pg/L ,..-------l---------'r"'-----+ 32.,"8.81 18.3 ±2.5 39 ±l.l 3.7

# ±~ 9.0 ±3.412.4±l.O

1.9 ±0.3 2.3 ±0.2 1.3 ±0.2 O.S ±O.l 

# i 1,0 ±0.2 

## i 0.9 ±O.I 

# I #prey

. taxa'/fish 

# l- stomach

Taxadenotes order classIficatIon orabove

3Table 3. Analysis ofthe numberofindividuals ofa given insect order represented in stomachs of adult bluegill from experimental ponds (two ponds at each treatment level). Values are individual pond means ± standard error (n=25). From Kettle Table 3. 

### I I I Insect Order i 

## o /-igiL i 20/-ig1L 500/-ig1L

..**~.** ...**_-~** i

## EPhemeroP~8.3 ±5.1 ! 13.4 ±2.1 i 1.8 ::to.S 3.7 ±2.0 8.3 .2±1 

## Odon'ta' -+"_6 ±O.3 3.2 ±O.9 0 I 0 0 0 

### I .. ! Coleopteri

. 1.0 ±0.4 0.9 ±0.3 <0.1 0 <0.1 <0.1
! Diptera3 I 13.1 ±5.2 i <0.1 <0.1 

### I I I 'Treatments are different from controls atp<O.OOI 2Treatments are different from controls at p<O.O 1 3Treatments are not different from controls (p>O.05)



### Data Summary Table: Effect Magnitude Nominal Day of Time Did Comments of Effect Atrazine Study Weighted Recovery relative to Concentration Effect Average Occur

- if controls (/-igIL) Occurred Atrazine so, when Concentration (see from Day 0 to comment)?

When Effect Occurred in 20
(# 136 425 j.lg/L
Calculated Decrease in 136 17 from means bluegill diet (# in Table 2. food itemsl Excludes stomach) 500 Ilg/L 136 425 Ilg/L no crustacean zoo lankton. Calculated 136 17j.lg/L no from means in Table 2.

4**~.~ :-:c.~_**  ..**---r-c~---:-...--:-----:---r-------,r--------r--~.~~~-** ..**~------,** Effect Magnitude Nominal Day of Time Did Comments of Effect Atrazine Study Weighted Recovery relative to Concentration Effect Average Occur

- 

### if controls (jig/I) Occurred Atrazine so, when Concentration (see fi'om Day0 to comment)?

When Effect Occurred prey taxa/ , Excludes stomach) , crustacean I I 52% zooplankton. 

# I Taxa

(different 500 !J.g/L 136 425 J,tg/L no denotes atp<O.OOI) order**~8** 

### I I classification

. orabove.**i---~. 85%---+----t~~--t--------+------+---'-----··-**  Decrease III I (d't"" t 

# I g/ bluegill diet (# •

### a/p~~~;o J) , 20 j..I. L , 136 17 !J.g/ L no Calculated Ephemeroptera r-"-7'-8 o '-X---'-+------t--------1f--------1-----l from means individuals/ (di;erent 500 !J.g/ L 136 425 Jlg/L 

# I no in Table 3. stomach) atp<O.OOI)

no

60%

90% i

# ~:i~erent i 20 !J.glL Ii 136 17 j..I.g/L Ino Calculated 

## i**;~~:o.OOU~__ l--~-----l**  from means 

## I I in Table 3.

I,**(differ~nt** '500 J.lg/L 136 425 /lg/L no

.'!!.F<O.OOI 

### i------I-- 

### L----+-----i 85%

. 

### I \ (different 

### I 20 f-lg/L i 136 17 J.lg/L I no Calculated ./!!Jl..<_O• ...;.O...;.IL.,)**-+------+------I----~-----i** from means 78% 

# I in Table 3. (different 500 J.lg/L 136 I 425 /lg/L

. no at p<O.Ol)

.

Based on 20 Jlg/L 60 18.7/lg/L I no visual 

# I**---t---~--1'------_1-----_l** estimates

.. verified by 500 j..I.g/L 60 466.9 )lg/L no qualitative 

### I

Decrease in 90% 20 j..I.g/L I, 300 134 "giL""

# £;:~~~~. emerged,


### 1 '------1------11-----+------+---_-; photographs submerged, taken after and floating

, -'-_>_9_5_°/°

__-111"

.. 500 Ilg/L

## ~3_0_--:0-=-_-=:--,--3-=3_4-:-.6_/l_g_I_L-c-=--,--_n_o--::::---:=--,-:,::p.::.;o:.:;n.::.:d.::.:s..;:;w-,:-'e_r_e---J1 I**~acrophytes** I '-:- i drained.;

Bolded values are those data pomts specIfied mthe "Cosm Data Pomts and Exposure Profiles" spreadsheet. 85% (not

. 

# 1'·---· Decrease in 1 different, 20 Jlg/L 136 17 j..I.g/L I no bluegill diet (# p>0.05) Calculated Diptera**~-:-::'-..:...::..L--+------t--------lf___-----\-~----l**from means

, d' 'd I / 78% (not in Table 3. 

### I , m IVI ua s different, 500 j..I.g/L 136 425 )lg/L

stomach)

, -+:...rp>O:05) 

# I O""a" i"

emergent macrophyte cover Decrease in

1 bluegill diet (# 

# I I Odonata

individuals/ stomach)
Decrease in bluegill diet (# Coleoptera individuals/

1 stomach)

5Time weighted averages are calculatedbased on the text's statementthat pond concentrations ofatrazine at Day 136 were 70% ofthe initial atrazine concentration. (This was the only informationprovided in the study) It is assumed that Day 0 concentrations were equal to the nominal concentrations, andthat aconstant linear decline occurred overthe study period. This is likely areasonable assumption forthe Day 60 effect, butprobably not agood assumption for the Day 300 effect. Calculations are shown below; see also "Kettle 1987 tables.xls".

No Chemograph available

- using 70% at Day 136 as reported in the text with linear decline in between**~.. ··~··-r·---~_~·i·_···_·_·~·I~_·~·~~··_·**  Assumed I 

### I Modeled TWA

Day 0 , Day of i Effect Day C t 

### r Nominal Atrazine Concentration Concentration i Effect i Concentration i oncen ra Ion (fl9/L) (jJg/L) I (jJg/L) 

### I (jJg/L)

# 18.7~ 20 20 60 17.4 i**.~-_** ..**_.~_** ..**f-..-....~** 500 500 60 433.8 466.9 :

20 20 136 14 

### .7nl 17.0 , 500 500 136 350 425.0

20 20 300 6.8 13.4

._.- . 500 500 300 169.1 334.6 '

Reviewer Comments: Some aspects ofthe methodology are not fully explained. As with other studies by this group, the reasoning for and timing ofdraining and refilling the experimental ponds is not described, and it is not stated whether the reservoir is known to be free ofherbicide contamination. The fish are taken from local sources, and it may be thatthose sources have at least some history ofherbicide contamination; however, this issue is not addressed. The results may be invalid ifthe pond water or some ofthe organisms taken for stock have prior atrazine orotherherbicide exposure. The atrazine application method is not described in detail. It is unclear whetherorhow the atrazine was completely dissolved and uniformly distributed throughout the ponds. Additionally, minimal information regarding how atrazine concentrations changed over time is provided, making the calculation oftime weighted averages more uncertain. The statistical methods are only partially described, and are weakened by the study design. The one-way ANOYA method is not described and so may not be appropriate. The Kruskal-Wallis test is well described in terms ofimplementation, but is interpreted somewhat crudely. This test detennines whetherthe treatment samples come from the same distribution as the control samples; showing that they do not is not the same as proving atrend. In fact, in most cases, the data do not show a cleartrend with increasing

6concentration overthe two treatment groups. Between-pond variation at the same treatment level was strong and more than two ponds should have been used at each treatment level to getbetterevidence for atrend. Despite these weaknesses, however, even cursory examination ofthe raw data show thatthe atrazine-treated ponds provided a much differenthabitatfor the bluegill than the control ponds. Given the methods used to samplethe macrophyte populations, the reduction endpoints should be interpreted only as qualitative measurements. In addition, the predicted atrazine concentrations at 60 and 300 days are uncertain. Description ofUse in Document(QUAL, QUAN, INV): Qualitative

Rationale for Use: Although the study results are weakened by the small number of replicates and treatments and by a lack ofinformation aboutthe atrazine concentration overtime, the studyprovides reasonable evidence thatbluegill diet and reproduction are altered by atrazine exposure even at 20 Ilg/L concentrations. The study also provides a certain amount ofqualitative evidence and ecological reasoning to supportthe mechanism ofatrazine-induced decreases in macrophyte habitat affecting bluegill diet and reproductive success.

Limitations ofStudy: The limitations ofthe study are discussed as part ofthe Reviewer's comments.
Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc
Secondary Reviewer: Anita Pease, M.S., SeniorBiologist, Environmental Fate and Effects Division, U.S. Environmental Protection Agency.

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