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 of bluegill recovered from experimental ponds treated with atrazine.

Bull. Environ. Contam. Toxicol. 38:47-52 (MRID 47543506).

Purpose ofReview:

Determine the 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). Specifically, the study reports the effects of atraiine

on macrophyte and bluegill sunfish populations and considers direct and indirect

responses to the herbicide.

Summary of Study 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, six ponds (0.045 ha each) were drained and then refilled with water from an

adjacent reservoir to a depth of 2.1 m (no further details about dates or timing of draining

and refilling, or water quality in reservoir). Three species of fish were added to each

pond, in the following quantities: bluegill sunfish (Lepomis macrochirus), 50 fish of

average total length (TL) 85 mm; channel catfish (Ictalurus punctatus), 20 fish of average

TL 116 mm; and gizzard shad (Dorosoma cepedianum), 7 fish of average TL 185 mm.

All fish were obtained from local ponds and reservoirs.

I 11.1.11111

2087853
On July 24, atrazine in its commercially available herbicide formulation (CO-OP liquid,

EPA registration number 1990-381,40.8% active ingredient) was added (no details on

application method or the extent of complete 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 of atrazine active ingredient). Atrazine levels were monitored

throughout the study (complete data not provided; 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 of photographs of individual ponds. Individual plants were collected from rake

hauls for species identification.

After 136 days had passed since atrazine addition, the ponds were drained overnight to a

depth of 1 m and immediately seined to recover all fish, including young that had been

produced. Fish were kiIled and preserved in 10% formaline. Stomachs of adult bluegill

recaptured at the end ofthe study were removed and the contents analyzed for relative

differences in diet at that time.

Statistical significance of differences in young produced in treatment vs. control ponds

were analyzed using one way ANaYA (no description of particular 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 different pond 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 content results showed significant differences in diet between fish in control and

treatment ponds; 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 compared to averages of 3.8 and 5.7 items/stomach from 20 flg/L and 500 Ilg/L

treatment ponds, respectively.

The food items in bluegill stomachs were from a significantly greater number of

taxonomical groups in the control ponds compared to the treatment ponds (p-value <

0.001). Analysis of bluegill stomach contents by insect order (see Table 3) found a

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significantly greater number of Ephemeropterans (p-value < 0.00 1) and Coleopterans (pvalue < 0.01) in control pond fish than in both levels oftreatment pond fish, while

Odanata and Diptera were regularly found in control pond fish but very rare or absent in

treatment pond fish (no significance analyses reported). 86% of fish from control ponds

had Ephemeroptera in their stomachs and 42% had Odonata, whereas in 20 /lg/L and 500

/lg/L treatment ponds the percentages were 50% and 38% for Ephemeroptera and near

zero at both treatment levels for Odonata.

The macrophyte cover was noticeably reduced in treatment ponds (no statistical analyses

of macrophyte observations were provided). Visual estimates found roughly a 60%

decline at the 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 verified these

relative differences. 10 months after atrazine addition the ponds were drained

completely, in May (when macrophytes are normally well established in ponds subject to

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 Potamogeton pusillus, P. nodosus, and Najas guadalupensis with

small amounts of Chara 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 refugia providing substrate and food for the insect prey ofthe fish,

with some specific possible mechanisms described.

Table L Reproduction of adult 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

20 pg/L

llB

#food

itemsljish

stomach

Table 2. Analysis ofstomach contents of adult bluegill from experimental ponds (two ponds at each

treatment level). Values are individual pond means ± standard error (n=25). Excludes consideration

of crustacean zooplankton. Treatments are different from controls at p<O.OOl From Kettle Table 2.

opg/L 20 pg/L I 500 pg/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

Taxa denotes order classIficatIon or above

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Table 3. Analysis of the number ofindividuals of a 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 at p<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

ofEffect 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 17 j.lg/L no from means

in Table 2.

4
~.~ :-:c.~_..---r-c~---:-...--:-----:---r-------,r--------r--~.~~~- ..~------,

Effect Magnitude Nominal Day of Time Did Comments

ofEffect Atrazine Study Weighted Recovery

relative to Concentration Effect Average Occur - if

controls (jig/I) Occurred Atrazine so, when

Concentration (see

fi'om Day 0 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 . or above. 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'-8o

'-X---'-+------t--------1f--------1-----l from means

individuals/ (di;erent 500 !J.g/L 136 425 Jlg/L I no in Table 3.

stomach) at p<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

emerged,

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

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 m the "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___-----\-~----lfrom 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)

IO""a" i" emergent

macrophyte

cover

Decrease in

1 bluegill diet (#

I

I Odonata

individuals/

stomach)

Decrease in

bluegill diet (#

Coleoptera

individuals/

1 stomach)

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Time weighted averages are calculated based on the text's statement that pond

concentrations of atrazine at Day 136 were 70% ofthe initial atrazine concentration.

(This was the only information provided in the study) It is assumed that Day 0

concentrations were equal to the nominal concentrations, and that a constant linear

decline occurred over the study period. This is likely a reasonable assumption for the

Day 60 effect, but probably not a good 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)

20 20 60 

..

17.4 .. f-..-....~

i 18.7~ 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 of draining and refilling the experimental ponds is

not described, and it is not stated whether the reservoir is known to be free of herbicide

contamination. The fish are taken from local sources, and it may be that those sources

have at least some history of herbicide 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 or other herbicide exposure.

The atrazine application method is not described in detail. It is unclear whether or how

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 of implementation, but is interpreted

somewhat crudely. This test detennines whether the treatment samples come from the

same distribution as the control samples; showing that they do not is not the same as

proving a trend. In fact, in most cases, the data do not show a clear trend with increasing

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concentration over the 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 get better evidence for a trend. Despite these weaknesses, however,

even cursory examination ofthe raw data show that the atrazine-treated ponds provided a

much different habitat for the bluegill than the control ponds.

Given the methods used to sample the 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 of Use 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 about the atrazine concentration

over time, the study provides reasonable evidence that bluegill diet and reproduction are

altered by atrazine exposure even at 20 Ilg/L concentrations. The study also provides a

certain amount of qualitative evidence and ecological reasoning to support the

mechanism of atrazine-induced decreases in macrophyte habitat affecting bluegill diet

and reproductive success.

Limitations of Study: The limitations ofthe study are discussed as part ofthe

Reviewer's comments.

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.

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