### Open Literature Review Summary

Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation: Dewey, S.L. 1986. Effects ofthe herbicide atrazine on aquatic insectcommunity structure and emergence. Ecology. 67:148-162 (MRID 47543611).

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 (particularly benthic insects) in experimental ponds (mesocosms).

Summary ofStudy Findings:

Methods: The study was part ofthe 80S-day experimental pond study described in deNoyelles etai., 

### 1989, and also described in Carney, 1983 and deNoyelles etai., 1994.



### Eight experimental ponds ofsurface areas 0.045 ha each atthe University ofKansas Nelson Environmental Study Area were used. See Figure 1 for adiagram ofthe experimental pond system. Theponds were left full, uncontaminated, andundisturbed for 17 months before being drained on May 20, 1981 and refilled from the storage reservoiron May 30, 1981 to adepth of2.1 meters. At thattimeponds were stocked with bluegill sunfish (Lepomis macrochirus), 

### gizzard shad (Dorosoma cepedianum), 

### and channel catfish (lctaluruspunctatus). 

### In May 1982, four adult grass carp (Ctenopharyngodon idella) 

### were added to each pond, and in June 1982, 10% ofeach pond's surface areawas fenced offto excludethe grass carp. Sincethe exclusion areas were established afterthis study started, they are notpart ofthis study's experimental design.



# 11111••111 2087825

### In 1981, two replicate ponds at 3 atrazine treatment levels (0, 20, and 100 Ilg/L) were prepared using reagent grade 97% atrazine, andtwo additional ponds were prepared at 20 Ilg/L using commercial atrazine (CO-OP liquid atrazine, EPA registration number 1990- 318,41 % active ingredient). The ponds with commercial atrazine were convertedto 500 Ilg/L treatment using reagent grade atrazine on May 30, 1982. Atthis time all other ponds received a second annual atrazine application, and additionally the ponds were all broughtbackto 2.1 mdepth by adding well water. 1 

### Periodic atrazine concentration measurements were made. Insect emergence samples were taken from the 0 (control), 20, 100, and 500 Ilg/L treatmentponds over 16 weeks beginning on June 8, 1982. Two additional samples were taken in May before the 20 Ilg/L commercial treatmentponds
were converted to 500 Ilg/L, and one preliminary control pond sample was taken on April 24. Watertemperature was measured between 1600 and 1800,30 cm above the pond bottom in the center ofeach pond, on the first day ofeach sampling period. Additionally waterquality measurements, taken "periodically" (no further details provided) as partof the largermesocosm study, included water depth, morning oxygen concentration at 0.5 m below the water surface, andturbidity.

Adult insect emergence was sampled using partially submerged funnel emergence traps. The traps (modifications ofearlierdesigns - 6papers from 1954-1980 are cited) consisted oftransparentrigid vinyl funnels attached to 3.76-L semitransparent polyethylenejars, eachjarwith two ventilation windows covered with 175-11 

### nylon mesh. In each pond, four emergence traps were suspended from the access pier, and were setfor 48 hours (except for one 24 hour sampleperiodthat occurredon June 12-13) before collection. Following collection,jarcontents were preserved in 80% ethanol, then later sorted and identifiedto lowestpossible taxon (usually species) using keys and descriptions from several sources.2 

### Several chironomid species required slide mounting in Euparal for identification to species. Females ofOrthocladiinae, Chironomini, and Tanytarsini (Chironomidae) groups could not be identifiedto species due to the lackof comprehensivekeys available. Females and severely damaged males ofmost families couldnotbe identified furtherthan tribe orgenus.

Several measures were used to describe the insect communities: percentage similarity (PS), 

### abundance (the number ofindividuals in a community or sample), species diversity (H'), 

### species richness (S, 

### the total number ofspecies in a sampled community), andthe species evenness orequitability index, J'. 

### Definitions ofPS, H', 

### andJ'

### are:

1Reviewernote: the 200 IlgiL treatmentponds were also created atthis time, according to Carney 1983, butDewey does notmention them, presumably because they were not sampled in the insect emergence study.
2 Ross, 1944 ("The caddis flies, orTrichoptera, ofIllinois" from the Illinois Natural History Survey Bulletin); Townes, 1945 ("The nearctic species ofTendipedini [Diptera, Tendipedidae (=Chironomidae)]" from American Midland Naturalist); Burks, 1953 ("The mayflies, or Ephemeroptera, ofIllinois" from the Illinois Natural History Survey); Roback, 1971 ("The adults ofthe subfamily Tanypodinae in North America (Diptera: Chironomidae)" from Monographs ofthe Academy ofNatural Sciences of Philadelphia); and Oliver, 1981 ("Chironomidae" in pages 423-458 ofthe Manual ofnearctic Diptera, vol. I, CanadaDepartment ofAgriculture Research Branch Monograph 27).

2s' PS

## =1- 0.5· L!Pai

- Phil**i~1** s H'=

- 

### LPi logpi**i~1** 

### J'= H'/H' = 

### H'/log(S) max 

### where

### Pi is the

### pro~ortion ofindividuals comprisedby the ith 

### species 

### (Pai and

### phi, the proportion ofthe 

## l species in community 

### a or b, 

### respectively), and s' in the PS

### equation is the total number ofspecies in both communities being compared.

Statistical tests were conducted for significance and trends. One-way analysis of variance (ANOVA) was used with critical levels at a 

### = 

### 0.10 for the Ftests to test for significant differences, and linearregression ofmean values with logarithm atrazine concentration used to evaluate trends. Ifavariable had a significant ANOVA result, the Student-Newman-Keuls (SNK) a posteriori testwas used to determine significant differences among all treatments. Othermethods were dendrogram cluster analysis of ponds based on percentage similarities, and the Smimov two-sample test (on temporal emergence-frequency curves, to test for significant distributional similarity). Sample totals from both replicate ponds were used for between-treatmentcomparisons testing for significant differences.



### 3

### o 500 

### o 20 T 20 500



# < N

20 n

Figure 1. Experimental pond system layout. Numbers below ponds referto nominal atrazine treatment level (p.1g/L); ponds with 5 and 200 p.1g/L were not used in this study. T = varied treatment; D = pond was drained for much ofthe study period. Numbers inside ponds are the unique pond labels, 1-8, used in some data tables.



### Results: Atrazine concentrations decreased in all ponds overthe study period by an average of0.2 flg/L/day after addition on May 30, 1982. Pond water depths decreased due to evaporative loss at an average rate of5 cm/wk; however, water depths did not differ significantly between replicates (ANOYA). Mean pondtemperatures changed seasonally (low ofl7.5°C on May 8, high of30.4°C on August 1) and again did not differ significantly between replicate pairs (ANOYA) (a graph oftemperature overtime is presented in the paperas Figure 2, butnot reproducedhere). Oxygen concentrations did not significantly vary among treatment levels (ANOYA) or change with time (linear regression) and ranged from 5.1 to 11.0 mg/L (mean 

### = 7.46 mg/L). Mean turbidity varied significantly with treatment level (ANOYA, p-value < 

### 0.10), and increased with increasing atrazine concentration up to 100 flg/L (see Table 2) (regression results: r2 

### = 0.85, p-value 

### < 0.10 according to the table, butthe text states r 

### = 0.82, which is inconsistent). Species richness and total abundance ofemerging benthic insects were regressed to turbidity to see ifturbidity was a significantpredictorofthese variables, but results were not significant. The study author attributes changes in turbidity with atrazine-related elimination ofemergent and submergent vegetation resulting in windinduced mixing ofbenthic and limnetic zones.

4

### Phytoplankton and macrophyte communityresponses are discussedbriefly and qualitatively, with references to deNoyelles etal., 

### 1982, deNoyelles and Kettle, 1983, and Carney 1983. Qualitative personal observations ofperiphyton are noted: on the permanent walkway supports, periphyton in control ponds was dense and green with thicknesses ofa few mm up to 4-5 cm, butwas absent on structures in 100 

### Ilg/L and 500 

### Ilg/L ponds.

Forty-seven (47) insect species were collected from emergence traps in all ponds between May 8 and September26, 1982. All ofthese species except for Chaoboruspunctipennis 

### have benthic or epiphytic habitat as larvae, and for this reason 

### C. punctipennis 

### was considered separately from the benthic insect community (not included in the benthic community measures). For all species collected, the trophic habitat, microhabitat, and lifestyle are listed with references in the study's Table 1 (notreproducedhere). The traps used in the study did not sample the Odonata anddytiscid beetles, which crawl out ofthe water to emerge, orthe baetid mayflies and ceratopogonid flies, although both groups are known to be presentin the pond benthos and to emerge in the pelagic zone.

The abundances (total numbers) ofthe chironomidLabrundiniapilosella 

### varied significantly among treatments (ANOYA, p-value < 

### 0.005), declining drastically even at atrazine concentrations of20 

### Ilg/L (SNK, no p-value provided, see Table 2). Emergence rates ofCricotopus 

### cf. trifasciatus, Parakieferiella 

### cf. coronata, Parachironomus chaetoalus, P. tenuicaudatus, 

### and theremainderofthe Chironomini (unidentified) showed similarresponses atthis low treatment level (raw dataprovided inthe appendix;
see Table 3), but significantheterogeneity among treatments was not detected, probably because ofthe small numbers involved. Species equitability, J', 

### andtotal abundance of emerging benthic insects both varied significantly among treatment levels (ANOYA, pvalue = 

### 0.05). J'

### increasedbetween controls and 20 

### Ilg/L, but declined on the higher treatment levels (statistical significanceresults were mixed; see Table 2). Mean values of total abundance and species richness S

### declined significantly with atrazine addition (linearregression, r2

## =0.90 and 0.93, p-value < 

### 0.10 and < 

### 0.15, respectively; see Figure 2 and Table 2) and were significantlyreduced atthe 20 

### Ilg/L level (SNK, no p-value provided, Table 2). H', 

### the diversity index, declined somewhat with treatment, but results were not significant (see Table 1).

Clusteranalysis was performed on all eightponds based on taxonomical percentage similarity (Figure 3). Differences between controls and treatments were clear, butthe treatment levels did not show a clearidentification ofreplicate pairs. In particular, pond 8 (the 500 Ilg/L treatmentpond, physically located between the two controls) was shown to be most similarto a 20 Ilg/L pond, which may indicate confounding effects such as immigration and egg deposition occurring from proximity to controls. The tendency for pond 8to provide samples similarto a lower-concentrationpond is further demonstrated by examination ofthe raw data in Table 3.

5To further investigate the trends, insects were grouped into two feeding guilds: predatory and nonpredatory. The former group includes omnivores and is expected to have the least dependence on periphyton for food supply. The lattergroup consists ofherbivores, including collector-gatherers, scrapers, selective grazers, macrophyte leaf-miners, and filterers ofbenthic particles, and is expected to depend on periphyton for food supply. As
can be seen in Table 2, the nonpredatory insect community declined significantly in abundance and richness significantly with any atrazine treatment level (ANOVA with pvalue 

### < 0.1 0 [abundance] and 

### < 0.10 [richness], SNK, and linearregression with r2 

### = 0.94 [abundance] and = 0.90 [richness] and p-value < 0.10 [abundance] and < 0.05 [richness]) (see also Figure 4). The nonpredator abundance and richness appeared more sensitive to atrazine treatmentthan the predatory insect community (see Table 1, no statistics provided). The author also describes the raw data (see Table 3; predators include

### Ablabesmyia, Procladius, and the pelagic predator 

### Chaoboruspunctipennis, and nonpredators include

### Labrundiniapilosella, species of

### Cricotopus, most Chironomini, Caenidae, 

### Hexagena limbata, and 

### Oxythirapallida) as supporting this differential atrazine sensitivity by feeding guild, although no statistics are provided. Various mechanisms for the differential sensitivity are proposed.

Temporal emergence trends were investigated for the 11 species collected that emerged from all treatments in sufficient numbers to allow statistical (Smirnov two-sided, twosample test) and/or visual comparison oftemporal emergence distribution curves. Three ofthe 11 species are predatory and eight are nonpredatory (herbivorous). Ofthe predatory species, two 

### (Procladius bellus and 

### Chaoboruspunctipennis) were very abundant, and showed no statistically significant difference between control and any atrazine treatment level, while

### Ablabesmyia annulata was not abundant enough for statistical comparison and did not visually appear different (see Figure 5). Ofthe herbivorous species, 

### Cladotanytarsus viridiventris showed no statistically significant difference between control and any treatment level. 

### Labrundiniapilosella and 

### Oxyethira pallida were analyzed by comparing the controls with all atrazine treatments combined, finding statistical significance in the latterbutnot in the former species (see Figure 6; no p-value provided). Visual inspections only were possible on

### Dicrotendipes modestus, Parachironomus chaetoalus, and 

### Cricotopus cf. 

### sylvestris temporal emergence distributions, but these species all clearly showed earlieremergence in the atrazine treated ponds (see Figure 7). The single

### Parachironomous tenuicaudatus from a treatmentpond emerged towards the early side ofthe distribution for controls, and 

### Caenis simulans showed no clearchange in emergence pattern under atrazine treatment (Figure 7). The author speculates that early emergence may occur as a stress response to atrazine treatment (perhaps indirectly as a result ofdecreased food supply for the herbivores), although some herbivorous species showed no change in emergence time due to treatment.

640

...... V) 35
(/) (/) 30 S 

### = 18.40

- 2.85 log C w Z I 25 

# \ () a:: rJ) 20 W () 15 

# +,,--- 

# j W ll. (/) 10 

# f

5

400



### « 300 W A 127.0

- 32.7 log C () z <: a 200 z :::l co <: 100 

# t + 

## +
0 0 100 200 300 400 SOD

ATRAZINE CONCENTRATION ()jg/L)

Figure 2. Benthic insectcommunity response to atrazine concentration in eight experimental ponds overthe total samplingperiod. Vertical bars represent range ot'two replicate samples. Lines fitted by the linear regression equation shown.

7ATRAZINE POND NO. CONC.(ug/U

7
5

6

3

8
4

2 500

100

100

20

500

20

a

a

30 40 50 60 70 80 90 100
PERCENT FAUNAL SIMILARITY Figure 3. Dendrogram cluster analysis ofeightexperimental ponds based on percentage similarity (PS) ofthe benthic insect community. 

# =1

250

200

UJ U Z 150 « 0 z ::l ltl « 100

50

0 NONPREDATORS

NP 

### = 84. 1 - 3O. 1 log C

o 100 200 300 400 500

ATAAZINE CONCENTRATION ()Jgl L) Figure4. Nonpredatory benthic insect community response to atrazine concentration in eight experimental ponds overthe total sampling period. Vertical bars represent range oftwo replicate samples. Line fitted by the linear regression equation shown.



### 8A. Prock/diUI> bellvs**5~**

45 u.J 0 Z 35 q a 

### z :)**~~** m «
1 5 B. CtI'JoborU$ PiA'lCflpor/nt$

c. Ablac/!!imp'o oflfll.Jla!c

MAY JUNE JULY DATE AUGUST SEPTEMBER

Figure 5. Emergence frequency distributions ofthree abundantpredatory insect species. Solid: the combined numbers ofindividuals emerging from the six atrazine-treated ponds. Hollow: the combined numbers ofindividuals emerging from the two control ponds.

925

# 1
<r ,,, Cl Z

c,

# ~_ •• L- 

# ""'.""'.L...1JLi, .t. C'I'JdManylan'l5**vir"'d/~J6\.,tl'is**



## o

l.!AY .JUNE .JULY DATE AUGUST**~;** [P 1 L f,' BEll

Figure 6. Emergence frequency distributions ofthree abundant herbivorous insect species. Solid: the combined numbers ofindividuals emerging from the six atrazine-treated ponds. Hollow: the combined numbers ofindividuals emerging from the two control ponds.

10A Dicro/endi pt'5 modest"s

EI**Por{}~h..,on()m()s** ch'Jelooliis 10

w U 7: « C'l 2: :; ro <l'. 

# n C G,icotop"s ct, Sy,Wislr .•,

# ~~u.-..L..\.--U--l.J,l.,I....,ll-iJ,---- __

o 1''a1(J{:nirONJf!)I.,'S**'f!''''ili:~iJ(Joh./$**

{; COf.'tlis !i!mvlonS

ij 14 MAY B**'~7** 10 ir,t 2GZS**JU~Y** DATE AUGUST 11 i i 19 :?e**~;EPl** EMBER

Figure 7. Emergencefrequency distributions offive moderately abundant herbivorous insect species. Solid: the combined numbers ofindividuals emerging from the six atrazine-treated ponds. Hollow: the combined numbers ofindividuals emerging from the two control ponds.

11Table 1. Benthic insect predators (excluding Chaoboruspunctipennis) vs. nonpredators emerging from experimental ponds (from Table 2 ofstudy). Diversity indices also provided (taken from study text).

Nominal atrazine concentration 

### oflg/L 20 flg/L 100 flg/L 500 flg/L Pond number I 2 3 4 5 6 7 8 Predators Abundance 62 51 32 17 28 46 40 58 % ofindividuals 17 29 57 41 90 74 87 50 No. taxa 7 4 5 4 5 5 3 5 % oftotal taxa 19 19 31 31 71 50 43 29 Nonpredators Abundance 294 127 24 24 3 16 6 58 % ofindividuals 83 71 43 58 10 26 13 50 No. taxa 30 17 II 9 2 5 4 12 % oftotal taxa 81 81 69 69 29 50 57 71 Diversity Index (H') 1.02 0.9 0.86 0.84 0.52 0.7 0.48 0.71

Table 2. Statistical results for variables for which one-way ANDVA showed significant differences among treatments at p<O.10 or for which there was a significant regression (p<O.10). SNK results
are shown via underbars: means spanned by the range barwere found to not differ significantly (no p-values provided for significant differences found). Chaoboruspunctipennis is excluded from the community measures. From study table 3.

Treatment Means Nominal atrazine concentration ANOVA R . I egreSS1?n Variable 

## oflg/L 20 flg/L 100 Ilg/L 500 flg/L p-value 0 p-value r- Turbiditl 30.36 39.02 49.06 50.05 <0.10 0.85 <0.10 Abundance Total 267 48.5 46.5 81 <0.10 0.90 <0.10 Nonpredators 210.5 24 9.5 32 <0.10 0.94 <0.05 Labrundiapilosella 35 3.5 1.5 3.5 <0.005 0.96 <0.25 Species richness (S) Total 29 14.5 8.5 12 <0.15 0.93 <0.05 Nonpredators 23.5 10 3.5 8 <0.10 0.90 0.05 Species evenness (J')3 0.65 0.72 0.62 0.55 0.05 0.79 >0.10

I Linearregression oftreatment means on the logarithm ofatrazine concentration (equations appear in the study table but are not reproduced here)
2Turbidity in Nelson Turbidity Units
3 The mean J'value for the control treatment is not significantly different from the 100 or 500 flg/L treatments

12Table 3. Cumulative total number ofinsects caught in emergence traps in experimental ponds, May 8 - September26, 1982. Study Appendix table At.

Nominal atrazine concentration 

## oJ.!g/L 20 J.!g/L 100 J.!g/L 500 J.!g/L Pond number Taxon 2 3 4 5 6 7 8 Diptera Chironomidae Tanypodinae Tanypodini Tanypus concavus 1 0 0 0 0 0 0 0 T. stellatus 3 1 0 1 0 0 0 2 Macropelopiini Procladius bellus 35 46 25 13 24 35 38 50 P. subletti 1 0 1 1 1 1 I 0 Pentaneurini Ablabesmyia annulata 4 0 2 2 0 4 0 I A. mallochi 0 0 0 0 1 1 0 0 A. peleensis 3 0 1 0 1 0 0 0 A.!>pp. 6 2 2 0 0 2 0 0 Labrundiniapilosella 31 39 5 2 0 3 0 7 Larsia cf. decolorata 0 1 0 0 0 0 0 0 Paramerina smithae 13 0 0 0 0 5 0 1 Orthocladiinae Corynoneura celeripes 1 0 0 0 0 0 0 0 Cricotopus cf. sylvestris 0 15 I 0 0 0 0 0 C. cf. trifasciatus 9 4 0 0 0 0 0 0 

## c. sp. 7 0 2 0 0 0 0 0 Nanocladius cf. mallochi 2 1 0 0 0 0 0 0 Parakieferiella cf. coronata 3 2 0 0 0 0 0 0 Orthocladiinae unidentified 0 5 0 0 0 0 1 2 Chironominae Chironomini Chironomus decorus 1 0 0 0 0 0 0 0 C. staegeri 0 0 0 0 0 0 0 1 Cladopelma collator 1 4 0 0 0 0 0 4 C. edwardsi 6 0 0 0 0 0 0 0 C. viradula 0 0 0 0 0 0 0 2 Cryptochironomusfulvus 0 0 0 1 0 0 0 3 Cryptotendipes emorsus 1 0 0 0 0 0 1 3 Dicrotendipes modestus 11 2 1 0 1 0 0 I D. nervosus 1 0 0 0 0 3 0 0 Endochironomus nigricans 3 0 0 0 0 0 0 0 Harnischia incidata 0 0 0 0 0 0 1 0 Parachironomus sp. B 3 0 0 0 0 0 0 0 P. carinatus 1 0 0 0 0 0 0 0 P. chaetoalus 38 7 4 0 I 0 0 0 P. directus 1 0 0 0 0 0 0 0 Pmonochromis 0 1 0 1 0 0 0 0

13Nominal atrazine concentration 

## oflg/L 20 flg/L 100 flg/L 500 flg/L Pond number Taxon I 2 3 4 5 6 7 8 P. potamogeti 1 0 0 0 0 0 0 0 P. tenuicaudatus 14 3 0 0 0 0 I 0 Polypedilum simulans 0 1 0 0 0 0 0 0 P. sp. 0 0 0 

## a a a 1 

## a Pseudochironomouspseudoviridus 

## a a 2 1 

## a a 0 

## a P. richardsoni 1 

## a a 0 

## a a 0 0 Chironomini unidentified 92 23 0 2 1 2 

## a 5 Tanytarsini Cladotanytarsus viridiventris 

## 2a 3 3 4 

## a 1 0 26 Paratanytarsus sp. 2 

## a 0 

## a 0 

## a 0 0 Stempellina sp. 

## a 1 2 0 0 0 0 1 Tanytarsus sp. 5 

## a 0 

## a 0 0 0 0 Tanytarsini unidentified 7 2 2 1 0 3 0 1 Chaoboridae Chaoboruspunctipennis 24 22 20 19 15 19 17 86 Ephemeroptera Caenidae Caenis delicata 2 2 I 2 0 0 0 1 C. simulans 4 7 

## a 0 0 2 2 0 C. spp. 8 0 1 1 0 0 0 1 Ephemeridae Hexagenia limbata 0 3 0 0 0 Trichoptera Hydroptilidae Oxyethirapallida 13 5 6 0 0 2 TOTAL 380 200 76 60 46 81 63 202

14

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

- if controls (fJg/L) Concentration so, when from Day 0 to (see

When Effect comment)? Occurred Total emerging insects3 Nonpredatory benthic insect abundance4 Labrundinia 90% 20 ).tg/L integrated 8.1 ).tg/L no Calculated pilosella 96% 100 flg/L over 16 88.1 flg/L no from study abundances 90% 500 ).tg/L weeks 488.1 ).tg/L no Table 3. Periphyton on NR 20 flg/L NR not calculable not Dewey artificial applicable personal surfaces 100% 100 flg/L NR not calculable no observation (walkway described in supportS)6 100% 500 flg/L NR not calculable no study text. Increase in +30% 20 ).tg/L integrated 8.1 ).tg/L no Calculated turbidity +60% 100 flg/L over 16 88.1 flg/L no from study (NTU)7 +65% 500 ).tg/L weeks 488.1 ).tg/L no Table 3. Benthic insect 50% 20 flg/L integrated 8.1 flg/L no Calculated community 70% 100 flg/L over 16 88.1 flg/L no from study species richness weeks Table 3. (S)8 60% 500 flg/L 488.1 flg/L no Benthic insect +10% 20 flg/L 8.1 flg/L Calculated (increase) integrated no from study community species 5% 100 flg/L over 16 88.1 flg/L Table 3. weeks no evenness (],)9 15% 500 ).tg/L 488.1 ).tg/L no Benthic insect 10% 20 flg/L integrated 8.1 ).tg/L no Calculated community 15% 100 flg/L over 16 88.1 flg/L no from review diversity index weeks Table 1. (H,)IO 17% 500 flg/L 488.1 flg/L no Note: NR=Not Reported 

### Time weighted average atrazine concentrations were estimated from the limited information available inthe study: the date ofannual atrazine addition in 1982, andthe

3 See deNoyelles etal., 1994 data summary table. Effectmagnitudes could be more precisely determined using the information in this study.
4 See deNoyelles etaI., 1994 data summary table, entries for Parachironomus, Endochironomus, and Ablabesmyia (all nonpredatory). This study has precise measured totals for the nonpredatory guild, but there is significant overlap with the 1994 study endpoints.
SANOVA significant atp<0.005, regression significant atp<0.025
6 A similarendpoint appears in Attachment 2 (EndpointNo. 44), butdoes not listthis study. 7ANOVA and regression significant atp<O.1 0
8 ANOVA significant atp<0.15, regression significant atp<0.05. Statistically comparable results found for nonpredatory community, which is not shown due to overlapping data sets.
9 ANOVA significant atp=0.05, regression not significantwith p>O.1O.
10 Results were not statistically significant (ANOVA).



### 15

### observation that atrazine concentration decreased "in all ponds overthis study period by
an average of0.2 Ilg/L perday after it was added on 30 May 1982." Although at the time ofthe annual addition, one ofthe 500 Ilg/L treatmentponds was created from a20 Ilg/L commercial treatmentpond, and all ponds would have had quite low atrazine concentrations relative to nominal before the addition, time weighted averages were calculated ignoring the fact that a few samples were taken priorto May 30. The reason was a lack ofinformation and need for transparency and computational simplicity. Assuming that concentrations on May 30 were nominal and extrapolating September26 concentrations from the stated average 0.2 Ilg/L perday reduction rate, time weighted averages were calculated as shown below. (Note that concentrations ofthe 20 Ilg/L ponds went negative by September26; clearly the average reduction rate is apoor predictor oflow-treatmentpond concentrations.)
Atrazine, Ilg/L Date Day Conc_20 Conc_100 Conc_500 30-May-82 0 20 100 500 26-Sep-82 119 -3.8 76.2 476.2 Time weighted average: 8.1 88.1 488.1



### Reviewer Comments: This study was part ofthe larger 1981-1983 mesocosm study by deNoyelles and colleagues. As such, many fundamental problems exist with the basic experimental design, and the experiment cannotbe consideredwell-controlled. See reviews of deNoyelles 

### etal., 1989 and Carney, 1983 for details.

The study's sampling period overlapped with the timing ofannual atrazine addition to the ponds (sampling began May 8, while atrazine was added on May 30). The time weighted average concentrations do not account for concentrations priorthe addition, since the study providedno information on these concentrations. Also, a 500 Ilg/L pond was created outofthe 20 Ilg/L commercial treatmentpond at the time ofthe addition, so results from this pond (the study did not identify which pond itwas) are confounded by the change in treatment level. It 

### would have aided interpretation ofthe results ifsamples priorto May 30, 1982 had been left out ofthe total abundances and other community
measures.

In terms ofthe adult emerging insect sampling method, it appears that some insects are better at evading or escaping from the traps. The authormentions that baetid mayflies and ceratopogonid flies were not caughtby the method, despite knowledge thatthese insects were present in the pondbenthos and emerged in the pelagic zone. Some ofthe differences in abundances could be explainedby limitations ofthe trapping method.

16The author notes that Pond 8 was an outlier in terms ofdisplaying unexpectedly high levels ofinsects, andthat this may have been due to its physical location in between the two control ponds. It is also possible that ifPond 8 was the pond converted from 20 Ilg/L treatment, that would be another explanation for the unexpected behavior. The problems with Pond 8 highlight the uncontrollednature ofthe experiment design.

The author used a wide range ofstatistical methods and data visualizations to analyze the results, which are well described, appropriate, and correctly implemented. However, it would have been informative to include p-values for the SNK multiple comparison analyses and for the Smirnov tests for differences in emergence-frequency curves. The author's conclusion about the different effects on predatory and nonpredatory feeding guilds in the benthic insect community is not well supported by the statistical results.



### Description ofUse 

### in Document (QUAL, QUAN, INV): Qualitative

Rationale for Use: The macrophyte and phytoplankton data reported in this study were part ofthe 80S-day experimental pond study described in deNoyelles 

### etaf., 1989 and Carney, 1983. 

### In addition to the reported aquatic plant data, the study has some utility in that itprovides evidence ofpotential indirecteffects to nonpredatory insects at atrazine concentrations as low as 20 ug/L, presumably through reduction in food (periphyton and macrophytes), and habitat (macrophytes).



### Limitations ofStudy: Despite the well-done quantitative analyses ofthe dataset, problems with the experimental design (see Reviewer's comments) limitthe usability of the results. Similarto the deNoyelles 

### etal. (1989) an Carney (1983) reports, other limitations ofthe study are small sample size (two replicates at each oftwo treatment levels plus two controls) and lack ofmeasured atrazine exposure concentrations overthe duration ofthe study. No effort was made to documentwhether other chemicals (including atrazine) were present in the treatment and/or control ponds priorto study initiation. Pre-sampling was not conducted to determine whether aquatic communities
were similarto one anotherpriorto study initiation. Controls exhibited wide variability and given that there were only two controls and two replicates for each treatmentlevel, the ability ofthe studyto detect treatment-related effects is limited. Given these limitations, results should be taken as suggestive indications ofpossible effects, not as well-controlled evidence.



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



### Secondary Reviewer: Anita Pease, M.S., SeniorBiologist, Environmental Fate and Effeels Division, U.S. Environmental Protection Agency. 

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