### Open Literature Review Summary Chemical Name: Atrazine 

### CAS No: 1912-24-9 

### ECOTOX Record Number and Citation: Guasch, H., N. Ivorra, V. Lehmann, M. Paulsson, M. Real, and S. Sabater. 1998. Community composition and sensitivity ofperiphyton to atrazine in flowing waters: the role of environmental factors. Journal ofApplied Phycology 10(2): 203-213. (MRID 48261113). 

### Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plantcommunities and associated determination ofa Level ofConcern (LOC).


### Date ofReview: 9/4/09 

### Purpose ofStudy: This study examines the association ofenvironmental variables and community composition with sensitivityto atrazine inperiphyton communities from stream and river sites across a latitudinal gradient in Europe. 

### Summary ofStudy Findings: Methods: Twenty locations were sampled along a latitudinal gradient: five in Sweden; six in The Netherlands; and nine in Spain. Both agricultural and industrial sites were sampled. The sampling was performed once in the "spring-summer" of 1996, the period ofpredicted maximum agricultural runoffofatrazine. For each site, watertemperature, pH and conductivity were measured 

### in situ, and a sample ofwater was taken for chemical analysis. The water samples were keptrefrigerated until analysis, which was performed within 24 hours ofsampling. Waters were also tested for dissolved nutrients, alkalinity and dissolved organic carbon (DOC) following standard methods. The physiographic description ofthe sites is reported in Table 1. Turbidity and degree ofvegetative coverwere visually determined. Analysis for atrazine was performed on the test water after collection (forresidual atrazine contamination) and on the atrazine set ofstandards for toxicity testing. Identification and quantification ofatrazine was performed viareversed-phase HPLC equipped with a diode array
1 

# III1IIIIII 2087843following the procedures described by Durand and Barcelo (1990). Priorto HPLC analysis, samples were filtered and processed via solid extraction with 500 mg C18 cartridges. HPLC analysis was performed using the following conditions: ODS 5-micron Hypersil column (250 mm length); gradient mobile phase consisting ofA) acetonitrile, B) methanol and C) distilled water; flow rate 1 mL/min.; and column pressure 1200-1300 psi. Atrazine was identified via retention time and spectra comparison to reference standard atrazine. The detection limitwas 0.049 Jlg L- 1*. (*All atrazine concentrations reported in this review were converted from /lM to /lg L' l *by reviewer. Periphyton commUflities were allowed to colonize the surface ofsand-blasted glass substrata (1.4-1.5 cm2) for 3-4 weeks. The glass squares were gluedto stream stones and placed in shallowriffle areas at each study site. Ifthis horizontal positioning was problematic, the glass substratawere alternatively placed on both sides ofracks during colonization. The racks were placed vertically at 0.1-0.5 m depth and parallel to the main flow. Samples for short-term toxicity tests were used either in the field ortransported to the laboratory in cool-boxes filled with site water. Incubations atthe laboratory were performed within 3-6 hours ofsampling.
Atrazine toxicity tests were performed under field light and temperature conditions (not further specified) in the field or an open site near the laboratory. Throughoutthe experiment, the glass substrata were shacked to avoidthe formation ofgradients. Colonized glass substrata were preincubated for one hour with one ofeight atrazine concentrations (0 4.31e3Jlg L- 1). "Pure" atrazine was dissolved by sonication to prepare the dosing solutions. The use ofa solventwas not described. After pre-incubation, sodium 14C-HC03 solution (specific activity 0.5-1 JlCi per sample) was added and photosynthesis was measured another hour later (two hours postexposure). ECso values were estimated using log-linear interpolation ofphotosynthetic activity in atrazine-treated samples, expressed as the percentage ofthe mean activity ofthe controls (set to 100%).
Colonized glass substrata were preserved in 4% formaldehyde. Glass substratawere cleaned using a sonication bath following the procedure described in Guasch and Sabater (1998). Light microscope (lOOOx) was used to determine the algal taxonomic composition using permanent slides ofdiatom frustules; nondiatom cells were identified and quantified via observation ofthe sonicated material. For each sample, 250-300 cells were counted (Sabater 

### eta!., 1996).
Direct gradient analyses related the species data to environmental variables using the program CANOCO 3.11 (Ter Braak, 1987). In this technique, the ordination axes extracted from the species data are constrained to be linear combinations ofthe environmental variables measured. Theresulting diagrams provide a simultaneous ordination ofspecies, sites, and environmental variables. Redundancy analysis (RDA) on a correlation matrix ofthe species was performed withtwo data sets: the diatom species matrix and the algal groups matrix. The statistical significance ofthe relationship between each variable or group ofvariables and the species matrix was assessed by Monte Carlo permutation tests (999 unrestricted permutations). Diatom species and algal group percentages were In(x 

### + 1) transformed. Diatom species with a relative

2

### abundance of

### < 1% 

### and those occurring in only one site were excluded. The algal group Chrysophyceae was also excluded because it only occurred in two Swedish sites and was linked to high values ofDOC and low pH. Datafor 18 environmental variables were available for each stream site. Afterthe subset ofthe bestexplanatory variables had been selected in the ordinations 

### (p

# :s 0.07 and variance inflation factor 

### < 20), linearregression analyses were applied to determine relationships between the ordination axes obtained and the ECso values. Foreach European country, univariate regression analyses were also performed to find significant relationships between ECso values and single algal groups or single environmental variables.

Results: 

### Waterchemistry The dissolved oxygen content (DOC) ofthe stream and river sites increased withincreasing latitude. The pH and alkalinity decreased withincreasing latitude. The DOC ranged 4-25 mg/L in Sweden, 3-5 mg/L in The Netherlands and 1-4 mg/L in Spain. ThepH and alkalinity ranged 4.9-7.3 and 0-0.5 meq/L, respectively, in the Swedish sites and> 7.5 and 1-5 meq/L, respectively, in the Dutch and Spanish sites. All sites in Sweden, two sites in Spain (Avenc6 and Castanyadell) and one in The Netherlands (Keersop) were nutrient-poor (phosphate concentration 

### < 12 Ilg/L P04

### -P). Available data indicatedthat the abundance organic phosphorous was nearly negligible in all sites (Paulsson, unpublished data). Three sites in The Netherlands (Keizersveer, Geul, Eijsden) and four sites in Spain (Gaia, Ter, Ebre, Congost) had conductivity ranges of300-1000 llS/cm, highphosphate (> 100 llg/L P04

### -P), nitrate (0.8-2 mg/L N03

### -N) and high ammonium concentration (up to 2.4 mg/L**N~-N).** 

### Atrazine contamination was below 0.23 x 10-3 

### llmoVL in most sites, except for the sites near agricultural areas in Spain and The Netherlands (0.27 x 10-3 

### llmol/L in Ebre, 0.58 x 10-3 

### Ilmol/L in Keizersveer and 0.92 x 10-3 

### llmollL in Eijsden). 

### Ordination analysis A total of37 diatomtaxa were identified in the artificial glass substrata and included in the redundancy analysis. Algal groups whichwere identified were quantified as relative percentages. The list ofthe identified diatom taxaand algal groups (withrelative percentages) is reported in Table 2.
In the redundancy analysis ofthe diatom taxa, latitude explained 11.9% ofthe diatom species variance, while 7 ofthe 18 environmental variables explained45.3% ofthe taxonomic variance. First axis was positively correlated with atrazine concentration and nutrient content (P04

### -P, NH4

### -N and N03

### -N). Also, first axis separated sites with high concentrations ofatrazine and nutrients (two ifthe Dutch sites) from the less polluted ones (LG and Hu, Swedish sites). There was a strongrelationship between the abundance ofa particulartaxa and environmental variables. The results ofthe diatom taxa ordination analysis are listed in Table 3 and shown in Figure 1.

3

### In the redundancy analysis ofthe algal groups, 6 ofthe 18 environmental variables explained 50.8% ofthe variability in the Bacill, Cyan and Chlor algal groups. Geographicallocation explained only 4.7% ofthe variance. Firstaxis was positively correlated with temperature; the second axis was positively correlated with altitude and negatively correlated with industrial catchmentuse. Cyanobacteriawere most common in industrial areas at lower altitudes (polluted sites such as Kz 

### from The Netherlands). The results ofthe algal groups ordination analysis are listed in Table 3 and shown in Figure 2.

EC 50

### values and factors affectingsensitivity to atrazine Forthe Swedish sites, the effective atrazine concentrations (ECso) were 86.3, 107.8, 280.4, 280.4, and 194.1 Ilg/L for Hultablicken, Langemossen, Lillan, Gota Alv and Uirjean, respectively. For sites in TheNetherlands, ECso values were 427.1, 302.0,552.1,429.2,278.2, and 407.6 Ilg/L for Keizersveer, Geul, Eijsden, Eijsden (shaded), Keersop and Keersop (shaded), respectively. Forthe Spanish sites, ECso values were 295.5,552.1,291.2,522.0,522.0,327.8, 90.6, and 269.6 Ilg/L for Avenc6, Avenc6 (shaded), Castanyadell, Castanyadell (shaded), Riera Major, Gaia, Ter and Ebre (not detected for Congost). The mean ECso values were lower inthe Swedish sites (189.8 ± 

### 41.0 (s.e.m.) than in the Spanish (362.3 ± 

### 161.8 s.e.m.) and Dutch sites (399.0 ± 

### 41.0 s.e.m.) Ilmol/L. The lowestECso values (86.3-107.8 Ilmol/L) were observed in one nutrient-rich site in Spain and two low-nutrient sites in Sweden. The highest ECso values (> 

### 431.3 Ilg/L) were obtained inthe shaded sites ofSpain and in one open nutrient-rich site in The Netherlands. Univariate regression analysis for the Spanish sites (n = 7) showed a significant relationship between ECso values and the percentage ofdifferent algal groups (R2 

### = 0.681, slope 0.022,p 

### < 0.01 for diatoms and R2 = 

### 0.421, slope -O.013,p 

### < 0.05 for green algae) and light (R2 

### 0.651, slope = -0.731,p 

### < 0.01). Axis 1 ofthe RDA based on diatom taxa was a significant regressor (p 

### < 0.05) ofECso. Axis 1 ofthe RDA based on algal groups showed a marginally significantrelationship (p 

### < 0.1) with ECso values. Overall, the study authors determined thatthe ECso values were influencedpositively by light conditions (based on data from the Spanish sites). Additionally, the study authors concludedthat ECso values were influencedby the percentage ofdifferent algal groups (ECso values were positively correlated with the percentage ofdiatoms and negatively correlated with the percentage ofgreen algae). However, the study authors determinedthatthe results ofthe multivariate analyses indicatedthatthe most significant factor effecting ECso was the gradient of pollution. The study authors observedthat periphyton communities inthe mostpolluted sites were less sensitive to atrazine. The significantrelationship between axis 1 ofthe RDAbased on diatomtaxa and ECso values showed that diatom taxa occurring underdifferent levels of eutrophication and pollution had differing sensitivity to atrazine. The diatom taxa which was dominated inthose sites observedto be tolerant ofatrazine had been shown to be tolerant in previous literature (Navicula Menisculus, Navicula lanceolata, Gomphonemaparvulum. Nitzschiapalea 

### and Fragilaria capucina 

### var. vaucheriae). 

### Regarding algal groups,

4Bacillariophyceae-dominatedperiph}1on communities were more tolerantthan Chlorophyceae and Chrysophyceae-dominated communities. Table 1. Physiographic description ofstreams and rivers. Location (longitude); altitude (AU); catchment use (Urban urban use; Agr

### =Agricultural use; Ind industrial use); and site condition (Cov 

### =canopy cover; Turb water turbidity; Subs = type ofsubstratum in the river bed). (Guasch et al. 1998) Location I Alt m Catchment use Site condition Site abbr a.s.l. Urban Agr Ind Cov Turb Subs ! Hultablicken- Hu 57° 47' 55 

### - + 

## - +++ !



### - 3 

## I

La.ngemossen1 LG 57° 48' 105 

### - 

## - - ++ 

## - 4 i I Lilhin1 Li 57° 56' 59 i ++++ 

### - 

## - + ++ 5

. Gota Alv1 GA 57° 46' 5 +++ ++++ +++ 

### - 4 I Uirjean1 Lj 57° 48' 35 ++ ++++ 

## - - ++ 5 L-

. Keizersveerz Kz 51° 45' 5 +++ ++ ++ 

## - ++ 5 i I GeuI" Ge 50° 45' 170 ++ + I +++ 

## - ++ 2
i EijsdenZ Ejo 50° 45' 50 +++ ++ +++ 

## - - 4 ! EijsdenS,z Ejs 50° 45' 50 +++ ++ +++ ++ 

### - 5
! Keersop2 Kso 51 ° 20' 25 ++ +++ + + 

## - 5 I l-- ! Keersop··2 Kss 51°20' 25 ++ +++ + ++ 

### - 2 ! 

### I Avenc63 Avo 41° 46' 500 

### - . 

### - 

# - + 

### - ! 2
I Avenc6s,3 Avs 41°46' 500 

## - - - ++++ 

## - 3 ! CastanyadeU3 Co i 41 ° 55' 710 

### - - 

### - 

## - - 3 I Castanyadens,3 Cs 41°55' 710 

## - - - ++++ 

## - 2

RieraMajor3 Rrn 41° 56' 700 + + 

### - ++++ 

### - 2 I I Gaii'i3 G 41 ° 15' 110 ++ ++ 

# - - + 3
i Ter3 T 42° 465 ++ ++ + 

# - ++ 3 

## I Ebre3 E 40° 55' 13 + +++ + 

### - + 5 ! Congost3 Cg 41°41' 260 ++ ++ 

## - ++ 3LS Shaded sectIOn. Catchment use and site condition codes:

- = absent; + low; ++ =moderate; +++ =high; ++++ = very high; 5 = only sand; 4 mostly sand; 3 sand and cobbles; 2 mostly cobbles; 1 only rock. I From Sweden. 2From The Netherlands. 3 From Spain.

5Table 2. Dominantdiatom taxa and relative percentage ofidentified algal groups in those sites included in the multivariate analyses. The abbreviations correspond to those ofthe ordination analysis (Based on Guasch et al. 1998, Tables 2 and 3; see also Figures 1 and 2). Dominant diatom species Algal groups (%) Site abbr (abbr) Chlor Cyan Chrys Bacill Hultablieken1 Fragilaria construens (Fcon) Hu Achnanthes lanceolata (Alan) 43 0 56 1.2 Achnanthes minutissima (Amin) Eunotia sp. pI. & Pinnularia sp. pI. Langemossen1 (Eun)

- A. minutissima (Amin) LG Tabellariaflocculosa (Tab) 6.1 0 81 13 Frailaria capucina (Fcap) A. minutissima (Amin)

- T Lillan1 flocculosa (Tab) c..Yclotella sp. pI. Li (Cye)- Aulacoseira sp. pI. (Aul) 13 7.5 0 80 Asterionella Formosa (Ast) F. capucina (Fcap) Gota A.lv1 Tflocculosa (Tab)

- A. minutissima GA (Amin)
.. Fragilaria capucina var. 59 26 0 11 vaucheriae (Fvau) Uirjean1 A. minutissima (Amin)

- Lj Gomphonemaparvulum (Gpar)- 31 5.6 0 56**ll/J!~s..chia** valea (Nzpal) Keizersveer2 Thalassiosira sp.pl. (Tal)

- G. Kz parvulum (Gpar) Gymbella minuta 15 75 0 10

. (Cmin) Nitzschia dissipata (Nzd) Geuf Ge IV. palea (Nzpal)

- G. parvulum 27 41 0 32 (Gpar) Synedra ulna (Syn) G. parvulum (Gpar)

- Navicula Eijsden2 menisculus (Nmen)

-N. palea Ejo (Nzpal) Rhoicosphenia abbreviata 17 12 0 71 (Rhab) G. parvulum (Gpar) N. menisculus (Nmen) Cocconeisplacentula Eijsden,·2 Ejs (Coce) Navicula lanceolata (Nlan) 27 13 0 50 Navicula cryptocephala (Nert)- Melosira varians (Mvar) Nitzschia amvhibian (Nza) 2 A. lanceolata (Alan)

-A. Keersop Kso minutissima (Amin) Eunotia (Eun) 31 25 0 44 

### :9..: vlacentula (Coee) KeersopS,2 A. lanceolata (Alan) Nitzschia sp. Kss (Nitz)

- Navicula radiosa (Nrad) 56 20 0 23 Navicula sp. (Nav) Avene63 Avo C. placentula (Coce) Cymbella 62 4 0 35 sinuate (Csin)

-A. lanceolata (Alan) Avene6',3 Avs C. placentula (Coce) C. sinuata 0 34 0 66 (Csin) Castanyade1l3 Co Cymbella sp. pI. (Cym)

- C. I 48 10 0 42 placentula (Coce) Castanyadell,,3 Cs C. placentula (Coce)

- A. 2.4 39 0 59 minutissima (Amin) RieraMajor3 Rm C. placentula (Coce) C. sinuata 38 8.5 0 54 (Csin)

- N. dissipqtq(Nzd) 

### 6

## I Site Dominant diatom species Algal groups (%) 

## I abbr (abbr) Chlor I Cyan 

## i Chrys Bacill 

### I Gaia' C. placentula (Cocc)

- A. i I G minutissima (Amin)

- Cymbella sp. 84 0 0 16 

## I i (Cym) I 

# , C. placentula (Cocc)

- C. sinuata i iTer T (Csin) -Gomphonema olivaceum 87 2.6 0 II i (Goli) i l-- i Ebre' Nitzschia sp. (Nitz)

- Cyclotella sp. i E 6 81 0 13 

### I Congose (Cyc)**~N** palea (Nzpal) Cg C. placentula (Cocc) 68 3.4 0 

### I 30 

## I S Shaded section. 1 From Sweden. 2 FromThe Netherlands. 3 From Spain. Algal groups: Chlor= Chlorophyceae; Cyan = Cyanobacteria; Chrys = Chrysophyceae; Bacill Bacyllariophyceae.

Table 3. Inter set correlations ofenvironmental variables with the first two axes for RnA after fitting latitude as a covariable (Guasch et at 1998, Table 4). Only significantvariables for each analysis are included. Subs = type ofsubstratum in the river bed; Turb = waterturbidity; N03-N 

### =nitrate; NH4-N = ammonium; P04-P

### = phosphate; Atr

### =atrazine; Urban 

### =urban use; Ind 

### =industrial use; Ait = altitude; Temp 

### =water temperature; and Alk = alkalinity. Diatoms Algal groups RnA1 RDA2 RnAI RnA2 

# , Subs 0.036 0.197 Turb -0.216 0.052 I N03-N 0.510 -0.107

! NH4-N 0.572 -0.047 

## I P04-P 0.608 -0.112 I AIr 0.766 -0.240 -0.054 0.060
i Urban 0.456 0.426 0.152 0.117 I Ind ! -0.025 I -0.278 

### I Alt -0.026 i 0.548 I Temp 0.465 -0.094 

### I Alk 

## i 0.096 -0.097

7

### b

Ncrt 

### Urban

FOCInAs! Fcap Au! Tall eye 1 

### a

3 4 0 RDA 1 (17%) 

### 2 1 

### -2 4

-


### 2

- 

### 5--,--------;-------.., I eli I 

### i I AmIn I NIlld Avs I Goli,." 

### Sub N:zI:l Alan 1

- Cs'" AVo
..... : 

## e! KSo ARm EiMl '" , HIli "**~K'** EJ 

### 0 

## T a.; 

## o_·:--U:;.**~r ~_** 

# .._ .._ ..**~-** _.5 

## uru Cym -1 - Hu. 

# l'LJ 1 -2 -+--.,-----j---,---....,--..,.--...-..1 I I I I I -1 0 

### _ 3- 

### '#. ('II) ..- 

# -

Figure 1. Biplot based on redundancy analysis ofthe diatom species in the European sites studied: A = Spanish sites; 0 

### =Dutch sites;

_ Swedish sites (Guasch et al. 1998). Latitude was included as a covariable. Site scores (a) and diatom species scores (b) for axes 1 and 2. Axis units are standard deviations. Percent of variance in the taxonomic data for each axis is specified in brackets. The abbreviation Nutr is used to represent nitrate, ammonium and phosphate. Significance ofaxis 1 is P

### =0.03, significance ofoverall test is P

0.04. 

### 3--,--------;-- --. b

1 

### Urban



# o Alt

Ind diat 

### Atr cyan



### -1 1



# o

-1


### 3 .a.T

I 2 I 

### 1 I 

## o AAvo • Ge KSs 

### a (l) Li :_KSo 

### 1

_

### .E~~LG 6.Avs • 

### Els 6.Rm AG

, ACG 

# o - "
. .a..Cs , ; 6.,CQ
.. , . 

### 2

-

-2 - 6.E -3 I I 

### -3 -2 -1 

# - '#.

# eN -1 - 0::

RDA 1 (34%) Figure 2. Biplotbased on redundancy analysis ofthe algal groups in the European sites studied: A = Spanish sites; 0 Dutch sites; _ = Swedish sites (Guasch et aI. 1998). Latitude was included as a covariable. Site scores (a) and diatom species scores (b) for axes 1 and 2. Axis units are standard deviations. Percent of variance in the taxonomic data for each axis is specified in brackets. The abbreviation Nutr is used to represent nitrate, ammonium and phosphate. Significance ofaxis 1 is P = 0.07, significance ofoverall test is P

0.02.



### 8

### Data Summary Table: No data table is presented because there were no reported endpoints that are relevantto the current analysis ofatrazine. 

### Reviewer Comments: The intent ofthis study was to evaluate the effects ofexperimental atrazine exposure on periphyton communities obtained from sources with a broad range ofenvironmental characteristics. Acute toxicity results are only presented as they relate to large-scale trends, such as geographical location, pollution, and cultural aspects (eg., industrial catchmentuse). The results are thus useful groundwork for the eventual development ofamethodto classify the potential sensitivity ofsites with specific characteristics to atrazine exposure. 

### However, individual atrazine concentrations and toxicity test results were not provided. Atrazine was described as "pure" butwas not further characterized with regard to purity or source. The use ofa solvent and/or solvent control was not described. The collection ofthe water samples was incompletely described (sampling depth and apparatus). This study was similarin methodology and purpose to Guasch and Sabater (1998). That study examined acute toxicity ofatrazine withrespectto lighthistories of7 ofthe 9 Spanish sites used inthis study. The location ofthe Gaia site was slightly different. 

### Description ofUse in Document: Invalid 

### Rationale for Use: This study does not provide useful endpoints for the evaluation ofpotential community level effects ofatrazine. Although EC50 values were reported based onphotosynthetic activity, individual atrazine concentrations tested and toxicity testresults at each concentration were not provided. 

### Limitations ofStudy: Empirical atrazine purity and the results ofreported atrazine measurements are not described. The use ofa solventis unknown. Precise atrazine concentrations for acute toxicity testing are not given, andresults are not explicitly described. 

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

### Secondary Reviewer: Catherine Aubee, Biologist, Environmental Fate and Effects Division U.S. Environmental Protection Agency 

### Tertiary Reviewer: Brian Anderson, Biologist, Environmental Fate andEffects Division

# 4--- U.S. Environmental Protection Agency 

## /f2..**~t,** ( ,_ r/r,

9