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 of periphyton to atrazine in flowing waters: the role of

environmental factors. Journal ofApplied Phycology 10(2): 203-213. (MRID 48261113).

Purpose of Review:

Determine 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 ofReview: 9/4/09

Purpose of Study:

This study examines the association of environmental variables and community composition

with sensitivity to atrazine in periphyton communities from stream and river sites across a

latitudinal gradient in Europe.

Summary of Study 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 of predicted maximum

agricultural runoff of atrazine. For each site, water temperature, pH and conductivity were

measured in situ, and a sample of water was taken for chemical analysis. The water samples

were kept refrigerated 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 of vegetative cover were visually determined.

Analysis for atrazine was performed on the test water after collection (for residual atrazine

contamination) and on the atrazine set ofstandards for toxicity testing. Identification and

quantification of atrazine was performed via reversed-phase HPLC equipped with a diode array

1

III1IIIIII

2087843
following the procedures described by Durand and Barcelo (1990). Prior to 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 of A) 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 limit was

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 cm

2

) for 3-4 weeks. The glass squares were glued to stream stones and placed in

shallow riffle areas at each study site. Ifthis horizontal positioning was problematic, the glass

substrata were 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 or transported to the laboratory in cool-boxes filled

with site water. Incubations at the 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. Throughout the experiment, the glass

substrata were shacked to avoid the formation of gradients. Colonized glass substrata were preincubated for one hour with one of eight atrazine concentrations (0 4.31e3 Jlg L-1

). "Pure"

atrazine was dissolved by sonication to prepare the dosing solutions. The use of a solvent was

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 of photosynthetic 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 substrata were 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 of diatom frustules; nondiatom cells were identified and quantified via observation ofthe

sonicated material. For each sample, 250-300 cells were counted (Sabater et a!., 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.

The resulting diagrams provide a simultaneous ordination ofspecies, sites, and environmental

variables. Redundancy analysis (RDA) on a correlation matrix ofthe species was performed

with two data sets: the diatom species matrix and the algal groups matrix. The statistical

significance ofthe relationship between each variable or group of variables 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

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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 of DOC and low pH. Data for 18 environmental variables were available for each

stream site. After the subset ofthe best explanatory variables had been selected in the

ordinations (p :s 0.07 and variance inflation factor < 20), linear regression analyses were applied

to determine relationships between the ordination axes obtained and the ECso values. For each

European country, univariate regression analyses were also performed to find significant

relationships between ECso values and single algal groups or single environmental variables.

Results:

Water chemistry

The dissolved oxygen content (DOC) ofthe stream and river sites increased with increasing

latitude. The pH and alkalinity decreased with increasing latitude. The DOC ranged 4-25 mg/L

in Sweden, 3-5 mg/L in The Netherlands and 1-4 mg/L in Spain. The pH 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 indicated that 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 of 300-1000 llS/cm, high phosphate (> 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 of 37 diatom taxa were identified in the artificial glass substrata and included in the

redundancy analysis. Algal groups which were identified were quantified as relative

percentages. The list of the identified diatom taxa and algal groups (with relative 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 explained 45.3% 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 of atrazine and

nutrients (two ifthe Dutch sites) from the less polluted ones (LG and Hu, Swedish sites). There

was a strong relationship between the abundance of a particular taxa and environmental

variables. The results ofthe diatom taxa ordination analysis are listed in Table 3 and shown in

Figure 1.

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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. First axis was positively correlated with temperature; the

second axis was positively correlated with altitude and negatively correlated with industrial

catchment use. Cyanobacteria were 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.

EC50 values andfactors affecting sensitivity to atrazine

For the 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 The Netherlands, 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. For the 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 in the

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 lowest ECso 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 in the shaded sites of Spain 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 of different 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) of ECso. Axis 1

ofthe RDA based on algal groups showed a marginally significant relationship (p < 0.1) with

ECso values.

Overall, the study authors determined that the ECso values were influenced positively by light

conditions (based on data from the Spanish sites). Additionally, the study authors concluded that

ECso values were influenced by the percentage of different algal groups (ECso values were

positively correlated with the percentage of diatoms and negatively correlated with the

percentage of green algae). However, the study authors determined that the results ofthe

multivariate analyses indicated that the most significant factor effecting ECso was the gradient of

pollution. The study authors observed that periphyton communities in the most polluted sites

were less sensitive to atrazine. The significant relationship between axis 1 ofthe RDAbased on

diatom taxa and ECso values showed that diatom taxa occurring under different levels of

eutrophication and pollution had differing sensitivity to atrazine. The diatom taxa which was

dominated in those sites observed to be tolerant of atrazine had been shown to be tolerant in

previous literature (Navicula Menisculus, Navicula lanceolata, Gomphonema parvulum.

Nitzschia palea and Fragilaria capucina var. vaucheriae). Regarding algal groups,

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Bacillariophyceae-dominated periph}1on communities were more tolerant than Chlorophyceae

and Chrysophyceae-dominated communities.

Table 1. Physiographic description of streams 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

Riera Major3

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 ++ ++ - ++ 3 LS 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.

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Table 2. Dominant diatom taxa and relative percentage of identified algal groups in those sites included in the

multivariate analyses. The abbreviations correspond to those of the 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 T flocculosa (Tab) - A. minutissima

GA (Amin) .. Fragilaria capucina var. 59 26 0 11

vaucheriae (Fvau)

Uirjean1 A. minutissima (Amin) -

Lj Gomphonema parvulum (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) Cocconeis placentula

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)

Riera Major3

Rm C. placentula (Coce) C. sinuata 38 8.5 0 54 (Csin) - N. dissipqtq(Nzd)

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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

iTer T (Csin) -Gomphonema olivaceum 87 2.6 0 II i

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 From The Netherlands.

3 From Spain.

Algal groups: Chlor = Chlorophyceae; Cyan = Cyanobacteria; Chrys = Chrysophyceae; Bacill Bacyllariophyceae.

Table 3. Inter set correlations of environmental variables with the first two axes for RnA after fitting latitude

as a covariable (Guasch et at 1998, Table 4). Only significant variables for each analysis are included. Subs =

type ofsubstratum in the river bed; Turb = water turbidity; 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

FOCIn

As!

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 of axis 1 is P =0.03, significance of overall 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. Biplot based on redundancy analysis of the 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 of axis 1 is P = 0.07, significance of overall test is P

0.02.

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Data Summary Table:

No data table is presented because there were no reported endpoints that are relevant to the

current analysis of atrazine.

Reviewer Comments:

The intent ofthis study was to evaluate the effects of experimental atrazine exposure on

periphyton communities obtained from sources with a broad range of environmental

characteristics. Acute toxicity results are only presented as they relate to large-scale trends, such

as geographical location, pollution, and cultural aspects (eg., industrial catchment use). The

results are thus useful groundwork for the eventual development of a method to 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" but was not further characterized with regard to purity or

source. The use of a solvent and/or solvent control was not described.

The collection ofthe water samples was incompletely described (sampling depth and apparatus).

This study was similar in methodology and purpose to Guasch and Sabater (1998). That study

examined acute toxicity of atrazine with respect to light histories of 7 ofthe 9 Spanish sites used

in this study. The location ofthe Gaia site was slightly different.

Description of Use in Document: Invalid

Rationale for Use:

This study does not provide useful endpoints for the evaluation of potential community level

effects of atrazine. Although EC50 values were reported based on photosynthetic activity,

individual atrazine concentrations tested and toxicity test results at each concentration were not

provided.

Limitations of Study: Empirical atrazine purity and the results ofreported atrazine

measurements are not described. The use of a solvent is unknown. Precise atrazine

concentrations for acute toxicity testing are not given, and results are not explicitly described.

Primary Reviewer: Lisa Muto, M.S., Staff Scientist, 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 and Effects Division 4---

U.S. Environmental Protection Agency /f2.. ~t, ( ,_

r/r,

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