### Open Literature Review Summary
Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Guasch, H. and S. Sabater. 1998. Light history influences the sensitivity to atrazine in periphytic algae. Journal ofPhycology 34(2): 233-241. (MRID 48261116).


### 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 plant communities and associated determination ofa Level ofConcern (LOC).



### Date ofReview: 9/4/09


### Purpose ofStudy: This study examines the influence oflight history (light conditions during colonization) onthe sensitivity to atrazine ofperiphyton communities from stream and river sites with different water chemistry and lightregimes.


### Summary ofStudy Findings:

Methods: The study included seven sites located in five streams and rivers: Avenco (Avo, Avenco open; Avs, Avenco closed/shaded); Castanyadell (Co, Castanyadell open; Cs, Castanyadell closed/shaded); Gaia (G); Riera Major (RM); and Ter (T). In the Avenco and Castanyadell systems, open canopy sites were located 100-200 m downstream from shaded sites. The physiographical description ofthe streams and rivers are provided in Table 1.
Periphyton communities were allowed to colonize the surface ofetched glass squares (1.4 cm2) throughout July 1996. The glass squares were glued to stream stones and placed in riffle areas at each study site. Colonization time was shortto avoid periphyton self-shading: 3 weeks in Avo, Avs, Co, Cs and RM (sites with low nutrients and/or light availability) and 2 weeks in G and T (open sites with high nutrients). Water and environmental conditions were measured at each sampling date; values are reported in Table 2.

1 

# 1II1IIIIII 2087864

### After colonization, the glass substrates were removed from the sites and transported in coolboxes filled with site water. Ten to twenty liters ofsite water was also collected at each sampling. Periphyton samples for atrazine testing (100-200 colonized glass substrates) were stored in the refrigerator at 4°C until use after receipt at the laboratory.

The analysis ofthe photosynthetic pigments was performed by extracting the algal pigments in 90% acetone after sonication ofthe attached algae for 4 minutes. Total chlorophyll a 

### was measured via acidification corrections (Lorenzen 1967). The differentpigment groups, such as carotenoids, were identified and quantified via HPLC analysis using procedures described by Mantoura and Llewellyn (1983) and detailed in Guasch and Sabater (1995). The chromatographic results were used to estimate the percentage ofchI a, other forms ofchlorophyll (including chI a'

### and b) 

### and chlorophyll derivatives. Total carotenoids were calculated as the sum ofall carotenoid peaks.

The analysis ofthe algal taxonomic composition was performed by removing the cells from the glass substrates via sonication. Cells post-sonication were monitored via scanning electron microscope (SEM) in orderto ensure that no viable damage was done during removal. Successive short sonications (ca. 

### 20 s) were performed until complete removal ofalgae. After each sonication, the sonicated material was removed and 2 mL ofdistilled water was added. The sonicated material was preserved in 4% formaldehyde. Lightmicroscope (1000x) 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. Semiquantitative data on algal abundances was used to calculate the proportions ofdifferent algal groups and cyanobacteria.
Photosynthesis-irradiance (P-I) curves were studied using natural light (neutral-density filters were used to reduce incident lightto desired levels). ForAvo, Avs, G, Cs and T study sites, four replicate glass substrates were incubated at each offour different light intensities (10-60, 70-200, 250-300 and 1200-1700**~mol** 

### m-2 S-l). 

### For Co study site, four replicate glass substrates were incubated ateach oftwo different light intensities (10-60 and 1200-1700**~mol** 

### m-2 S·l). 

### For RM study site, four replicate glass substrates were incubated at one light intensity (10-60**~mol** 

### m-2 

### s1). Intact colonized substrates were put in glass vials containing 4 mL offiltered stream water. Incubations were performed outdoors in waterbath (± 

### 3°C, field temperature) with continuous shaking. Afterpre-incubation (1 h), photosynthesis was measured by the addition ofa 14C

_ source (a diluted 14C-HC03- solution, specific activity 1**~Ci** 

### per sample). After 1 h ofincubation at the same conditions, the reaction was stopped by the addition of0.4 mL of37% formaldehyde to each vial. P-I curves were fitted to the inhibition model ofPlatt et al. (1980). The fitted curves were used to estimate a (the initial slope ofthe P-I curve) and 

## p(the inhibition parameter). The fitting was done via the GraFitprogram using nonlinear regression. The light saturation and light inhibition parameters (Isat and Ib) 

### were calculated as Pmax/a and Pmax/P, respectively.

2

### Atrazine concentration-response curves were studied using the same procedures, conditions, sites and irradiances as described above for P-I curves. For each periphyton community and irradiance, replicate glass substrates were incubated at five atrazine concentrations prepared from a stock of"pure" atrazine (0, 10.78, 107.84,215.68, and 1078.40 /lg L·l; all atrazine concentrations converted from /lmol L-lto /lg L-l by reviewer). Diluted l4C-HC

### 0 3' 

### solution (specific activity 1 /lCi per sample) was added after one hourpre-incubation with atrazine. An 

### hour later (two hours after atrazine exposure), the reaction was stopped by the addition of0.4 mL of37% formaldehyde to each vial and photosynthesis was measured. ECso and EC 10 

### values were estimated using log-linear interpolation ofphotosynthetic activity in atrazine-treated samples, expressed as the percentage ofthe mean activity ofthe controls.
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 via HPLC equipped with a diode array following 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); gradientmobile phase consisting ofA) acetonitrile, B) methanol and C) distilled water; flow rate 1 mUmin.; and column pressure 1200-1300 psi. Atrazine was identified via spectracomparison to reference standard atrazine. The detection limit was 0.1 /lg L-l

.
The study authors used Pearson correlation analysis to determine relationships between the environmental data and biological variables and those concerning responses to atrazine. Athreeway ANOVA was used to test for effects oflightregime during colonization (Leol), atrazine exposure (A), and the irradiance ofincubation (line) on photosynthesis. Photosynthesis was expressed as the percentage ofthe maximum activity measured in each periphyton community. The analysis included three factors: A, atthe five concentrations; line, grouped into three levels (50-80,250-300 and 1200-1700 /lmol photons m·2 

### S·l); and light conditions during colonization, Leo\' with two levels (high light, HL, and low light, LL; based on the photosynthetic behavior of the corresponding periphyton communities).

Results: 

### Site characterization
Light extinction (Lext) 

### measurements indicated that canopy covers (shaded sites) caused a reduction ofirradiance, and stream sites which were open, undisturbed and had a low concentration ofnutrients had negligible reductions ofirradiance.

3

### Photosynthesis-irradiance (P-I) relationships

The P-l curves for periphyton from the three open sites (Avo, G and T) had similar initial slopes and saturation irradiances around 50 

### /lmol m2 

### S-l. Additionally, a slight photoinhibition at high intensity was observed. ForAvs, the initial slope ofthe P-l curve was similar to the open community, butphotoinhibition occurred around 200 

### /lmol m-2 

### S-I. For Cs, the initial slope of the P-l curve was the steepest ofthe test sites, and evidence for photoinhibition was observed around 100 

### /lmol m2 

### s-l. Forthe five sites, the correlation ofthe irradiance ofinhibition, lb, with light conditions during colonization, Leo], 

### resulted in the following values: r = 0.88, P < 0.05, 

### n = 5. 

### Two levels ofLeol 

### were established: high light, HL, 

### where Leol 

### were above 500 

### /lmol m-2 

### S-10r Lext < 50% 

### and low light, LL, 

### where Leol 

### were below 40 

### /lmol m-2 

### S-l or Lext 

### > 95%. 

### P-l relationships in the presence and absence ofatrazine are reported in Table 3 and illustrated in Figure 1. 

### For example, in the open-canopy sites, the respective mean ECso and EC IO 

### values for periphyton treated with atrazine were 295.5 

### and 97.06 

### /lg L-Ifor Avo, 291.2 

### and 127.3 

### /lg L-Ifor Co, 327.8 

### and 60.39 

### /lg L-1for G, and 90.59 

### and 6.47 

### /lg L-1for T, respectively. Forthe closedcanopy sites, the mean ECso and EC lO 

### values, respectively, for periphyton treated with atrazine were 552.1 

### and 155.3 

### Ilg L-1for Avs, 498.2 

### and 138.04 

### /lg L-Ifor Cs, and 521.9 

### and 157.5 

### /lg L- l 

### for RM, respectively.


### Chlorophyllcontentandpigmentcomposition
Algal communities on the glass substrates were generally thin biofilms due to the short amount ofcolonization time. The periphyton from open sites had statistically significantly greater mean chI a 

### amounts than those ofperiphyton from closed sites; alternatively, the periphyton from open sites had statistically significantly lower meantotal carotenoids than those ofperiphyton from closed sites. Pigment composition was correlated with Leol (% 

### chI 

### a: r 

### = 0.821, P 

### < 0.05, 

### n 

### = 6; % 

### TCar: r = -0.989, P < 0.05, 

### n = 6). 

### Therefore, differences in pigment composition coincided with differences inthe photosynthetic parameters ofeach algal community.


### Community structure

Green algae (Chlorophyceae) and cyanobacteria were more prevalent than diatoms in periphyton communities from open-canopy sites; and, alternatively, diatoms were the more dominant taxonomic group for periphyton from closed-canopy sites. 

### Cocconeisplacentula was the dominant group within the diatoms in all sites.



### Atrazine toxicity
Periphyton from open-canopy sites was more sensitive to atrazine than periphyton from closed canopy sites for incubations between 55 

### and 85 

### /lmol m2 

### S-I and for incubations at saturating irradiances. At saturating irradiances (PS % 

### sat 

### = 100, 

### see Table 6), 

### periphyton from open4canopy sites showed a slight increase inphotosynthesis at 0 and 10.78 Jlg L-! atrazine concentration and strong photosynthetic inhibition at atrazine concentrations above 21.57 Jlg/L. Forthe periphyton from closed-canopy sites, the increase ofphotosynthesis was not observed; however, the similarinhibition by atrazine at high atrazine concentrations was observed. The effect ofatrazine on the P-l relationships for open-canopy and closed-canopy sites is illustrated in Figures 2 and 3; the tabulated forms ofthese graphs are provided in Tables 4 and 5, respectively, where values for photosynthesis were estimated by the primary reviewer.
The mean EC

### so and EC IO values for periphyton treated with atrazine at open-canopy and closedcanopy sites at the various irradiances ofincubation are reported in Table 6.
A negative relationship between EC

### so values measured at saturating irradiance and Leo! was determined (r2 = 0.56, P = < 0.005, n = 5). Results ofthis correlation are illustrated in Figure 3.
A negative relationship between mean 

### ECso values for each site (mean data obtained at saturating irradiances, see Table 3) and the percentage ofchI a was determined (r = -0.857, 

### P = < 0.05, n = 6). 

### Three-wayANOVA

The three-way ANOVA for the observed effects on photosynthesis, using Pearson correlation analysis, determined the interdependence ofthe various treatment factors oflightregime during colonization (Leo], high light, HL, and low light, LL), algal sensitivity to atrazine (A, at the varied concentrations), and the irradiance ofincubation (line, 50-80,250-300 and 1200-1700 Jlmol photons m2 s-!). The Leo! x tne interaction was significant (df2; SS 32,254; F value 38.2; P <0.001), suggesting a photosynthetic dependence. A dependence was also suggested for atrazine and Leo], based on the significance ofthe Leo! x A interaction (df4; SS 10,223; F value 6.06; P <0.001). This explained the observed enhanced effect ofatrazine for open-canopy sites. Additionally, the Leo! x line x A interaction was significant (df8; SS 8375; F value 2.48; P <0.05), suggesting that the magnitude ofLeo1 and A was attenuated by line.
Overall, the study authors determined that the light conditions during colonization were shown to be an important factor in the subsequentphotosynthetic behavior ofperiphyton communities, including the photosynthetic behavior in the presence ofatrazine. The study authors also concluded that the results ofthe study (light history ofperiphyton effecting the response to atrazine) support the hypothesis that periphyton algal communities in low-light environments have a certain degree ofprotection against this herbicide. The study authors also proposed that the increased resistance to atrazine exhibited by low-lightperiphyton algal populations was due to the higher chlorophyll content.
Regardingecological implications, the study authors noted that field agricultural streams are generally sites with little shading, high nutrient concentrations, and, consequently, periphyton communities dominated by filamentous green algae. Based on the findings ofthis study, the

5

### study authors conclude that the periphyton offield agricultural streams are likely to be more sensitive to atrazine than other shaded, undisturbed sites.

Table 1. Physiographic description ofstreams and rivers (Guasch and Sabater 1998). Water- Dominant

shed Steam Stream Site Location order Lithology riparian Human use condition area vegetation (km2) Avenc6 41°46'N 35 3 Calcareous Alnus Recreational, Undisturbed, 2°19'E glutinosa water supply closed Castanyadell 41°55'N 16 3 Siliceous Alnus Recreational Undisturbed, 2°n'E glutinosa closed Gaia 41°n'N Sewage Eutrophic, 240 3 Calcareous Populus alba inputs, I020'E agriculture open 41°56'N Alnus Recreational, Slightly Riera Major 2°25'E 194 4 Siliceous glutinosa sewage eutrophic, inputs closed' Agriculture, Eutrophic, Ter 42°00'N 1380 5 Calcareous Arundo donax industry slightly 2°15'E sewage polluted, inputs open

Table 2. Water quality and canopy cover ofsites sampled in July 1996 (based on Guasch and Sabater 1998).
Lcol Condo DOC Phosphate, Light (Jimol Lex! Temp. pH (JiS (mg Alkalinity Nitrate, conditions m-2 S-I)" (%)b ( DC) cm-1)C L-1)d (meq L- 1) Ammonium Site (Jig L))" Avenc6 Open 1700 ND 17.8 8.12 228 1.68 1.89 < 12,56, < 14 Avenc6 Closed 38 98 17.8 8.12 228 1.68 1.89 < 12, 56, < 14 Castanyadell Open 1100 ND 17.6 8.10 182 1.04 1.69 < 12,378, < 14 Castanyadell Closed 35 97 17.6 8.10 182 1.04 1.69 < 12, 378, < 14 Gaia Open 740 37 20.5 8.27 923 2.95 3.58 < 12,2002,28 Riera Major Closed 20 98 18.3 8.36 241 1.37 2.17 28,518,28 Ter Open 600 50 18.2 8.17 313 1.35 2.22 109, 1078, 154 ND =Not determmed. a Irradiance at the substrate surfaces (Lco!)' b extinction oflight due to canopy cover and turbidity (Lext). c Conductivity. d Dissolved organic carbon. e Phosphate, P043-; Nitrate, N03- ; and Ammonium, NH4+ .

6Table 3. Photosynthetic parameters and atrazine toxicity (from Guasch and Sabater 1998). The initial slope (a) and inhibition parameter (P) are given in J,lgC cm-2 h-Ij J,lmol m-2 S-l (estimated parameter and standard deviation). Units for irradiance ofsaturation (Is•t) and irradiance ofinhibition (Ib) in J,lmol m-2 fl. Effective atrazine concentration (ECso and EClO) means and standard deviations evaluated from concentrationresponse curve performed at light saturation and converted by reviewer to J,lg L-I

.
Site Light a 

### p Is•t Ib ECso (J,lg L-I) ECIO conditions' (J,lg L-I)

Avenc6 (Avo) Open 1.89 ± 0.46 0.057 ± 50 1754 295.5 ± 144.51b 97.06 ± 1O.78b 0.024 Castanyadell Open ND ND ND ND 291.2 ± O.OOb 127.25 ± 114.31 b (Co) Gaia (G) Open 2.37 ± 0.57 0.048 ± 42 2083 327.8 ± O.OOb 60.39 ± O.OOb 0.016 Ter (T) Open 1.96 ± 0.50 0.058 ± 51 1724 90.59c 6.47c 0.031 Avenc6 (Avs) Closed 1.88 ± 0.68 0.54± 59 203 552.1 ± 153.13 d 155.29 ± 107.84d 0.18 Castanyadell Closed 6.32 ± 0.66 1.12 ± 17 99 498.2c 138.04c (Cs) 0.43 Riera Major Closed ND ND ND ND 521.9c 157.45c (RM) ND = Not detenmned. a Open or closed with respect to canopy cover. bn=2. c n = 1. dn = 3.



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7

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### atrazine concentration (log /lM) 10-01 10-3 10-2 10'1 100 10' 

### atrazine concentration (log**~M)** Figure 2 .The relationship between logarithm ofatrazine concentration (,...M) and photosynthesis (% of controls) for periphyton from sites with open canopy cover (Guasch and Sabater 1998). Four irradiance levels for Avenco open, Gaia and Ter; two irradiance levels for Castanyadell open.

8Table 4. Effect ofatrazine exposure on the photosynthesis ofperiphyton communities using various irradiation intensity, from sites with open canopy cover ECso and EClOa from Guasch and Sabater (1998, Table 6).
Site Irradiation ECso (/lg L-l) ECIO (J-Lg L-l) (J-Lmol m-2 S·l) 56 120.78 ± 30.20 10.568 ± 2.804 192 399.01 ± 79.80 104.820 ± 21.137 Avenc6 (open) 270 191.96±47.45 90.370 ± 2.588 1575 45.29 ± 21.57 0.431 ± 0.216 23 159.60 ± 25.88 11.647 ± 1.941 125 327.83 ± 88.43 61.037 ± 16.392 Gaia 304 327.83 ± 51.76 59.959 ± 8.627 1703 364.50 ± 196.27 86.49 ± 46.37

82 362.34± 88.43 207.484 ± 51.116 Castanyadell (open) 1375 222.15 ± 45.29 45.077 ± 8.627 9.3 NI NI 51 332.15 ± 69.02 64.06 ± 13.80 Ter 189 90.59 ± 21.57 7.117 ± 1.725 1241 131.56 ± 32.35 7.980 ± 1.941 NI = No inhibition by atrazine detected. 'Converted to Jlg L- l by reviewer (see attached Worksheet).



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### atrazine concentration (log 

### J,.iM)

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### atrazine concentration- (log**~M)** Figure 3. The relationship between logarithm ofatrazine concentration (JIM) and photosynthesis (% of controls) for periphyton from sites with closed canopy cover (Guasch and Sabater 1998). Four irradiance levels for Avenc6 closed and Castanyadell closed; one irradiance level for Riera Major.

10Table 5. Effect ofatrazine exposure on the photosynthesis ofperiphyton communities aftervaried irradiation, from sites with closed canopy cover, ECso and EClOa from Guasch and Sabater (1998, Table 6).
Site Irradiation ECso (J.lg L-ll EC IO (J.lg L-l) (J.lIDol m-2 S-l) 56 716.06 ± 183.33 270.247 ± 69.449 192 414.11 ± 99.21 55.430 ± 14.235 Avenc6 (closed) 270 526.26 ± 120.78 141.055 ± 33.352 1575 NI Nt Riera Major 51 521.95 ± 120.78 158.309 ± 18.764 17 129.41 ± 30.20 47.665 ± 11.215 82 498.22 ± 10.78 138.251 ± 3.235 Castanyadell (closed) 258 601.75 ± 159.60 139.761 ± 36.881 1375 Nt Nt Nt = No inhibition by atrazine detected. aConverted to /lg L- l by reviewer.

11

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12

### Data Summary Table: Time Weighted Magnitude of Nominal Average Atrazine Day of Study Atrazine DidRecovery Effect Effect Concen- Effect Concentration Occur

- 

### if so, Comments relative to controls tration Occurred from Day Oto when? (Ilg/ L

) When Effect Occurred Up to 62% Magnitude ofeffects increase, 58% estimated from decrease, or Not Figures 2 and 3. no effect applicable Reasons for different depending on 11 1 (2 hours) Not calculable (only one responses across test experimental time point sites are uncertain, but conditions measured) are likely influenced (site and light by different regime) environmental Up to 42% conditions and increase, 58% presence ofdifferent decrease, or Not species. Atrazineno effect applicable related effects are depending on 108 1 (2 hours) Not calculable (only one considered to have experimental time point occurred at all conditions measured) treatment (site and light concentrations. regime) Change in Up to 200% Statistical test between photosynthesis increase, 83% treatment and control decrease, or Not not provided. no effect applicable Treatment and control depending on 216 1 (2 hours) Not calculable (only one responses with experimental time point overlapping errorbars conditions measured) are not considered (site and light effects (the study regime) authors do not define whatthe errorbars Up to 160% represent). increase, 95% Magnitude ofeffects decreased, or Not was calculated for no effect applicable each site and light depending on 1078 1 (2 hours) Not calculable (only one regime combination as experimental time point photosynthesis (% of conditions measured) controls) inthe (site and light atrazine control (0 regime) Ilg/L) compared to the atrazine treatment. Values not proVIded for madlances at less than lIght saturatIOn.



### Reviewer Comments: Atrazine was described as "pure," but no percentage was provided.

13The use ofa solvent was not reported.

The authors reported analyzing atrazine concentrations in the stream water and nominal concentrations used in the toxicity test, however, no measurements were provided for the toxicity tests (treatments or controls). Atrazine concentration was below the limit ofdetection «0.0005**~mol/L** or <0.1 07**~g/L)** for all sampling sites. Samples were taken for atrazine measurement after the periphyton colonization period.

For each light regime, photosynthesis in the atrazine treated and non-atrazine treated communities (i.e. no atrazine controls) is expressed as a percentage ofthe controls. It is unclear what constitutes a control for this purpose. It is assumed that these controls did not contain atrazine. Because photosynthesis (as a % ofcontrols) was 100% or lower for the non-atrazine control communities (see figures 2 and 3), photosynthesis may have been normalized to measurements taken at light saturation conditions (i.e., maximum photosynthesis) in samples without atrazine. Not enough information was provided to be certain.

Saturation irradiance was not clearly presented. The study authors report that saturation irradiance for all sites was around 50**~mol** m,2 S·1 (table 3) but for the toxicity tests, saturation irradiance was higher for most ofthe sites (PS % sat as presented in tables 4 and 5). It is not clear ifatrazine exposure affected saturation irradiance or ifthe difference in reported saturation irradiances for each site had a different meaning. Additionally, the "PS % sat" data for some sites appears to be inconsistent with results plotted in figures 2 and 3. For instance, table 5 states that the 56, 192, and 279**~mol** m·2 S·1 irradiation regimes were saturating irradiances (100%) but dataplotted in figure 3 suggests that only the 279**~mol** m,2 S·1 irradiation regime was saturating (100% ofcontrols). Not enough information was provided to determine what this difference indicates.

14

### Description ofUse in Document: Invalid for the purpose ofsetting an LOC for atrazine



### Rationale for Use: Exposure durations were approximately 2 hours. The purpose ofthis analysis is to estimate atrazine levels that produce community level effects. The ecological relevance ofa reduction in photosynthesis for a 2-hour duration is uncertain. Therefore, this study was not used for the purpose <;>fsetting a community level effectbased LOC because the time duration was insufficient to determine ifcommunity level effects could occur from exposures used in this study.



### Limitations ofStudy: Atrazine purity was not reported. The method ofatrazine dissolution in the treatments was not described (eg, solvent or no solvent). No measurements were reported for atrazine concentrations in the toxicity tests (treatments or controls). Finally, the duration of exposure was extremely short (2hrs).


### 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 and Effects Division U.S. Environmental Protection Agency

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15