Open Literature Review Summary

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

ECOTOX Record Number and Citation: Guasch, H., V. Lehmann, B. Van Beusekom, S. Sabater, and W. Admiraal. 2007. Influence ofphosphate on the response ofperiphyton to atrazine exposure. Archives of Environmental Contamination and Toxicology. 52(1): 32-37. (MRID 48261114)

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: September 16, 2009

Purpose ofStudy: This study examines the influence ofphosphate on atrazine sensitivity ofperiphyton communities from a river site atthe Netherlands-Belgium border.

Summary ofStudy Findings

Methods: The study site was the River Keersop in The Netherlands (51 ° 20'N, 5° 24'E). Keersop is a tributary ofthe polluted River Dommel. The authors state thatthe site was "relatively clean" comparedto the polluted Dommel. The sampling was performed in July and September 1996 for Experiment 1 and Experiment 2, respectively (experiments described below). Periphyton communities were allowed to colonize the surface ofsandblasted glass discs (1.5 cm2) mounted on racks which were suspended in the river for 1 week. Colonized samples were transported to the laboratory in cool-boxes and transferred to the experimental aquariain the laboratory.

Experimental units were lO-L indoor aquaria described in Invorra 

### et

### at. (2002). Each aquarium was filled with 6 L ofriver water and contained 18 polyethylene holders of 10 glass substrata (total of 180). The aquaria were maintained with the following

1 

# '111111111 2087842conditions: stirring (ca. 0.1 m/sec) with paddles; watertemperature maintained at 12°C; light/dark cycle, 12 h/12 h (110 Ilmol photons/m2 sec). For Experiment 1, six aquariawere used: two controls (C), two with 300 Ilg 

### P/L (P-l) and two with 600 Ilg 

### P/L (P-2). For Experiment 2, eight aquariawere used: two controls (C), two with 300 Ilg 

### P/L (P), two with 100 Ilg atrazine/L (ATR), and two with 300 Ilg 

### P/L and 100 Ilg atrazine/L (P 

### + ATR). Phosphate was added inthe form ofK2HP04; atrazine was added from a concentrated stock solution (no details provided) dissolved in acetone. The final concentration ofacetone in the treated aquariawas 

### < 0.001% (v/v).

Treatment effects were determined by weekly measurements ofchlorophyll concentration and measurement ofphotosynthetic activity and tolerance induction after 3 weeks of treatment (study termination). Tolerance induction was measured in short-term tests following the pollution-inducedcommunity tolerance (PICT) approach ofPaulsson (2000).

Before and after eachrenewal oftest solutions inthe laboratory (no details provided), samples were analyzed for actual phosphate and atrazine concentrations; the pH was also determined atthese intervals. Phosphate concentration was determined according to Murphy and Riley (1962). Atrazine concentration was determined by HPLC using the following conditions: Intersil ODS analytical column (4.6 x 250 mm, 2.5 11m) and UV detection (220 nm).

Algal biomass was determined weekly by the measurement ofchlorophyll-a from five colonized glass substrataper aquarium. Chlorophyll-ameasurements were performed photometrically after sonication for 4 minutes and extraction in 90% acetone.

Short-term atrazine toxicity tests were performed withthe samples ofExperiment 1 and 2 using the procedure described by Nystrom 

### etal. (2000). At experimenttermination (3 weeks), 40-50 periphyton glass discs were removed from each aquarium and separated from the polyethylene holders. The periphyton glass discs were storedunfiltered control water during handling. The glass discs were distributed among 20-mL glass vials with 2 mL ofriver water (previously strainedthrough a glass-fiber filter, GF/F). An equal volume ofatrazine solution was distributed from double-concentrated stock solutions (prepared with filtered river water) in a geometric concentration series (0,0.43,21.6, 43.1, 216, 431, 2157 and 4314 Ilg atrazine/L). Five replicates were prepared for each concentration. Samples were incubated in an incubatorwith continuous shaking and light and temperature conditions matching those ofthe aquaria. After 60 minutes ofpreexposure, 100 ilL of14C-HC03 solution was added; the 14C-HC03 solution was prepared from a 200-fold dilution ofa stock solution ofspecific activity 2 mCi/mL using milli-Q water adjusted to a pH of9 using 0.1 M NaOH. After 1 hour ofincubation, the reaction
was quenched by the addition offormaldehyde to a final concentration of2% (v/v). To eliminateremaining inorganic carbon, 200 ilL ofconcentrated acetic acid was added, and the samples were dried at 60°C under a stream ofair. Release ofincorporated 14C carbon

2from periphyton was enhanced by the addition of 1 mL ofdimethyl sulfoxide. The radioactivity was measured in the samples using LSC. EC

### so values were calculated from DPM data using a nonlinear fit procedure. The photosynthetic rate from the controls was measured as the incorporation of14C-HC03

. C-uptake**~er** incubated glass was calculated taking into account the incorporation of14C, the added 4C-HC03 and dissolved inorganic carbon (DIC). The controls (no atrazine addition) from the short-term toxicity testing
were used to characterize the 'long-term toxicity effects' onphotosynthetic activity of periphyton communities after 3 weeks in the experimental aquaria (treatments & controls). In contrast, the full concentration series (7 atrazine concentrations and 1 control) was used to determine the short-termtoxicity (EC50 for photosynthetic activity) ofatrazine onperiphyton communities after 3 weeks inthe experimental aquaria (treatments & controls)

..

Differences between treatments in phosphate and atrazine concentration, algal biomass, photosynthesis and EC50 values were examined by analysis ofvariance (ANOVA) using the STATISTICA program. Between-factors ANOVA ofrepeated measures was used to determine treatment effects overtime (time as a repeated factor). Differences between treatments for each sampling date were also determined by between-factors ANOVA. Forthe first experiment, the factors included inthe ANOVA were time (with three levels) and phosphate (also with three levels). Forthe second experiment, three factors were included: time (with three levels); phosphate (with two levels); and atrazine (also with two levels). For each date, treatment effects were then analyzed

### posthoc by a Turkey HSD test.



### Results: Concentrations of DissolvedCompounds ThebackgroUfld concentrations (means ± SD) ofphosphate were slightly lower (74 ± 23 j.!g 

### P/L) and more stable during Experiment 1 than Experiment 2 (114 ± 106 j.!g 

### P/L). Stream waterpH (7.7 ± 0.3) and conductivity (481 ± 33 j.!S/cm) were similarin both periods. Total inorganic carbon (TIC), total organic carbon, DIC and dissolved organic carbon were 22,8,21 and 7 mg/L, respectively. A good agreement between nominal and actual phosphate concentration was always found after waterrenewal; phosphate concentrations before waterrenewals were 20-45% ofthe nominal values. Atrazine concentration after waterrenewal was 46% above the nominal concentration and showed small changes overtime. Before waterrenewal, atrazine concentration was always above 80% ofthe initial measured concentration. Atrazine was not measured inthe controls. Changes inpH were also observed after and before waterrenewal. Analysis ofthe waters ofExperiments 1 and 2 after waterrenewal (initial conditions) and 2-3 days later before waterrenewal (final conditions) is reported in Table 1; phosphate concentrationresults
are illustrated in 

### Figure 1. The water renewal procedure is not explained and UfIclear.

3Table 1. Measured phosphate and atrazine concentration and pH in the water ofdifferent (duplicate) treatments after water renewal and phosphate and/or atrazine addition (initial conditions), and 2-3 days later before water renewal (final conditions). Values reported as means (standard deviation) throughout the exneriment (seven times). Experiment 1 Parameter Control P-l (300 Jig PIL) P-2 (600 Jig PIL)
Initial Final Initial Final Initial Final Phosphate (Ilg P/L) 74 (23) 33 (6) 320 62 (10) 579 185 (74) (37) (34) pH 7.6 8.7 7.7 9.5 7.7 9.7 (0.2) (0.2) (0.2) (0.4) (0.2) (0.4) Experiment 2 Control P (300 Jig PIL) ATR (l00 Jig/L) P+ATR Initial Final Initial Final Initial Final Initial Final Phosphate (Ilg P/L) 114 45 (35) 341 95 (65) 114 46 (38) 349 135 (106) (118) (103) (114) (67) Atrazine (Ilg /L) 146 138 (8) 146 137



### -- -- -- -- (25) (24) (12) pH 7.6 9.3 7.7 9.5 7.7 8.8 7.7 8.8 (0.3) (Ll) (0.3) (1.2) (0.3) (1.0) (0.3) (1.0)

4200 

### 400



### ATR P+ATR b b

P 

## c exp2



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200 

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## c 600 

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### ATR P+ATR Figure 1. Phosphate concentration after water renewal in experiment 1 and experiment 2. Values are means (standard deviation) ofseven samples taken throughout the experiment (upper graphs). Algal biomass (measured as chlorophyll concentration) and photosynthesis (measured as the incorporation of14C-HC03). Values are means (standard error) of10 replicates obtained from two aquaria after3 weeks oftreatment. Different letters indicate significant differences between treatments analyzed post hoc by one- and two-way analysis ofvariance.



### Periphyton communities In Experiment 1, the addition ofphosphate (three weeks oftreatment) did not affectthe community response to atrazine. After 2 hours exposure to atrazine (eight concentrations) the EC50 values for photosynthetic activity (ranging between 216 and

5308 !lg/L) did not show any response to the experiment 1 phosphate additions compared to the controls. The following results from experiment 1 do not involve addition of atrazine but are included as supplemental info. The addition ofphosphate affected the algal growth and carbon uptake (photosynthesis). The chlorophyll concentration ofthe control group did not change significantly overtime (from 0.41-0.59 !lg chl-a/cm2to ca. 1.0 !lg chl-a/cm2; day

## achlorophyll concentration 0.29 ± 0.12 !lg chl-a/cm2; p-value not reported); however athree- to four-fold increase overtime was observed in both levels of phosphate treatment (from 0.61-0.81 to 3.3-4.0 !lg chl-a/cm2 for P-1 and from 0.20-0.43 to 2.4-3.5 !lg chl-a/cm2 for P-2). The ANOVA results for significant changes in and chlorophyll overtime in phosphate treatments were the following: factor time: 

### F

### = 116.,89,p 

### < 0.00001; factor phosphate: F= 60.2,p 

### < 0.00001). Post-hoc comparison between treatments for each sampling date indicatedthat chlorophyll was not enhanced by P-addition after 1 week oftreatment, but was higher in P-1 and P-2 groups in week 2 and 3 (week 2: factor phosphate: F

### = 37.7,p < O.00001; week 3: factor phosphate: F = 32.3,p < 0.00001). After 3 weeks ofexposure, phosphate addition also caused a significant enhancement (three- to four-fold increase) ofphotosynthesis (factor phosphate: F= 48.5,p < 0.00001). The algal biomass and photosynthesis results of Experiment 1 are reported in Figure 1 and Table 2.

Table 2. Algal biomass (chlorophyll concentration, chI) in f.lg chl-a/cm2 offive colonized substrata in the different aquaria after 1,2, and 3 weeks ofexposure (wkl, wk2 and wk3, respectively). Photosynthetic activity (PS) in f.lg C/cm2 h at the end ofthe experiment (after 3 weeks ofexposure). Values reported as means (standard deviation). Experiment 1 Algal biomass and Control P-l (300 f.lg PIL) P-2 (600 f.lg PIL)

photosynthesis Aqu. I Aqu.2 Aqu.1 Aqu.2 Aqu.l Aqu.2 Ch1-wk1 0.59 0.41 0.81 0.61 0.43 0.20 (0.3) (0.1) (0.3) (0.3) (0.2) (0.1) Chl-wk2 1.0 1.1 2.5 2.4 2.5 2.1 (0.1) (0.1) (0.5) (0.6) (0.3) (0.3) Chl-wk3 0.95 0.93 4.0 3.3 3.5 2.4 (0.2) (0.1) (1.1) (0.6) (1.2) (0.8) PS-wk3 0.98 1.03 3.6 4.3 2.9 3.2 (0.1) (0.1) (0.4) (0.5) (0.4) (0.7) Experiment 2 Control P (300 f.lg PIL) ATR (100 f.lglL) P+ATR Aqu.l Aqu.2 Aqu.l Aqu.2 Aqu.l Aqu.2 Aqu.l Aqu.2 Chl-wkl 1.5 2.1 1.6 1.8 1.3 1.1 1.3 1.0 (0.1) (0.4) (0.2) (0.0) (0.2) (0.2) (0.1) (0.3) Chl-wk2 5.0 3.6 3.9 3.6 2.6 3.3 3.1 2.6 (0.5) (0.3) (0.5) (0.3) (0.5) (0.4) (0.4) (0.1) Chl-wk3 5.4 5.2 3.4 5.4 3.0 3.0 2.2 3.2 (0.6) (0.8) (0.6) (1.1) (0.6) (0.3) (0.7) (0.9) PS-wk3 2.2 3.3 2.7 5.6 1.9 2.4 2.1 3.2 (0.3) (0.5) (0.4) (1.0) (0.4) (0.4) (0.6) (0.7)

6In Experiment 2, algal biomass was affected by addition ofatrazine. The chlorophyll concentration increased in all treatments and controls overtime. Chlorophyll concentration did not change in response to phosphate treatment. A smallerincrease in chlorophyll (compared to the controls) was observed in the samples with atrazine treatment, leading to a 40% reduction (compared to the controls) in algal biomass after 3 weeks 

### (Table 2, 

### Figure 1). The ANOVA results confirming these observations were: factor time: F= 87.8,p < 0.00001; factor phosphate: F= 4.63,p = 0.03 and factor atrazine: F= 65.6,p 

### < 0.00001). Post-hoc comparison showed that the effect ofatrazine
was significant from week 1 onwards (week 1: F= 21.8,p 

### < 0.001; week 2: F= 25.2,p < 0.00001; week 3: F= 46,p < 0.0001). After 3 weeks ofexposure, phosphate addition caused a significant enhancement ofphotosynthesis (factorphosphate: F = 8.44,p 

### = 0.006) 

### (Table 2, 

### Figure 1)

.. Afterthree weeks, photosynthesis was reduced by atrazine in the aquaria with P supply (2.7-5.6 IJ,g C/cm2 h in P and 2.1-3.2 IJ,g C/cm2 h inATR

### + P; p-value notreported), butthe effects ofatrazine were not significant in the aquaria without P supply (2.2-3.3 IJ,g C/cm2 h in C and 1.9-2.4 IJ,g C/cm2 h in ATR; p-value not reported). For short-term atrazine exposure (2hr), the EC50 values ofExperiment 2 communities notpreviously exposed to experimental addition ofatrazine did not differ significantly (control: 293 IJ,g/L; P treatments: 235 IJ,g/L ; p-value notreported) and were similarto those obtained for Experiment 1. The EC50 values for the ATR and ATR

### +P treatments indicated that long-term addition ofatrazine reduced short-term toxicity; EC50 values were 

### ca. 500-750 IJ,g/L for ATR and 

### ca. 650 IJ,g/L for ATR 

### +P (values estimated by the primary reviewer from 

### Figure 2). The algal biomass and photosynthesis results of Experiment 2 are reported in 

### Table 2 and 

### Figure 1. The results ofthe short-termtests measuring the effective atrazine concentration for inhibiting the photosynthesis of periphyton are illustrated in 

### Figure 2.

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# c p ATR P+ATR Figure 2. Effective atrazine concentration (EC50) obtained from short-term tests (2 h) ofatrazine (eight concentrations) versus the photosynthesis ofperiphyton (measured as the incorporation of14C_ HC03) after 3 weeks oftreatment. Blackand white bars correspond to periphyton communities from different aquaria. Different letters indicate significant differences between treatments obtained by analysis ofvariance.

7Overall, the study authors determined thatthe enhancement ofphotosynthesis or increase in algal biomass after P-addition was indicative ofP-limitation since the conflicting results ofExperiment 1 and 2 appeared to be due to a difference inthe background phosphate levels and algal biomass. Phosphate supply could notreduce the inhibitory effect ofatrazine onperiphyton and did not increase atrazine tolerance (measuredby EC50 in acute toxicity tests). Long-term exposure to atrazine inducedtolerance ofthe periphyton community to atrazine, butthis was not influenced by phosphate addition.

Data Summary Table: Time Nominal Weighted Magnitude of Atrazine Day of Average Did Effect Effectrelative Concen- Study Effect Atrazine Recovery Comments to controls tration Occurred Concentration Occur-if (pg/L) from Day Oto so, when? When Effect

Occurred Calculated from values in table 2. Algal biomass**~35%** 1 week ATRtreatment 100/lg/L (assume 7 Not calculable No (chlorophyll a) reduction days) Significant effect of atrazine (p < 0.001). ATR treatment, evaluated at 3 weeks. Photosynthetic Based on activity 

### C 4C No effect Not Not activity of uptake): long- 100/lglL Not applicable term effects observed applicable applicable communities after 3 weeks exposure to 100 /lg atrazine/L (Figure 1 and Table 2) EC50s of controls and ATR Photosynthetic treatments activity 

### C 4C 50% reduction**~250** /lgiL 2 hours Not calculable Not ranged from uptake): short- (EC50) applicable**~250** to**~750** term effects /lgiL
Estimated from Figure 2.

8Reviewer Comments: Atrazine purity was notreported.

In anotherreviewed study, Guasch et al. (1998) also performed experiments on periphyton in the Keersop.

The measured concentration ofatrazine was**~40%** above nominal (Table 1). It was unclear whenthese**measurem~nts** were made as the study does notprovide sufficient details about the renewal oftest solutions that were performed. Atrazine concentrations
were not reported for any aquariathat did nothave experimental addition ofatrazine. The authors report thatthe communities and water used for the experiments carne from a "relatively clean streamthat is atributary ofapollutedriver" although no datawas provided as to background concentrations ofatrazine in the stream. As part ofthe discussion the author's reference background concentrations of< 0.5 /lg/L measured somewhere in the same river system (the details are not clear and may be from the literature) and thatthese levels were considered "an indication ofepisodes ofhigher concentrations that were notrecorded because oflimited sampling". Given the lackof information about atrazine in the stream or in the controls it is uncertain ifpart or all of the 40% increased atrazine concentration overnominal may be explained by background contamination.

The small size ofthe experimental aquaria (10 L tank filled with 6 liters river water) may have introduced container effects.

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

Rationale for Use: Water was obtained from a site withknown contamination, and there is evidence that atrazine levels in controls may have been sufficientto affectthe results of this study (see discussion in the "Limitations ofStudy" sectionbelow).

Limitations ofStudy: The results have good applicability to mesocosms ornatural aquatic systems. However, there is uncertainty aboutthe background levels ofatrazine in the controls and treatments. No effort was made to document whetherbackground concentrations ofother chemicals (including atrazine) were present in the treatment and/or control microcosms priorto experiment initiation. The authors indicated thatthe waterused in the study is from a watershed with known contamination ofatrazine, although itis not clear whatbackground levels may have been atthe study site. Measured atrazine levels in the atrazine-treated aquaria were well above nominal values (146 ug/L measured compared with 100 ug/L nominal). This discrepancy may have been

9caused by errors in dosing the aquaria; however, it may have also beenthe result ofusing contaminated water. Therefore, the results ofthis study may have been confounded by possible atrazine contamination in the control groups.

The methodology includedrenewal oftest solutions butthe details ofthe procedure were notreported. Water chemistry ofthe microcosms was not adequately monitored priorto or following treatment.


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

### Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA

# jf~":**i:~ c~~**  Tertiary Reviewer: BrianAnderson, Biologist, EPA 

## ;:

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