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 of periphyton to atrazine exposure. Archives of

Environmental Contamination and Toxicology. 52(1): 32-37. (MRID 48261114)

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 of Review:

September 16, 2009

Purpose of Study:

This study examines the influence of phosphate on atrazine sensitivity ofperiphyton

communities from a river site at the Netherlands-Belgium border.

Summary of Study 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 that the site was

"relatively clean" compared to 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 cm

2

) 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 aquaria in 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

2087842
conditions: stirring (ca. 0.1 m/sec) with paddles; water temperature maintained at 12°C;

light/dark cycle, 12 h/12 h (110 Ilmol photons/m2 sec).

For Experiment 1, six aquaria were 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 aquaria were 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 in the form ofK2HP04;

atrazine was added from a concentrated stock solution (no details provided) dissolved in

acetone. The final concentration of acetone in the treated aquaria was < 0.001% (v/v).

Treatment effects were determined by weekly measurements of chlorophyll concentration

and measurement of photosynthetic activity and tolerance induction after 3 weeks of

treatment (study termination). Tolerance induction was measured in short-term tests

following the pollution-induced community tolerance (PICT) approach of Paulsson

(2000).

Before and after each renewal oftest solutions in the laboratory (no details provided),

samples were analyzed for actual phosphate and atrazine concentrations; the pH was also

determined at these 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 of chlorophyll-a from five

colonized glass substrata per aquarium. Chlorophyll-a measurements were performed

photometrically after sonication for 4 minutes and extraction in 90% acetone.

Short-term atrazine toxicity tests were performed with the samples of Experiment 1 and 2

using the procedure described by Nystrom et al. (2000). At experiment termination (3

weeks), 40-50 periphyton glass discs were removed from each aquarium and separated

from the polyethylene holders. The periphyton glass discs were stored unfiltered control

water during handling. The glass discs were distributed among 20-mL glass vials with 2

mL ofriver water (previously strained through a glass-fiber filter, GF/F). An equal

volume of atrazine 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 incubator with continuous shaking and light

and temperature conditions matching those ofthe aquaria. After 60 minutes of preexposure, 100 ilL of 14C-HC03 solution was added; the 14C-HC03 solution was prepared

from a 200-fold dilution of a stock solution ofspecific activity 2 mCi/mL using milli-Q

water adjusted to a pH of 9 using 0.1 M NaOH. After 1 hour of incubation, the reaction

was quenched by the addition offormaldehyde to a final concentration of2% (v/v). To

eliminate remaining inorganic carbon, 200 ilL of concentrated acetic acid was added, and

the samples were dried at 60°C under a stream of air. Release of incorporated 14C carbon

2
from periphyton was enhanced by the addition of 1 mL of dimethyl sulfoxide. The

radioactivity was measured in the samples using LSC. ECso values were calculated from

DPM data using a nonlinear fit procedure. The photosynthetic rate from the controls was

measured as the incorporation of 14C-HC03. C-uptake ~er incubated glass was calculated

taking into account the incorporation of 14C, 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' on photosynthetic 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-term toxicity (EC50 for photosynthetic activity)

of atrazine on periphyton communities after 3 weeks in the experimental aquaria

(treatments & controls)..

Differences between treatments in phosphate and atrazine concentration, algal biomass,

photosynthesis and EC50 values were examined by analysis of variance (ANOVA) using

the STATISTICA program. Between-factors ANOVA ofrepeated measures was used to

determine treatment effects over time (time as a repeated factor). Differences between

treatments for each sampling date were also determined by between-factors ANOVA.

For the first experiment, the factors included in the ANOVA were time (with three levels)

and phosphate (also with three levels). For the 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 post hoc by a Turkey

HSD test.

Results:

Concentrations ofDissolved Compounds

ThebackgroUfld concentrations (means ± SD) of phosphate 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 water pH (7.7 ± 0.3) and conductivity (481 ± 33 j.!S/cm) were similar in 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 water renewal; phosphate

concentrations before water renewals were 20-45% ofthe nominal values. Atrazine

concentration after water renewal was 46% above the nominal concentration and showed

small changes over time. Before water renewal, atrazine concentration was always above

80% ofthe initial measured concentration. Atrazine was not measured in the controls.

Changes in pH were also observed after and before water renewal. Analysis ofthe waters

ofExperiments 1 and 2 after water renewal (initial conditions) and 2-3 days later before

water renewal (final conditions) is reported in Table 1; phosphate concentration results

are illustrated in Figure 1. The water renewal procedure is not explained and UfIclear.

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Table 1. Measured phosphate and atrazine concentration and pH in the water of different (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)

4
200

400

ATR P+ATR

b b

c P

exp2

o

600

200

400

c

c P-1 P-2

600

!

{

.c

c.

5....------------------,

5..-----------------,

c P ATR P+ATR

2

exp2

8 a

o

8..-------------------. P-2

b

c P-1

exp 1

OL-_-

exp 1

0"'----

c

b

P-1 P-2

5

3

2

o

exp2

c

b

P 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

of 14C-HC03). Values are means (standard error) of 10 replicates obtained from two aquaria after 3

weeks of treatment. Different letters indicate significant differences between treatments analyzed

post hoc by one- and two-way analysis of variance.

Periphyton communities

In Experiment 1, the addition of phosphate (three weeks oftreatment) did not affect the

community response to atrazine. After 2 hours exposure to atrazine (eight

concentrations) the EC50 values for photosynthetic activity (ranging between 216 and

5
308 !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 of phosphate affected the

algal growth and carbon uptake (photosynthesis). The chlorophyll concentration ofthe

control group did not change significantly over time (from 0.41-0.59 !lg chl-a/cm2 to ca.

1.0 !lg chl-a/cm2

; day achlorophyll concentration 0.29 ± 0.12 !lg chl-a/cm2

; p-value not

reported); however a three- to four-fold increase over time 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 over time 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 indicated that 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 of exposure, phosphate addition also caused a

significant enhancement (three- to four-fold increase) of photosynthesis (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 of five colonized substrata in

the different aquaria after 1,2, and 3 weeks of exposure (wkl, wk2 and wk3, respectively).

Photosynthetic activity (PS) in f.lg C/cm2 h at the end of the experiment (after 3 weeks of exposure).

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)

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In Experiment 2, algal biomass was affected by addition of atrazine. The chlorophyll

concentration increased in all treatments and controls over time. Chlorophyll

concentration did not change in response to phosphate treatment. A smaller increase 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 of exposure, phosphate addition

caused a significant enhancement of photosynthesis (factor phosphate: F = 8.44,p =

0.006) (Table 2, Figure 1).. After three 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 in ATR +

P; p-value not reported), but the effects of atrazine 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 not previously exposed to experimental addition of atrazine did not differ

significantly (control: 293 IJ,g/L; P treatments: 235 IJ,g/L ; p-value not reported) and were

similar to those obtained for Experiment 1. The EC50 values for the ATR and ATR + P

treatments indicated that long-term addition of atrazine 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-term tests

measuring the effective atrazine concentration for inhibiting the photosynthesis of

periphyton are illustrated in Figure 2.

I tI

, e.p2 I b

I :! ,

II i

I

I

n

I

.a I

r I

I

!

I I ,

l i •

m'l '''-<''''~,,',,------.....,

cooj exp1

J

1:

-~. .DD

0 II 1lI i1

III >:L_ ,'"

i

C P-1 P..2; c p ATR P+ATR

Figure 2. Effective atrazine concentration (EC50) obtained from short-term tests (2 h) of atrazine

(eight concentrations) versus the photosynthesis ofperiphyton (measured as the incorporation of 14C_

HC03) after 3 weeks of treatment. Black and white bars correspond to periphyton communities

from different aquaria. Different letters indicate significant differences between treatments obtained

by analysis of variance.

7
Overall, the study authors determined that the enhancement of photosynthesis or increase

in algal biomass after P-addition was indicative of P-limitation since the conflicting

results of Experiment 1 and 2 appeared to be due to a difference in the background

phosphate levels and algal biomass. Phosphate supply could not reduce the inhibitory

effect of atrazine on periphyton and did not increase atrazine tolerance (measured by

EC50 in acute toxicity tests). Long-term exposure to atrazine induced tolerance ofthe

periphyton community to atrazine, but this was not influenced by phosphate addition.

Data Summary Table:

Time

Nominal Weighted

Magnitude of Atrazine Day of Average Did

Effect Effect relative 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 ATR treatment 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 C4

C 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 C4

C 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.

8
Reviewer Comments:

Atrazine purity was not reported.

In another reviewed study, Guasch et al. (1998) also performed experiments on

periphyton in the Keersop.

The measured concentration of atrazine was ~40% above nominal (Table 1). It was

unclear when these measurem~nts were made as the study does not provide sufficient

details about the renewal oftest solutions that were performed. Atrazine concentrations

were not reported for any aquaria that did not have experimental addition of atrazine.

The authors report that the communities and water used for the experiments carne from a

"relatively clean stream that is a tributary of a polluted river" although no data was

provided as to background concentrations of atrazine 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 that these levels were considered "an indication of episodes of higher

concentrations that were not recorded because oflimited sampling". Given the lack of

information about atrazine in the stream or in the controls it is uncertain if part or all of

the 40% increased atrazine concentration over nominal 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 of Use in Document: Invalid for the purpose ofsetting an LOC.

Rationale for Use: Water was obtained from a site with known contamination, and there

is evidence that atrazine levels in controls may have been sufficient to affect the results of

this study (see discussion in the "Limitations of Study" section below).

Limitations of Study: The results have good applicability to mesocosms or natural

aquatic systems. However, there is uncertainty about the background levels of atrazine in

the controls and treatments. No effort was made to document whether background

concentrations of other chemicals (including atrazine) were present in the treatment

and/or control microcosms prior to experiment initiation. The authors indicated that the

water used in the study is from a watershed with known contamination of atrazine,

although it is not clear what background levels may have been at the 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

9
caused by errors in dosing the aquaria; however, it may have also been the result of using

contaminated water. Therefore, the results ofthis study may have been confounded by

possible atrazine contamination in the control groups.

The methodology included renewal oftest solutions but the details ofthe procedure were

not reported. Water chemistry ofthe microcosms was not adequately monitored prior to

or following treatment.

Primary Reviewer: Lisa Muto, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA jf~": i:~c~~

Tertiary Reviewer: Brian Anderson, Biologist, EPA

;:

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