Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Krieger, K.A., D.B. Baker, and lW. Kramer. 1988. Effects of herbicides on stream

Aufivuchs productivity and nutrient uptake. Arch. Environ. Contam. Toxicol. 17:299-

306. (MRID 47543509).

Purpose of Review:

Determine potential effects of atrazine on an aquatic community. Endpoints from this

study were also used to parameterize the Comprehensive Aquatic System Model (CASM)

and establish the model-associated LOC for atrazine.

Date of Review: November 3, 2008

Purpose of Study: This study examines the potential effects of atrazine to attached

aquatic plants and animals (Aufivuchs) in experimental artificial streams.

Summary of Study Findings:

Methods: Two experiments were conducted, the first examining constant exposure to

atrazine, and the second examining pulsed exposure to a mixture of four herbicides. Only

the methods relevant to the first experiment will be described here.

Six replicate streams were used concurrently, each consisting of a 7.5 em outer diameter,

123 cm long Pyrex glass tube stoppered at each end, with outlets and inlets passing

through the rubber stoppers to allow for recirculation ofthe water, addition of fresh

water, nutrients, and herbicide, and discharge of overflow water (see Figure I). The

stoppers were covered by plastic end caps tightened in place with a union bolt along each

side ofthe tube. The .inner walls ofthe partially-filled tubes were the substrate for the

Aufivuchs community. The tubes were arranged horizontally and parallel to each other,

with empty glass tubes on each side to maintain similar lighting patterns for all six

streams. The streams were illuminated by eight 1.22 m, 40 watt, "cool-white"

fluorescent bulbs aligned perpendicularly to the long axis ofthe streams at a height of 50

em above the water surface.

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The atrazine experiment was conducted twice, once at 25°C and once at lOoC, each time

testing the effects of nominal levels of 50 ~g/L and 200 ~g/L atrazine. (Average

concentrations over time were determined to be 24 ~g/L and 134 ~g/L atrazine,

respectively, and these measured concentrations are used throughout the study to refer to

the two levels of exposure.) Each experiment lasted 20 days. During the experiments,

fresh creek water from Rock Creek (a third-order agricultural stream in the Sandusky

Basin, Ohio) and macronutrients (Table 1) were added to each artificial stream, with a

peristaltic pump (Figure 1) supplying stock solutions of KN03, KH2P04, and NaSi03 via

return flow from the recirculating pump. The creek water was added at 7.8 mUmin and

nutrients were added at 1.0 mUmin, so according to the authors the turnover time in each

stream was about 7 hours (not sure ifturnover time is referring to nutrient spiraling, or if

so how it was computed from the provided information alone). The peristaltic pumping

rates declined by about 1.7% pcr day due to the tubing becoming worn, so the tubing was

replaced about every five days.

Daily average input rates for each stream were calculated by daily measurement ofthe

overflow volume and ofthe amount ofstock solution used over two-day intervals. Daily

samples ofthe overflow water were analyzed for nutrient concentrations. Overflow

water was collected and a portion filtered through a prerinsed Gelman Sciences Inc. 0.45-

~m membrane filter for analysis ofsoluble reactive P (SRP), N02 + N03 N (nitrite +

nitrate N), NH3 N (ammonia N), and Si02 (dissolved silica). Another portion was

retained unfiltered for analysis oftotal P and total Kjeldahl N (TKN). Both portions were

refrigerated without preservatives for up to two weeks before analysis. A composite

sample ofRock Creek water, collected continuously between times of overflow sample

collection, was processed similarly. Input and output quantities, respectively, wcrc

calculated by:

IT =Rs ·TCs +CR .(VT -RsT)

o =V .Cp+Cc

T T 2

where

Rs = stock solution pumping rate (Uhr),

Vr = total overflow volume (L) during the interval T,

Cs = stock concentration (mg/L),

CR = composite Rock Creek concentration (mg/L),

T = pumping interval (hr),

Cp = previous artificial stream overflow concentration, and

Cc = overflow concentration at end of present interval.

Processing rates for each nutrient were calculated as (lr - Or)/T. Total N was calculated

indirectly as the sum ofthe processing rates ofTKN (which included ammonia N) and

N02 + N03 N.

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For seven days pre-treatment, the streams were colonized by Aufivuchs entrained in the

creek water supply. Larger organisms were excluded by "the presence oftwo reservoirs"

(unclear what this means) and the small feed line diameters «2 mm) ofthe peristaltic

pump tubing. Starting on Day 8, streams 2 and 5 were continuously exposed to atrazine

at 24 Ilg/L, streams 3 and 6 to 134 Ilg/L, and streams 1 and 4 served as controls. The

herbicide was introduced in solution with the nutrients. Methanol, used to dissolve the

atrazine initially, was present in the high treatment streams at levels of 0.00057% by

volume, and the authors (citing two papers from the literature) do not believe any toxic

effect is likely from the methanol. Sufficient stock atrazine was added directly to each

stream at the start oftreatment to attain the nominal concentration. The stock was

prepared from 98.2% technical grade atrazine from CIBA-GEIGY Corp., Greensboro,

North Carolina.

During each experiment, samples were collected from the artificial streams and from

nutrient and pesticide stock bottles to verify the calculated chemical concentrations. At

the end of each 20-day experiment, the stream systems were dismantled and the

accumulated Aufivuchs inside each Pyrex tube was scraped into a plastic container,

homogenized in tap water at a known volume with a Tekmar Tissumizer, and four

replicate aliquots removed from each homogenate for analysis of chlorophyll a and ash

free dry weight (AFDW).

The nutrient samples were analyzed for total P, SRP, N02 + N03N, ammonia N, TKN,

and dissolved silica by the methods of USEPA, Methods for chemical analysis ofwater

and wastes, 1979. Total P analysis was modified by having samples digested with lIN

sulfuric acid and ammonium persulfate, and ifturbidity was present after digestion, the

samples were passed through a glass filter and processed according to the USEPA 1979

standard method. Instead of a single mixed reagent, the ascorbic acid was used as a

separate reagent and was added directly to the analytical cartridge in order to increase

reagent shelf-life. Distilled water was not used in the cartridge and the sample line size

was doubled to improve sensitivity.

Chlorophyll a was extracted by adding 3 mL of a 1% MgC03 suspension and 72 mL

acetone to 5 mL of fresh homogenized suspension, yielding a 90% acetone solution. The

samples in their polyethylene bottles were sonicated in the dark for 1 hour under

refrigeration and were stored at -10°C for up to 3 weeks. Just prior to analysis, the

samples were mixed and allowed to settle overnight at -lO°C. The supernate was

analyzed by the fluorometric method (American Public Health Association, 1981) using

an Aminco fluoro-colorimeter calibrated against chlorophyll a standards supplied by the

USEPA, Cincinnati, Ohio. Calculations were corrected for pheophytin a and interference

filters (660 nm and 440 nm) were used to eliminate interference from possible turbidity.

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Aliquots of 50 mL homogenized suspension were placed in pre-ashed and tared crucibles

and were analyzed for AFDW, following the method of WeberIUSEPA, Biologicalfield

and laboratory methodsfor measuring the quality ofsurface waters and effluents, 1973.

The method for extraction and analysis of atrazine and the quality control program are

described in detail in a separate paper by two ofthe three co-authors (Kramer and Baker,

American Society for Testing and Materials STP 867, 1985). Extracts were refrigerated

and analyzed within two weeks of preparation, using a temperature-programmed gas

chromatograph (Varian 3700 GC) equipped with a capillary column and nitrogenphosphorus detector. Two standards were analyzed after each set of 10 samples to

maintain calibration. Reagent blanks and spiked samples were also analyzed

"frequently" to check for contaminants and determine the percent recovery. Method

precision was measured by splitting samples in two immediately before extraction and

processing both portions. Approximate detection limits and percent recoveries are

presented in the results. All herbicide concentrations reported throughout the study have

been corrected for percent recovery.

Results for the control and treatments streams were compared by two-sample t tests or by

analysis of variance.

Results: Two experiments were conducted, the first examining constant exposure to

atrazine, and the second examining pulsed exposure to a mixture of four herbicides. Only

the results relevant to the first experiment will be described here.

Atrazine GC analyses were determined to have a detection limit of 0.075 Ilg/L

(uncorrected for percent recovery) and a mean recovery from field spikes of 86%

(standard deviation 15%). As mentioned in the Methods section, the average measured

atrazine concentrations in the treated streams were 24 Ilg/L and 134 Ilg/L in the 50 Ilg/L

and 200 Ilg/L nominal concentration streams, respectively.

AFDW and chlorophyll a showed highly significant (p<O.OO 1, specific test not stated)

inhibition of biomass accumulation at both temperatures during the 12 days oftreatment

at 134 Ilg/L, and both measurement endpoints were also significantly (p<O.O1, no test

stated)) inhibited by 24 Ilg/L at 25°C. At lOoC, however, AFDW and chlorophyll a in

the 24 Ilg/L streams were roughly equal to controls. See Table 2 and Table 3 for

tabulated results.

For nutrient uptake rates, larger differences were found between the two experiments at

different temperatures than were found within each experiment due to atrazine exposure

(no statistical tests cited). There were, however, consistently lower rates of uptake for

SRP, N02 + N03 N and dissolved silica in the 134 Ilg/L streams beginning several days

after atrazine application - after nine days, the N02 + N03 N uptake rate averaged 35%

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lower than controls at lOoe and 25% lower at 25°e, the dissolved silica uptake rate

averaged 31 % lower at lOoe and 58% lower at 25°e, and SRP uptake rate averaged 14%

lower at lOoe and 8% lower at 25°e, although "few ofthese differences were statistically

significant (p<O.05) because ofrelatively large within-treatment variation" (no details on

precisely which values were significant or with what tests). In the 24 llg/L atrazine

concentration streams, uptake rates of SRP and dissolved silica remained as high or

higher than control rates, while average N02 + N03 N uptake rates were depressed

relative to controls by about 10% at 10°C and 21 % at 25°C after nine days since addition,

but these differences were not statistically significant. The changing uptake rates showed

distinct curves for the two experiments at different temperatures (see Figure 2), and the

authors speculate that this is due to temperature effects on the development rates ofthe

algal communities.

. " •.",...--.- ......; •• ~.~ ...._, -,....... _..... .... .,.~ .. .. ......,."..'..,....-.,,_ •...,

;JtA

Figure 1. Schematic of artificial stream system showing one stream, nutrient and herbicide

containers (NP) and sample bottles (SB) receiving stream overflow water. RP = recirculating pump,

PP = peristaltic pump, CP = centrifugal pump, HB = constant head box for gravity feed of creek

water, AEP = air exchange port. Arrows show directions of flow.

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25 C

o "-........8--I...{)-...-I..11---H---'-I...Oc......

t

10 C

SRP

.I)e

.j).

,0:

• '"' ..

0 SiO~ .c;

2.~

'"'

~u

Experiment Day

Figure 2. Uptake rates of SRP, Si02 and N02+N03 in each of the six streams during the two

experiments at different temperatures, at constant exposures to atrazine at 0 (-),24 (----), and 134 (-

- - -) /-lg/L. Vertical arrows indicate onset of exposure. Note difference in vertical scale at the two

temperatures.

Table 1. Average measured nutrient concentrations (mg/L) in the input water to the artificial

streams, in addition to the ambient creek water concentrations. (from study table 2)

Nutrient

N02+N03 N

P04 P

Si02

8.2

0.15

7.7

4.1

0.23

9.3

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Table 2. Total accumulated AFDW (g) at the end of each experiment, including p-value when

significantly different from control. (from study table 3)

lO°C 25°C

%of %of

Treatment Mean Std Dev control p Mean Std Dev control p

oIlg/L (control) 1.92 0.22 3.85 0.14

241lg/L 1.84 0.12 96 2.92 0.43 76 <0.001

1341lg/L 1.01 0.08 53 <0.001 2.65 0.1 69 <0.001

Table 3. Total accumulated chlorophyll a (mg) at the end of each experiment, including p-value

when significantly different from control. (from study table 3)

lOOC 25°C

%of %of

Treatment Mean Std Dev control p Mean Std Dev control p

oIlg/L (control) 57.1 5.5 77.2 14

241lg/L 57.9 6.3 101 53.8 6.8 70 <0.01

1341lg/L 34.5 2.8 60 <0.001 43.6 5 56 <0.001

Data Summary Table:

£.lfect Magnitude of Nominal Day ofStudy Time Did Comments

Effect relative Atrazine Effect Weighted Recovery

to controls Concentration Occurred Average Occur- if

(/lgIL) Atrazine so, when

Concentration (see

from Day ato comment)?

When Effect

Occurred

Decline in

24% Unknown Results for

attached 50 12 24 (effect 25°C

community (p<O.OO I) measured experiment.

(Aufwuchs) at end of Declines were

biomass 31% study) significant at

(ash-free (p<O.OO I) 200 12 134 p-values

dry weight) shown

Decline in Unknown Results for

attached 30% (p<O.OI) 50 12 24 (effect 25°C

community measured experiment.

(Aufwuchs) at end of Declines were

biomass 44% study) significant at

(chlorophyll (p<O.OOI) 200 12 134 p-va1ues

a) shown

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Effect Magnitude of Nominal Day ofStudy Time Did Comments

Effect relative Atrazine Effect Weighted Recovery

to controls Concentration Occurred Average Occur -- if

(fig/L) Atrazine so, when

Concentratiun (see

ji-om Day 0 to comment)?

When Effect

Occurred

Decline in 21% (not 50 9 24 NR Results for N02+NOJ significant)

N uptake 25°C

25% 200 9 134 NR experiment. rate

Decline in no effect 50 9 24 NR Results for dissolved

silica 25°C

uptake rate

No decrease 200 9 134 NR experiment.

Decline in no effect 50 9 24 NR Results for

SRP uptake 8% 200 9 134 NR 25°C

rate experiment.

Note: NR=Not Reported

It was not necessary to compute time weighted averages, as the authors use measured

averages throughout the study to describe the exposure concentrations, and only twice

show the "nominal" values (i.e. study table 2.).

Reviewer Comments:

The source of natural stream water, Rock Creek, is only briefly and parenthetically

described. It would be useful to have more information regarding the watershed, the

stream biotic and abiotic environment, any potential for or measurements of background

concentrations of atrazine, and the time of year over which the experiments took place

and discussion ofthat timing relative to agricultural activities.

A few minor points of confusion affect the study interpretation: first, the definition

"turnover time" cited for the nutrients and how the 7 hour number was calculated, as it

did not appear to be an obvious quantity based on the information given; second, how the

"presence oftwo reservoirs" excludes macroinvertebrates and where these reservoirs

were (they are not shown in the Figure 1 diagram); and finally, the p-values are cited but

the methods description says that either two-sample t test or ANOVA may be used, and

the exact tests associated with a p-value are never provided.

Despite these minor limitations, overall the study appears well-reported, with a wellcontrolled experimental design and clear discussion of methods and results. Replication

was limited (two replicates at each treatment level), although for biomass endpoints

results were still statistically significant.

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Description of Use in Document (QUAL, QUAN, INV): QUAL

Rationale for Use: Study limitiations limit the utility ofthe study; however, it

contributes to the weight of evidence regarding the potential for atrazine to affect aquatic

communities.

Limitations of Study: This study provides evidence of a temperature-dependence in the

effects of atrazine on periphyton biomass. The results appear reliable since low

variability was found despite the poor replication, but they are only applicable to

similarly controlled environments, not naturallotic ecosystems. Also, not enough

information is provided about the source streamwater.

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer:

./p~/1 Brian Anderson.~1/;/.,/l

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