Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Carder, j .P. and KD. Hoagland. 1998. Combined effects of alachior and atrazine on

benthic algal communities in artificial streams. Environ. Toxicol. Chern. 17(7): 1415-

1420. (MKiO '1:rJ43b03 )

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:

December 12, 2008

Purpose of Study:

This study examines the potential effects of atrazine to aquatic plants (epipelic algae) in

experimental streams.

Summary of Study Findings:

Methods: The experiment was a duplicated, 3 x 3 factorial design testing effects of zero

(control), low, and high levels, ofatrazine, alachlor, and a combination of both, in 18

artificial streams. Details of alachlor-related methods that are not essential to

understanding the overall. design or the atrazine methods will not be reported.

Stream water and benthic substrate were collected from Wahoo Creek, 1.5 km southeast

of Wahoo, Nebraska, on March 25, 1993. Wahoo Creek is a third-order, sedimentdominated stream draining primarily agricultural land, and is subject to major runoff

events during the growing season. Analysis of Wahoo Creek water at the onset ofthe

experiment showed background atrazine concentrations of2.9 ~g/L. Water was collected

via centrifugal pump from the stream surface and was transported to the University of

Nebraska Laboratory Stream Facility in a 3,800 L polyethylene tank. Sediments

(epipelon) were collected from the upper 2 cm of benthic substrate using a garden trowel.

1••••1.111

2087871
Samples were placed in a bucket, homogenized by stirring with the trowel, and

transported back to the laboratory.

Eighteen recirculating artificial streams were constructed from 114 L, oval Rubbermaid®

plastic livestock tubs (Rubbermaid, Wooster, Ohio), with a lengthwise wooden divider

cut 25 cm from each end to allow for the passage of water, and lined with two layers of 4-

mil (1.016 mm) clear plastic. The authors cite a 1997 alachlor paper for a photograph of .&.1- '"' ~U~ l:1'~4.-~_ ........... _",.~,," ....,..J .cr.."............ f.\,.,,, ') O()(), T +.... _1"" ~_+ ..... +hlO n.....-i-~+:n~nl .... t,..,rH"l+vH·' ~....,. ......,'*'"'rls"'\"F'\"'\ LJl(; ~vL-U}J. vv dtC;:l \'Va.~ PUlllP~U II \.1111 l,.U\"; J,UVV .,l.....; LallI' lULU 1.11\,.1 U.l Lll-1\.dUI 0U ..... 44111..;:) 11.1 ,1 UBU-VBI

1/3 increments to ensure homogeneity among the streams' water, and all were filled to

within 10 cm ofthe top of each tub (about 86 L of water in each). The tubs were

arranged on three parallel tables, with an electric variable-speed motor attached to the end

of each table controlling a belt-driven shaft above the streams, driving a paddle wheel

dipping approximately 3 cm into the water surface of each stream to produce the current.

The electric motors were adjusted daily to achieve similar velocities in the streams (no

details on how similarity was determined). At four different times in the experiment

(timing not described), a Marsh-McBimey current meter (Marsh-McBimey, Frederick,

Maryland) was used to determine the water velocity in the center of each channel, at a

depth of one-halfthe water column. In the sending segment, velocities were 0.05-0.1

mis, and in the returning segment, they were 0.01-0.05 m/s. A 1,000 W metalarc lamp

was the light source (the authors parenthetically reference "~600 Ilmollm2/s" and it is

unclear what this refers to, as it is not in any standard illuminance or irradiance units),

and operated on a 12 hour light, 12 hour dark photoperiod. The ambient room

temperature was controlled (no details provided). Approximately 1-2 L of water was lost

from each stream daily due to evaporation, so stream water from the collection tank was

used to maintain water levels. The stream water was mixed in circulating holding

streams for at least 24 hours before being added. The initial batch of water was

insufficient, so additional stream water from Wahoo Creek was collected "on several

other occasions" (no details or background atrazine levels on these collections provided).

Three unglazed masonry tiles (20 cm x 20 cm x I cm) were placed on the bottom ofthe

return channel of each stream to shape the liner and provide a uniform surface for the

epipelon sampling units. Sampling units were 60 mm x IS mm plastic Petri dish bottoms

filled with Wahoo Creek benthic sediments. Units were initially placed immediately

after water was added to the artificial streams, four on each tile (12 in each stream).

Epipelon not used in the initial setup was stored in a circulating holding stream, and these

sampling units were used to replace the sampling units that were removed for sampling.

The replacement was only to ensure that stream flow around the units was not affected by

sampling unit removal- replacement units were not used for measurements. All streams

were allowed 6 weeks to acclimate to lab conditions before the 4 week experiment

commenced.

Four I L solutions of both high and low concentrations of alachlor and atrazine were

developed (methods or solvents not described) so that 100 mL ofsolution would create

the desired test concentrations in the 86 L streams. Atrazine was a commercial product

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(Farmland Industries, Kansas City, Missouri; 40.8% active ingredient). Atrazine test

concentrations were nominally 12.0 jlg/L ("low") and 150.0 jlg/L ("high"). The 3 x 3

factorial design cross-classified control, low, and high alachlor treatments with control,

low, and high atrazine treatments, with duplication. Treatments were randomly assigned

to the 18 streams (randomization method not described). Treatments (l00 mL solutions)

were added to the appropriate streams immediately after collecting the initial algal

samples on May 5, 1993.

Herbicide analyses were conducted on water samples taken from each stream 24 hours

after herbicide addition and at the end ofthe experiment. Samples were frozen until they

were analyzed using Ohmicron RaPID Assay® (Newton, Pennsylvania) immunoassay

kits for the detection of atrazine (coefficient of variation was <5% according to the

manufacturer). The ability ofthe immunoassay kit to accurately determine herbicide

concentration was also verified using gas chromatography on several randomly selected

samples.

On each sampling day prior to algal sampling, pH and stream temperature measurements

were taken using a Fisher Scientific Accumet® portable pH meter (model 1002; Fisher

Scientific, Pittsburgh, Pennsylvania), dissolved oxygen (DO) concentrations were

measured using a Yellow Springs Instruments portable DO meter and probe (model 57;

YSI, Yellow Springs, Ohio), and conductivity was measured using a portable Orion®

conductivity/total dissolved solids (TDS) meter (model 124; Orion, Boston,

Massachusetts). Additionally, water samples from "four randomly selected streams"

(treatments ofselected streams not described) were taken immediately before the

experiment, on May 4, 1993 and immediately after it ended, on June 3, 1993, for nutrient

analysis. Nutrients analyzed were total nitrogen, using the alkaline potassium persulfate

digestion (D'Elia et aI, 1976, Limnol Oceanogr 22) and ultraviolet absorption (American

Public Health Association's 1985 standards) methods, and total phosphorus, using the

ascorbic acid method (Lind, 1985, Handbook ofCommon Methods in Limnology).

For sampling of epipelon, all paddle wheels were stopped and a randomly selected

sampling unit was removed and a replacement unit put in immediately (as previously

described). In all cases, this occurred between 0900 and 1030, the time of day when

motile epipelic species would be expected to have migrated towards the surface for light

absorption. The removed sampling unit was subsampled with a sharpened 1 em copper

tube in a random (randomization not described) location, and the upper 2 mm of epipelon

from the inside ofthe tube was drawn off using O.S-cm tygon tubing attached to a

vacuum pressure system. The subsample was drawn directly into a graduated 120 mL

wide-mouth glass jar and 40 mL of 6:3:1 (water:ethanol:formalin) was used to rinse the

vacuum system and preserve the sample. Sampling occurred before herbicide addition

(May 5, 1993), 24 hours after herbicide addition, and 1, 2, and 4 weeks after herbicide

addition.

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Algal samples were analyzed for cell density (results not described), cell biovolume, and

relative abundance ofthe six dominant taxa (meaning those comprising >5% by

biovolume in the initial samples). Samples were homogenized by gently swirling the

sample jar, a 0.10 mL aliquot was pipetted into a Palmer counting cell and scanned at

400x using an Olympus BH-2 compound microscope (Olympus, Tokyo, Japan), and then

algae were counted until 300 viable (chloroplast intact) cells had been quantified, or 100

fields of view had been observed. Data were analyzed using analysis of variance

(ANOYA) with repeated measures, performed with SAS Version 6.0 software, with

statistical significance inferred for p-values:::; 0.05.

Results: To the extent possible, only atrazine results will be described. However, for

some results, either all streams or "randomly selected" streams were sampled; these

results include streams treated with alachlor in the factorial design.

Average atrazine concentrations in all streams were relatively close to nominal (see

Table 1), though they started out higher than nominal due to existing background levels of atrazine

in the stream water. The high atrazine treatment streams decreased in atrazine concentration by

13% over the four-week experiment. The low atrazine treatment streams actually increased in

concentration by nearly 15% over the course of the experiment, which probably reflects changes in

background contamination (as evidenced by increase in "control" concentrations in

Table 1). The authors state that physical and chemical water quality measurements did

not have any obvious trends or differences among the experimental streams (however,

there appeared to be some changes in nutrient levels and conductivities). Over all

streams, means (and standard deviations) were: dissolved oxygen, 9.0 (0.3) mg/L; pH, 8.8

(0.1); and temperature, 1704 (004) °C. Conductivities over all streams were 0.89 (0.04)

mS/cm initially and lAO (0.1) mS/cm at the end ofthe experiment. Nutrient

concentrations in four randomly selected streams changed somewhat over the course of

the experiment: total nitrogen increased from 1,007.5 (172.5) Ilg/L to 1,571.9 (82.8)

Ilg/L, while total phosphorus declined from 117.0 (10.0) Ilg/L to 81.8 (7.7) Ilg/L (these

results are not interpreted in the paper, just provided in a table it is difficult to interpret

them without knowing which streams were sampled).

Effects of both atrazine and alachlor on algal biovolume are shown in Figure 1. No

significant differences were found between pretreatment streams and control, or between

streams at 24 hours after addition. Time-independent analyses showed significant

atrazine effects at both high and low concentrations at week 1 (both with p=0.0005) and

week 4 (both with p=O.OOOI), with high concentration treatment effects at week 2

(p=0.0064). No significant difference between high and low treatments was found at

week 1, but there was a significant difference at week 4 (p=0.0006). Time-dependent

analyses found that both treatment levels induced significant changes in algal biovolume

throughout the experiment (p=0.0083), but the difference between high and low treatment

levels was not significant (p=0.3629).

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Relative abundances of dominant algae were not significantly affected by atrazine,

although two were affected by alachlor. The six dominant algae taxa were:

Fragilaria capucina (Chrysophyta, diatoms)

Fragilaria spp. (diatoms)

Cylindrospermum majus (Cyanophyta, blue-green algae)

GyroSlgma eXln1lum ldiatom)

Rhopalodia gibba (diatom)

Navicula spp. (diatom)

5
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;:: 120 9

100

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(..') :0 '- 0 ,"'"

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Initial 24h

lwk F 120 '",

'c ;oo-L_--

I~~ jmoOffi~n2wk -I" k 1,)0 1;0 .----1

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Figure 1. Percentage of mean control algal biovoJume for each treatment combination on each of the

five sampling dates. ("LO"=Jow, "HI"=high, "al"=alachlor, "at"=atrazine; for atrazine-only data,

see the third and fourth bars from the left on each subplot). t indicates a significant (p-value 0.05)

time-dependent effect based on repeated measures analysis; * indicates a significant (p-value 0.05)

time-independent effect based on ANOVA of the individual sampling period; and 'I' indicates a

significant interaction.

Table 1. Measured atrazine concentrations in the artificial streams. Initial concentrations were

taken 24 hours after atrazine addition, and final concentrations were taken 4 weeks later upon

termination of the experiment. Means (and standard deviations) over all streams are shown.

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Data Summary Table:

Effect Magnitude of Nominal Day of Time Did Comments

Effect Atrazine Study Effect Weighted Recovery

Relative to Concentration Occurred Average Occur- if

Controls (~g1L) Atrazine so, when

Concentration (see

from Day 0 to comment)?

When

O",-,Ui l"U

~50%

Decrease in (different 12 ~g1L Day 7 16.4 Ilg/L No (>28

from control days) benthic at p={) ()O{) 1. ) Magnitudes from algal

community ~60% Fig. 1.

biovolume (difTerent ISO 7 145.21lglL No (>28

from control

at n=O OO!)1) i

Time weighted averages are averages of the initial and final means given in

Table 1.

Reviewer Comments:

This study had a factorial design, including duplication, although the alachlor results are

not of interest from the perspective ofthis review. Most sampling and analytical methods

were reasonably well described and either followed standards or were cross-validated.

The only real endpoint presented by the study was changes in epipelon biovolume.

Water quality measurements that may have been affected by treatment, like dissolved

oxygen and nitrogen levels, were not taken in enough streams at enough sampling times

for proper examination.

The main complication ofthe study was the significant and apparently time-varying

background levels of atrazine in the stream water. Given that the average "control"

atrazine concentration started out at 2.9 Ilg/L and increased to 6.4 IlglL by the end ofthe

experiment, and the "low" treatment concentrations also increased somewhat, there was

clearly significant background atrazine. It would have been informative to learn what

kind ofrunoff events, herbicide applications, etc., were occurring in the area around the

times ofstream water collection. The fact that the epipelic communities clearly had prior

exposure to atrazine was not discussed, and could confound the results somewhat, as the

community may have some pre-developed atrazine tolerance. In addition. the "controls"

were not contaminant-free, so some effects may appear weaker relative to the "controls"

than they would if compared to truly contaminant-free controls.

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

Rationale for Use: The study was limited because ofthe presence ofatrazine in the

controls at levels that may influence the endpoints evaluated in this study.

Limitations of Study: Background water and "controis;; had 2-7 llg/L concentrations of

atrazine, so effects "relative to controls" are misleading. Only benthic algal communities

were present in the experimental streams, so the ecological dynamics were highly

simplified and may not be applicable to natural streams.

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

Secondary Reviewer: Brian Anderson, U.S. EPA

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