Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Kosinski, RJ. 1984. The effects ofterrestrial herbicides on the community structure ~f

stream periphyton. Environ: Pollut. (Series A). 36: 165-189. (vYl P..,l 0 4:;<; i.("3 $'"D·t)

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:

October 22, 2008

Purpose of Study:

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

experimental streams (lotic mesocosms).

Summary of Study Findings:

Methods: Two sets of experiments were conducted in 1981 and 1982. In each year,

experimental design was a factorial combination ofa or 0.01 mg/kg (0 or 10 /lg/L) of

"chronic" atrazine treatment during initial colonization, and 0, 0.1, 1.0, or 10 mg/kg (0,

100, 1,000, or 10,000 /lg/L) "acute" atrazine treatment 3 to 4 weeks after initial

colonization. (The factorial design also included three other pesticides, but this review

will be limited to the methods and results for the atrazine experiments.)

The 1981 experiments used apparently pristine water from Minter Spring, southern

Brazos County, Texas. The spring water was almost anoxic and had temperature nearly

constant at around 21°C, mean specific conductance at 25°C of 54.6 mS/m, alkalinity of

71 mg CaC03/L, 404 /lg/L nitrate, and 21 /lg/L soluble reactive phosphate (SRP). (No

information on frequency or timing of water quality measurements.) Cyanobacteria

(mostly Anabaena), diatoms (Nitzschia, Rhopalodia, and Navicula), and a few species of

protozoans, rotifers, ostracods, and chironomids were present on the walls or in the water.

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2087870
The author states that it is "improbable" that algae in the spring had prior atrazine

exposure (no further discussion or evidence oftesting for background levels).

The 1982 experiments used water from the Little Brazos River, western Brazos County,

Texas - an intensively farmed cotton and sorghum region. The river received high

volumes ofrunoff from adjacent fields, as evidenced by the gully erosion patterns in the

watershed terrain. Water and algae were taken from the hardpan clay riffle at a bridge

that crosses the river. At the sampling point, the river was about 30 cm deep and 7-10 m

wide (no information on flow rate or discharge). Dense growth of Cladophora glomerata

(chlorophyll a measured to be 250 mg/m2

) was observed, as well as a "diverse

invertebrate and fish community" (no further details). Water quality was highly

mineralized and nutrient-rich. Mean specific conductance was 95.5 mS/m, alkalinity was

153 mg CaC03/L, and nitrate and SRP were 1716 and 78 Ilg/L, respectively. Dissolved

oxygen varied between 75-150% ofsaturation. (No information on frequency or timing

of water quality measurements was reported.)

Experiments were performed in 32 artificial streams (4 herbicides x 4 acute treatments x

2 chronic treatments, 1 replicate for each combination), located on a flat roof on the

Texas A&M University campus. Each stream consisted of a wooden trough, 2.44 m long

and 12.6 cm wide, lined with plastic and filled with source water to a depth of 4 cm, with

a pump at one end circulating water through a garden hose beneath the trough and back

through the channel at a velocity of 3 cm/s. The substrate for colonization was 1-2 cm

diameter autoclaved, streamworn pebbles, which lined the middle 1.4 m of each stream.

A heavy glass plate with four horizontal microscope slides attached to it with a rubber

band was placed between the pebble zone and the pump in each artificial stream.

Translucent fiberglass covers protected the streams against excessive evaporation and the

elements, and automatic reservoirs filled with source water kept the stream depths

constant. Twice a week, 2 liters (15% ofthe stream volume) were discarded and replaced

with source water, and atrazine levels were brought to nominal concentrations at this

time. (No further details were reported on age and handling ofsource water or on

materials used;) Water-algae suspensions harvested from the source spring or river (no

dates provided) were homogenized in a blender to break up large filaments, and each

stream received 100 mL of homogenate for colonization. The colonization took place

over 3-4 weeks (no specific number of days cited) inside the artificial streams, with the

"chronic" herbicide treatment levels maintained by adding more herbicide with each 15%

water replacement. After colonization, (apparently single) "acute" treatments were

applied using commercial atrazine (Aatrex 80 WP) at nominal concentrations based on

the active ingredient concentration in the commercial preparation (specific application

method and dates not stated).

The rate of atrazine decay in the streams was determined via water samples from both

control and 10 mg/kg (10,000 Ilg/L) streams at time lags of a few minutes, 1 day, 3 days,

and 7 days after acute treatment application, which were frozen until analysis. Analysis

was performed at the TAMU Agricultural Analytic Service by a nitrogen-phosphorus

2
detector method modified from Purkayastha and Cochrane, J. agric. Food Chern. 21,

1973.

Algal samples were taken each week (except the week following acute treatment

application) by removing one colonization slide "from two streams per chronic x acute

treatment" (the meaning ofthis is unclear, since the study states there were 32 streams

total, which would leave one replicate for each herbicide/chronic/acute treatment

combination in the factorial experimental design). In the week following acute treatment

in 1982, six slides per chronic x acute treatment were harvested to allow between-slide

comparisons within a stream (again, the meaning of "per chronic x acute treatment" is

unclear and this appears inconsistent, since there are only 4 slides total per stream and

one replicate at each herbicide/chronic/acute combination). Organisms from the upper

side of each slide were scraped into "6:3: 1 preservative" (probably

water:ethanol:formalin, and apparently kept in some kind ofstorage vials, but details are

not provided). For cell counting, storage vial material was diluted and duplicate

Sedgewick-Rafter cells were made, and on each cell 10 fields were counted at 400 x on

an Olympus inverted microscope. In 1982, since aggregated growths like cyanobacterial

mats were common, large filaments and mat fragments were counted in an additional 10

fields per cell at 40 x. For unicells and "modular" colonies of definite size, such as

Scenedesmus, the counting unit was individuals or colonies. For spreading colonies, such

as Nostoc, the counting unit was the number of occupied second-order squares of a

Whipple grid. For filaments, the counting unit was length in terms ofthe size of a

second-order square. The volume of cellular contents in each counting unit was

determined by geometric approximation (no details on primitive shapes or volumes used),

and final results were expressed as 11m3 biovolume per mm

2 ofslide surface. Nondiatoms were identified from wet mounts of preserved material at 400 x and 1000 x,

while diatoms were identified at 1000 x from Hyrax mounts of cleaned frustules. Nine

references were used for identifications (not listed here).

Statistical analyses were performed on only the 15 most common species in each

experiment in 1981 and the 20 most common in 1982. The experimental design and

number ofreplicates limited the multivariate analysis to 7 dependent variables, so seven

"species groups" were defined, sometimes on taxonomic grounds and sometimes on less

strict associations (such as "filamentous diatoms"). See Table 1 for the species groups

defined in 1981 and 1982. The species group variables represent the sum ofthe

biovolumes ofthe component species. These variables were transformed by In(x+1) and

subjected to univariate ANOYA, as well as multivariate ANOYA (MANOYA) to process

several response variables at once and track correlations between them (implemented via

the GLM procedure in SAS software). Significance was determined at the 0.05 level by

Pillai 's trace, and computed eigenvectors were used for a principal components analysis

of selected data sets. Univariate ANOYA was additionally performed on ShannonWeiner diversity indices computed from the total biovolumes ofthe 15 or 20 dominant

species.

3
Results: Only the results for atrazine will be presented.

The half-life of atrazine in the artificial streams was calculated as 3.2 days, and no

interferences from plasticizers or other non-herbicide contaminants were detected in the

control samples.

Some effects did not yield significant results and so were not presented. Only main

effects rather than interactions (between chronic and acute treatments) were presented

due to the absence of interaction. The Shannon-Weiner index ranged from 0.721 to 2.385

but varied in response to succession rather than treatment, and therefore, was not further

discussed.

The authors claim that "between-stream error" was the main source of variation within a

treatment, since multivariate ANOYA found it significant 38 out of 44 times, while 3 of

the 4 tests on within-stream slides found insignificant results.

See Table 2 through Table 8 for the results ofthe atrazine comparisons. Weeks are

designated as weeks before (-) or after (+) application of acute atrazine treatment, with

Week 0 data taken one day after the application. The dominant species groups in 1981

were NITZ and FILDIA, with FILDIA as the second dominant group replaced by RHO at

Week 2. Species abbreviations are defined in Table 1. In 1982, OTHDIA and ACHNAN

dominated up to Week 0, whereas in Weeks 1-3 the dominant species groups were

ACHNAN and then RHO. Overall, no clear pattern suggesting resistance was found, and

for the most part there were no significant multivariate effects from treatment. Different

streams were found to have different communities (significant multivariate effects,

p<0.05 with MANOYA), but these effects were not significantly associated with different

treatments (with few exceptions). Exceptions were chronic effects during Week 0 in

1981 (p=0.038) and Week 3 in 1982 (p=0.032), acute effects during Week 1 in 1982

(p=0.026), and chronic x acute effects during Week 3 of 1982 (p=0.027). The author

does not suggest a pattern to these effects. Univariate effects on specific species groups

during specific weeks were found, particularly in 1982 when acute treatment effects were

found also as opposed to just chronic effects. It is unclear how much the lack of effects

(or of patterns therein) were due to the small number ofreplicates, from experiment

design as well as laboratory mistakes in 1981; see Reviewer Comments.

The author notes that reductions in algal crop in the two highest treatment streams were

obvious to the eye and persisted for the duration ofthe experiment. The observation was

confirmed by the results ofthe principal components analysis, shown in Figure 1 in

which treatments with different community structures would be expected to occupy

different areas ofthe plot. Figure 1 shows data from Week 1 of 1982, the time at which

the differences were most apparent.

4
1.0

,...

CJ

A. o•s

o

\

0.5

PC2

1

Figure 1. Principal components analysis of the algal counts one week after acute atrazine addition in

1982. Principal components (PCl, PC2) are linear combinations of the species group densities which

maximize separation between the acute treatment levels. Polygons enclose the points from the 0, 0.1,

1, and 10 mg/kg (0,100,1,000, and 10,000 p.lg/L) treatments. PCl and PC2 explained 89.4% and

7.1%, respectively, of the separation between the acute treatments.

Table 1. Composition ofspecies group variables in each year of experiments (from study tables 1-2)

1981

CALO

NFILBGS

GREENS

FILDIA

NAVSYN

NUZ

RHO

1982

PHOR

SCENCLA

CENTRLES

ACHNAN

OTHDIA

NITZ

RHO

Calothrix sp.

Chroococcus sp., Rhabdoderma sp.

Scenedesmus sp., S. bijuga alternans, Stichococcus bacillaris

Melosira sp., Navicula confervacea

Navicula cocconeiformis, Synedra delicatissima

Nitzschia ignorata, N. palea, N. palea tenuissima

Rhopalodia gibba ventricosa, R. Gibberula

Phormidium minnesotense, P. tenue

Cladophora glomerata, Scenedesmus quadricauda

Biddulphia laevis, Cyclotella meneghiniana, Melosira italica, M italica tenuissima

Achnanthes sp., A. lanceolata dubia, Cocconeis placentula lineate

Amphora veneta, Diploneis elliptica, Gomphonema angustatum intermedium, G.

parvulum, Surirella ovata

Nitzschia ignorata, N. linearis, N. Palea

Rhopalodia gibba, R. gibba ventricosa

5
Table 2. 1981 log-transformed Iln(x+I)] weekly mean total biovolumes in llm3/mm2. Week 1 and 3

data are missing due to loss ofsamples in a lab accident. Slide collection error required combining

the lower two and higher two acute treatment results (from study table 3)

Week

Atrazine Treatment -2 -1 0 1 2 3

Acute

Acute

Ch ' Control 12.137 12.857 13.502 13.438 romc . J~Jl:g~~ !?yj_~ }_~:~?~ }_~._~!! 1_4:~9_~ _ Control + 100 f.lg/L 12.400 12.097 13.403 14.017

1,000 + 10,000 f.lg/L 12.287 13.070 13.296 13.570

Table 3. 1982 log-transformed [In(x+1)] weekly mean total biovolumes in llm3/mm2. Different

superscript letters denote significant differences (no p-value provided) by Duncan's multiple range

test (from study table 3)

Week

Atrazine Treatment -2 -1 0 1 2 3

h . Control 12.885 12.155 13.170 a 13.382 13.948 14.492

__ __~~~~~ !9_tt_g/_L J}:?_~~ !J:??_~ !?:~?_I_ ~' }}._6~? J3:?_~? }?:?!? _ Control 11.913 12.080 13.721 a 14.662 a 14.899 ab 15.467 a

100 f.lg/L 11.737 11.778 13.099 a 14.837 a 14.674 ab 14.916 a

1,000f.lg/L 11.638 11.432 12.068 b 13.168 b 13.028 ab 13.767 ab

1O,000f.lg/L 11.929 12.122 12.196 b 11.353 c 12.605 b 12.272 b

Table 4. 1981 p-values for multivariate effects on 7 species groups of different independent variables

(MANOVA). Values in bold indicate significance at p < 0.05. Week 1 and 3 data are missing due to

loss of samples in a lab accident. (from study table 3)

Week

Independent Variable -2 -1 0 1 2 3

Chronic treatment 0.169 0.381 0.038 0.636

Acute treatment 0.800 0.334 0.761 0.720

Chronic x Acute 0.113 0.792 0.844 0.315

Streams 0.052 0.002 0.001 0.001

Table 5. 1982 p-values for multivariate effects on 7 species groups of different independent variables

(MANOVA). Values in bold indicate significance at p < 0.05. (from study table 3)

Week

Independent Variable -2 -1 0 1 2 3

Chronic treatment 0.284 0.238 0.554 0.190 0.068 0.032

Acute treatment 0.761 0.811 0.103 0.026 0.384 0.340

Chronic x Acute 0.522 0.849 0.840 0.274 0.426 0.027

Streams 0.001 0.001 0.005 0.027 0.005 0.007

Slides 0.001

6
Table 6. 1981 log-transformed [In(x+l)) mean biovolumes (/l-m3

/mmZ

) ofspecies groups that showed

statistically significant univariate effects (ANOYA, p<O.OS) from chronic atrazine treatment. (There

were no significant univariate effects from acute treatment in 1981.) (from study table 1 and 4)

Week

-2

-2

-I

o

Variable

NFILBGS

NAVSYN

RHO

RHO

Treatment Means

Control 10 t-JglL

1.867 4.804

1.245 5.384

7.958 2.755

9.841 3.472

Species most affected

Chroococcus sp.

Synedra delicatissima

Rhopalodia gibba ventricosa, R. gibberula

Rhopalodia gibberula

Table 7. 1982 log-transformed [In(x+l») mean biovolumes (/l-m3/mmZ) of species groups that showed

statistically significant univariate effects (ANOYA, p<O.OS) from chronic atrazine treatment. (from

study table 1 and 4)

Week

-2

-1

o

o

o

2

2

Variable

RHO

PHOR

PHOR

NITZ

RHO

PHOR

NITZ

Treatment Means

Control 10 fJglL

o 2.834

6.748 1.968

8.443 4.212

10.37 7.713

8.715 2.306

8.542 5.054

11.318 6.602

Species most affected

Rhopalodia gibba, R. gibba ventricosa

Phormidium minnesotense

Phormidium minnesotense, P. tenue

Nitzschia ignorata

Rhopalodia gibba

Phormidium minnesotense, P. tenue

Nitzschia palea

7
Table 8. 1982 log-transformed [In(x+1)) mean biovolumes (/-lm3

/mm2

) ofspecies groups that showed

statistically significant univariate effects (ANOVA, p<O.05) from acute atrazine treatment. (from

study table 1 and 4)

Treatment Means

Week Variable Control 100 pglL 1,000 flglL 10,000 flg!L Species most affected

0 PHOR 8.585 8.694 5.367 2.663 Phormidium minnesotense,

P. tenue

0 RHO 9.984 4.769 5.826 1.462 Rhopalodia gibba, R. gibba

ventricosa

CNTRLES 4.635 2.945 2.624 1.609 Biddulphia laevis

ACHNAN ] 1.546 12.439 11.062 9.573 Achnanthes sp.

RHO 14.350 14.426 9.414 3.683 Rhopalodia gibba, R. gibba

ventricosa

I OTHDlA 10.730 10.854 9.653 7.295 Amphora veneta

2 PHOR 10.970 5.997 3.773 6.755 Phormidium minnesotense

2 SCENCLA 10.833 7.494 0 3.984 Cladophora glomerata

2 ACHNAN 11.836 12.109 11.184 11.039 Achnanthes sp., A.

lanceolata dubia

3 SCENCLA 9.632 8.614 0 0 Cladophora glomerata

3 RHO 15.142 14.417 11.396 1.549 Rhopalodia gibba, R. gibba

ventricosa

Biddulphia laevis,

3 CNTRLES 4.120 2.683 0 0 Cyclotella meneghiniana

Data Summary Table:

Effect Magnitude of Nominal Day ofStudy Time Did Comments

Effect relative to Atrazine Effect Weighted Recovery

controls Concentrat Occurred Average Occur - if

ion (fig/L) Atrazine so, when

Concentratio (see

nFom Day 0 comment) ?

to When

Effect

Occurred

no effect (I % Maximum

increase, not sig. 100 I-lg/L N/A 53 I-lg/L effects at I

diff. from no week. No

control) recovery within

Reduction in 10% (sig. diff. 3 weeks after

periphyton from all other 1,000 I-lg/L Day 7 528/lg/L Yes, Day addition unless

biovolume treatments) 14 otherwise noted.

20-25% (sig. No p-value

10,000 provided. See diff. from all I-lg/L Day 7 5,284 I-lg/L no review table 3 other treatments) for source data.

Change in Maximum

periphyton no clear effect 100/lg/L NA 53 I-lg/L no effects at I

8
Effect Magnitude of Nominal Day ofStudy Time Did Comments

Effect relative to Atrazine Effect Weighted Recovery

controls Concentrat Occurred Average Occur - if

ion (Jlg/L) Atrazine so, when

Concentratio (see

nfrom Day 0 comment)?

to When

Effect

Occurred

community week. No

composition Some effect 1,000 Ilg/L Day 7 528 Ilg/L No recovery within

3 weeks after

addition. Source

qualitative

Clear effect 10,000 Day 7 5,284 Ilg/L No magnitudes: PC I

Ilg/L in Figure I;

MANOYApvalue was 0.026.

Time weighted averages were calculated using the only atrazine concentration

information provided: the half-life of atrazine in the artificial streams was calculated at

3.2 days. Daily concentrations were computed based on the assumption of initial

concentrations equal to nominal levels, and exponential decay with 3.2 day half-life.

Time weighted averages on Day 7 were simple averages ofthe computed concentrations

on Days 0-7. See the Excel-linked table below for calculations.

Reviewer Comments: This study suffers from deficiencies in methodology and

presentation that limit the usefulness of its results. However, details on study

methodology were reported in several publications including the following (all of which

were reviewed):

• Moorhead, D.L. and RJ. Kosinski. 1986. Effect of atrazine on the productivity

of artificial stream algal communities. Bull. Environ. Contam. Toxicol. 37:330-

336.

• Kosinki, RJ., and M.G. Merkle. 1984. The effect of four terrestrial herbicides on

the productivity of artificial stream algal communities. 1. Environ. Qual. 13:75-

82.

Details on the study design were reported in these publications that were not reported in

the current publication. Details from all publications that reported study design details

from the same series ofstudies are included in this review.

The study methodology deficiencies include problems with the data aggregation method,

and with the lack of initial steady state in the communities under study. Lab and

sampling errors also interfered with the analyses (see Table 3).

9
Statistics were all performed on species groups. Aggregation could introduce

misclassification errors, and effects to sensitive species within groups may not be

detected.

The final methodological issue is that succession dynamics appeared strong enough to

possibly overwhelm any treatment effects, as suggested by the Shannon-Weiner indices

(which the author does not discuss in detail). Succession could be a strong confounder.

Some ofthe observed changes in the periphyton community could result from the

interaction oftreatment with succession dynamics or even from succession alone, due to

insufficient homogenization ofthe initial colonization material. The artificial streams

should have been at something closer to a steady state in terms ofthe community

dynamics when the acute treatments were applied, in order to separate out the succession

and treatment effects.

In terms of presentation, several important pieces of information were unclear or missing

from the study. Background atrazine in source water was not analyzed. Determination of

background atrazine levels would allow for more accurate interpretation ofthe results.

No schedule or dates were provided for exactly when sampling occurred, when water was

replaced in the artificial streams, or when herbicide was applied. No p-value was

provided for the Duncan's multiple range test results, although this multiple comparison

method is not recommended due to its tendency to provide inflated estimates·of

significance. The "species most affected" in Tables 6-8 had no specific method presented

for determining them, so it is unclear what these species really represent. Finally, the

description of periphyton sampling from the artificial streams was apparently

inconsistent: it was not clear ifthere were only 1 or four replicates at each

herbicide/chronic/acute treatment combination level. However, a subsequent publication

based on the same data and methods provided additional clarifying information (Kosinski

and Merkle (1984). There was also some error in the description ofthe sampling

protocol.

Since most ofthe methodological issues would bias the study results towards the null, the

fact that some statistically significant effects were found is suggestive of actual treatment

effects. However, the magnitude of effects determined in the study are likely

underestimates of effects because ofthe aforementioned issues.

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

Rationale for Use: The study deficiencies limit the utility ofthis study. However, the

study appears sufficient to contribute to the weight of evidence regarding the

concentration-response relationship of atrazine and aquatic plants. Data were obtained

from several additional publications to allow for an evaluation ofstudy adequacy.

10
Limitations of Study:

The study methodology deficiencies include problems with the data aggregation method,

and with the lack of initial steady state in the communities under study. Lab and

sampling errors also interfered with the analyses. In addition, source water for controls

was not evaluated for the presence of atrazine, and there were several reporting

deficiencies

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

/1

i i

Secondary Reviewer: Brian Anderson, U.S. EPA

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