Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

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. (t11e.t 0 l..[7->4:-:;5l>f{'

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 27,2008

Purpose of Study:

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

experimental streams (lotic mesocosms).

Summary of Study Findings:

Methods: Methods are similar to those in Kosinski, 1984, though this study provides

more detail on most aspects. Only the methods pertaining to atrazine will be described

(three other pesticides were also examined).

Two sets of experiments were conducted in 1981 and 1982, April-May of both years. In

each year, experimental design was a factorial combination of 0, 0.1, 1.0, or 10 mg/kg (0,

100,1,000, or 10,000 /-lg/L) "acute" atrazine treatment, after 0 or 0.01 mg/kg (0 or 10

/-lg/L) "chronic" atrazine in the water during initial colonization. Acute studies were

conducted after chronic exposures to atrazine at amg/kg or 0.0 1 mg/kg atrazine for 3 to 4

weeks.

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

Brazos County, Texas. The water, emerging from a concrete standpipe in a pasture, was

almost anoxic and had constant temperature of around 21°C, mean specific conductance

at 25°C of 54.6 mS/m, alkalinity of 71 mg CaC03/L, 404 flg/L nitrate, and 21 flg/L

11111111111

2087869
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. The author states that it is

"improbable" that algae in the spring had prior atrazine exposure; however, this

conclusion was not supported by data

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

Texas - an intensively farmed cotton and grain sorghum region, with each crop

accounting for about 40% ofthe planted acreage in the watershed. Atrazine use in the

watershed was "in significant amounts". 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 a bridge that passes over the river. No information is provided

on river width, depth, or 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 78Ilg/L, respectively. Dissolved

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

of water quality measurements.)

Water and algae were collected at the source areas and transported back to the laboratory

in carboys, where they were either used immediately or stored in covered indoor tanks for

up to 10 days. Atrazine experiments were perfonned in 32 artificial streams (4 acute

treatments x 2 chronic treatments, 4 replicates for each combination - note that this is a

different description than in the Kosinski 1984 review as more information was provided

in this study), 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 0.15-mm plastic

sheeting 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,

streamwom pebbles, which lined the middle 1.4 m of each stream. A sheet of corrugated,

clear fiberglass overlain by a translucent white plastic sheet and a clear plastic sheet

provided covers to protect the streams against excessive evaporation and the elements.

The covers attenuated the intensity of photosynthetically active radiation by 60% (no

information on how this was measured), which reportedly moderated the greenhouse

effect on high irradiance days. 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.

Colonizationlinitialization ofthe streams is not described in detail, but is stated to have

taken place over "3 or 4 weeks" with regular additions with water replacement keeping

the non-control "chronic" treatment streams at 0.01 mg/kg atrazine concentration (10

Ilg/L). After colonization with or without the presence of 10 ug/L atrazine, single

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"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). Chronic treatment additions ceased

after acute treatment was applied.

"A small number" of analyses were performed on frozen streamwater samples to estimate

the rate of disappearance ofthe herbicides from the water (no further details provided).

Analysis was performed at the TAMU Agricultural Analytic Service by a nitrogenphosphorus detector method modified from Purkayastha and Cochrane, J. agric. Food

Chem. 21, 1973. No attempt was made to separately determine effects of emulsifiers and

other nonherbicidal components ofthe commercial atrazine product.

Productivity was measured with open-water oxygen methods, i.e. by estimating the sum

of photosynthesis and respiration (net community productivity or NCP) via correcting the

rate of change ofthe dissolved oxygen (DO) concentration for exchange with the

atmosphere. Oxygen saturation deficit was computed from temperature using equations

in Benson and Krause, Oxygen. Limnol. Oceanogr. 25, 1980. The reaeration coefficient

(K) was estimated by reducing DO in source water samples to 2-3 mg/kg (by adding 0.04

mg/L sodium bisulfite and a crystals/L cobaltous chloride), then circulating the water in

an uncolonized artificial stream channel and monitoring its DO and temperature until

oxygen saturation occurred. Multiplying the slope ofthe resulting DO vs. oxygen

saturation deficit by stream depth yielded K, which was then corrected for temperature at

a rate of 1.6% per degree Celsius. K was measured during most experiments, and the

resulting 60 measurements averaged 0.09 m/hr at 30°C.

During the experiment, DO was monitored in each ofthe 32 streams every 15 minutes,

starting 0.5 hours before dawn and ending 1 hour after sunset; then once per hour until

midnight; then once the following dawn. (It is unclear whether this regime occurred

every day.) DO and temperature were measured with two YSI-57 DO meters with

sensors in the turbulent area ofthe artificial streams where the hose entered the upstream

end. Meters were compared every 0.5 hours to Winkler titrations, which yielded

correction factors as coefficients for the raw meter readings (derived from the averages of

two streams' simultaneous readings). Corrections were made to the raw meter data in a

time-varying fashion (linear interpolation of correction factors over time). The

thiosulfate titrant was standardized with iodate every 2-3 weeks. Additionally, the meter

temperature readings were found to have inaccuracies up to 1.5°C, so these were also

calibrated, with a mercury thermometer every 0.5 hours.

Productivity was calculated with a computer program that corrected the temperature and

DO values, computed solubilities from the corrected temperatures, corrected the

reaeration coefficient for temperature, and then corrected the DO change in each time

interval for diffusion, ultimately yielding a time series ofNCP (net community

productivity) which was integrated over the day to estimate the balance of photosynthesis

3
and respiration (g 02/m\ (No further details provided on the computer program used or

written.) Photosynthesis (as gross primary productivity, or GPP) was estimated by

defining the pre-dawn and post-sunset NCP as morning and evening respiration,

assuming linear transition between these rates during the day, and correcting the NCP of

each interval for this interpolated respiratory rate. A midday estimate ofrespiration was

attempted by darkening the streams with black plastic sheeting for 1 hour at noon, but the

dissolution of bubbles trapped in the pebble-algae matrix interfered, so no midday NCP

was included in the respiration estimate. Figure 1 shows the time course ofNCP in a

productive stream, including the estimated respiratory rate, with GPP represented by the

area between the NCP curve and the respiratory rate line. Since the estimation method

sometimes, for highly unproductive streams, yielded light respiration in excess of dark

respiration and thus a negative GPP (which is, of course, impossible), the authors call

their estimate of GPP the "apparent GPP" or AGPP. The principal measure of interest

was then dawn-to-dusk AGPP in g 02/m2.

Uptake of CO2 was measured as increases in pH, with the highest pH obtained during the

day taken as a rough measure of cumulative C02 uptake. pH was recorded hourly with

an Analytical Instrument Modell 07 pH meter, temperature-corrected and calibrated with

buffers before each use. (The authors state that results were similar to the oxygen

productivity measurements, but do not provide data.)

Data analysis was primarily factorial analysis of variance (ANOYA) on dawn-to-dusk

AGPP, NCP, and respiration, and on the change in these values from the day before acute

treatment to the day ofthe treatments. Additionally, chi-square median tests were

performed on dawn-to-dusk AGPP to test for differences in chronic and acute treatments

relative to median productivity. ANaYA was also performed on highest pH data. (Not

all ofthese methods described in the study carried over into the results reported.)

Results: Results will be described for atrazine only. Several other herbicides were

tested. The authors note that productivity in the artificial streams was notably lower than

in productive natural streams, and this was especially true for the 1981 experiment. The

algae communities in both years were dominated by diatoms. In 1981, the five most

dominant algae were: Chroococcus minor (Kutz.), Scenedesmus bijuga alternans

(Reinsch), Achnanthes lanceolata (Breb.), Nitzschia palea (Kg.), and Rhopalodia

gibberula (Ehrbg.). In 1982, the five most dominant algae were: Phormidium

minnesotense (Tilden), Achnanthes lanceolata (Breb.), Achnanthes lanceolata v. dubia

(Grun.), Amphora ovalis (Kutz.), and Nitzschia palea (Kg.).

Atrazine was calculated to have a half-life in the artificial streams of 3.2 days. Streams

treated with 10 and 1 mg/kg (10,000 and 1,000 Ilg/L) experienced severe inhibition of

photosynthesis that lasted for at least 3 days (Figure 2). The 1982 algae were inhibited

less, but also did not recover as quickly as the 1981 algae. 0.1 mg/kg (100 Ilg/L)

significantly inhibited the 1982 algae for less than 3 days but did not affect the 1981

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algae. "The chi-square median test on the productivity data and the ANOVA on highest

pH confirmed these patterns" (no p-values or details provided). There were no

pronounced acute effects of atrazine treatment on respiration.

Chronic atrazine treatment was often associated with lower productivity (see Table 1 and

Table 2). In 1981, chronic treatment AGPP was lower than chronic control AGPP seven

out of eight times, while in 1982 it was lower eight out of eight times. The authors note

that ifthe chronic control mean AGPP was actually equal to the chronic treatment mean

AGPP, the chance ofthese events is <0.01 (1981) or 0.03 (1982) (these correspond to pvalues for the hypothesis that the means are different; ANOVA p-values, which are not as

definitive due to internal variability within a treatment level, are shown in Table 1 and

Table 2). The authors conclude that the results show "detectable" inhibition of

photosynthesis at 0.0 I mg/kg (10 /lg/L). The productivity differences were not caused by

respiration, since there was no consistent difference in respiration among the treatment

versus control streams.

Although significant acute x chronic interaction effects occurred twice with AGPP in

1982, there was no pattern consistent with herbicide resistance being developed in the

chronic treatment streams. (No data or p-values provided.)

0600 1200 1800

I i , I i , ,

150

-;

... 100 .J::.

N

I

E

N 50

0

m

E 0

l1.

()

z -50

2400 0600

i , iii

-100

----- --- ------ ------

Figure 1. Time course of net community productivity (NCP) in a productive stream on 7-8 July 1981.

Each histogram represents the average NCP for the time interval. The dashed line connects average

dawn, evening, and late-night estimates of respiration. The reduction of NCP in the middle of the

day was caused by darkening the streams with a black covering, but it was not used in the respiration

5
calculations. The area between the ends of the histogram and the dashed line represents AGPP.

(study figure 1)

MS

13

1.0 2

3

:J

2

13

0.5 HI 4

ill 4

4

('oj

I 0

E

('oj 4

0

C' A B C 0 E F G

I 1.5 2

a..

a.. LB

Cl

<J: 1.0

1 21

0.5 1 21 121

: I

:1 rjl 2

HI ~I 3

3

0

Figure 2. Response of AGPP to acute treatment in the atrazine experiments. Notation is as follows:

MS ="Minter Spring" 1981 experiment, LB ="Little Brazos" 1982 experiment; ABC D E F G

correspond to days -7, -1, 0, 3, 7,14,21 after acute treatment (negative implies days before

treatment); the numbers 1,2,3,4 correspond to acute atrazine treatments of 0 (control), 0.1 mg/kg

(100 /-lg/L), 1 mg/kg (1,000 /-lglL), and 10 mg/kg (10,000 /-lg/L). Mean AGPP across treatments is

shown with vertical lines spanning those found to be not significantly different at p<0.05 by Duncan's

multiple range test. Standard errors for an acute treatment were (left to right): 0.083, 0.102, 0.139,

0.081,0.210,0.164,0.201 (1981, MS); and 0.026, 0.040, 0.042, 0.063, 0.047, 0.098, 0.123 (1982, LB).

Table 1. 1981 Minter Spring experiment: Apparent gross primary productivity (AGPP) (mg 02/m2)

in the chronic atrazine treatments. P-values are shown where significant differences were detected

between treatment and control values. (from study table 2)

Days After Acute Treatment Application

Control

10 flg/L

p-value

-7

74

-1

-1 0 3 7 14

438 318 245 993 857

462 308 55 906 312

<0.01 <0.05

6

21

883

698
Table 2. 1982 Little Brazos River experiment: Apparent gross primary productivity (AGPP) (mg

Oz/m1

) in the chronic atrazine treatments. P-values are shown where significant differences were

detected between treatment and control values. (from study table 2)

Days After Acute Treatment Application

-7 -1 0 3 7 14 21

Control 163 340 319 291 315 830 1116

10 ug/L 90 220 314 223 187 467 679

p-value <0.05 <0.05 <0.01 <0.05 <0.05

Data Summary Table:

Effect Magnitude of Nominal Day ofStudy Time Did C9mments

Effect relative Atrazine Effect Weighted Recovery

to controls Concentration Occurred Average Occur- if

(fJg/L) Atrazine so, when

Concentration (see

from Day 0 to comment)?

When Effect

Occurred

14 (assuming 1982 effect

from chronic 3 week treatment (see

40% colonization) Table 2).

(different (Day 14 after Temporary 10 ~g/L chronic 7.9 ~g/L no from control

treatment; recovery on

at p<0.05) Day -7 after day of acute

treatment. Pacute value with

Decrease in treatment) ANOYA

"apparent" 30%

gross (different primary from control 100 ~g/L o(acute) 100 ~g/L yes (Day 3) 1982 effect

productivity at p<0.05) from acute

treatment 50% (estimated (different

from control 1,000 ~g/L o(acute) 1,000 ~g/L no from Figure

at p<0.05) 2). P-va1ue

70% with Duncan's

(different multiple range

from control 10,000 ~g/L o(acute) 10,000 ~g/L no test

at p<0.05)

Time weighted averages for acute treatments are equal to nominal, since effects occurred

on Day 0 and the study provides no information regarding actual concentration directly

following treatment. For chronic treatment, time weighted averages are computed

assuming a 3.2-day halflife and replenishment for return to nominal every 3 or 4 days

(corresponding to twice-weekly water replacements described in Methods). Computed

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daily concentrations and 14 day time weighted average chronic concentration are

tabulated below in a linked Excel table.

Day Replenish

OY

1

2

3Y

4

5

6

7Y

8

9

10 Y

11

12

13

14 Y

TWA Cone.:

Cone.

10

8.052452

6.484198

10

8.052452

6.484198

5.221369

10

8.052452

6.484198

10

8.052452

6.484198

5.221369

10

7.905956

Reviewer Comments:

More detail on the factorial design was provided in this study than in the Kosinski 1984

study, which implied that only one replicate was used per chronic x acute atrazine

treatment. In fact, 4 replicates were used at each combined treatment level.

The most important problem with this study was the open-water method used to estimate

gross primary productivity. Commonly accepted techniques such as 14C-uptake or even

sealed bottle methods were not applied. It appears (from the many and frequent recalibrations found necessary) that the instruments used were not reliable, and additionally

much human labor was involved in taking half-hourly measurements. This increases the

chance of human error. Aside from that, the GPP results were clearly not accurate, as

negative values were achieved which necessitated the adoption of "apparent" GPP

(AGPP) as the metric. Rather than simply re-naming their metric, it would have been

better for data interpretability ifthe authors had more thoroughly investigated the reasons

for their erroneous GPP measurements (for example, it appears that their measure of net

community productivity or NCP was relatively sound, but that the respiration estimate

was quite crude). It appears that they tried to take alternative GPP measurements by

measuring pH as an indirect indication of CO2 production, but these data are not provided

and the authors simply assert that they "confirm" the AGPP patterns. The AGPP data

should be interpreted with caution, as they cannot be considered directly comparable to

actual GPP.

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Some comments are similar to comments in the Kosinski 1984 review. Background

atrazine in source water was not analyzed, and should have been to allow more accurate

interpretation ofthe results. No schedule or dates were provided for exactly when

sampling occurred, water was replaced in the artificial streams, or herbicide was applied.

This issue was especially problematic for estimating the day after chronic treatment at

which decreases occurred. Duncan's multiple range test is not a recommended method

for multiple comparison due to its tendency to provide inflated estimates ofsignificance,

so the significance at p-value<O.05 vertical lines may be misleading.

Finally, the authors state that the productivities found in their experiment are notably

lower than those found in natural productive streams. This may be due to faulty

measurement methods, but it may also be due to experimental design (insufficient

colonization, etc.).

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.

Limitations of Study: The study is primarily limited by the use of "apparent" GPP (GPP

measured with an apparently faulty method), rather than GPP or NPP measured with a

modem and commonly accepted method, as the measure of productivity. Additionally,

the concentrations of atrazine over time and the precise schedules of when applications

and effects occurred are not provided and remain uncertain.

i/I;//(

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, cambri~e Environmental Inc

II

/

HI?

I

Secondary Reviewer: Brian Anderson, U.S. EPA

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