### 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 offour terrestrial herbicides onthe productivity ofartificial stream algal communities. 1. Environ. Qual. 13:75-82. 

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Purpose ofReview: Determine potential effects ofatrazine 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 ofReview: October27,2008



### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants (algae) in experimental streams (lotic mesocosms).



### Summary ofStudy Findings:

Methods: Methods are similarto those in Kosinski, 1984, though this study provides
more detail on most aspects. Only the methods pertaining to atrazine will be described (three otherpesticides were also examined).

Two sets ofexperiments were conducted in 1981 and 1982, April-May ofboth years. 

### In each year, experimental design was a factorial combination of0, 0.1, 1.0, or 10 mg/kg (0, 100,1,000, or 10,000 /-lg/L) "acute" atrazine treatment, after0 or 0.01 mg/kg (0 or 10 /-lg/L) "chronic" atrazine in the waterduring initial colonization. Acute studies were conducted afterchronic exposures to atrazine at

# amg/kg or 0.01 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 apasture, was almost anoxic and had constanttemperature ofaround 21°C, mean specific conductance at 25°C of54.6 mS/m, alkalinity of71 mg CaC03/L, 404 flg/L nitrate, and 21 flg/L 

# 11111111111 2087869soluble reactive phosphate (SRP). (No information on frequency ortiming ofwater quality measurements.) Cyanobacteria (mostlyAnabaena), diatoms (Nitzschia, Rhopalodia, andNavicula), and afew species ofprotozoans, rotifers, ostracods, and chironomids were present onthe 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 supportedby 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 riverreceived high volumes ofrunofffrom adjacent fields, as evidenced by the gully erosion patterns in the watershed terrain. Water and algae were taken from abridge that passes overthe river. No information is provided
on river width, depth, oron flow rate or discharge. Dense growth ofCladophora 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% ofsaturation. (No information on frequency ortiming ofwater quality measurements.)

Water and algae were collected atthe source areas and transported backto the laboratory in carboys, where they were eitherused immediately or stored in covered indoortanks for up to 10 days. Atrazine experiments were perfonned in 32 artificial streams (4 acute treatments x 2 chronic treatments, 4replicates for each combination

- note thatthis is a different description than in the Kosinski 1984review as more information was provided in this study), located on a flatroofonthe Texas A&M University campus. Each stream consisted ofawooden trough, 2.44 m long and 12.6 cm wide, lined with 0.15-mm plastic sheeting and filled with source waterto a depth of4 cm, with apump at one end circulating waterthrough a garden hose beneath the trough and backthrough the channel at avelocity of3 cm/s. The substrate for colonization was 1-2 cm diameter autoclaved, streamwompebbles, which linedthe middle 1.4 m ofeach stream. A sheet ofcorrugated, clearfiberglass overlainby atranslucentwhite plastic sheet and a clearplastic sheet provided covers to protectthe streams against excessive evaporation and the elements. The covers attenuatedthe intensity ofphotosynthetically active radiation by 60% (no information on how this was measured), which reportedly moderatedthe greenhouse effect on high irradiance days. Automatic reservoirs filled with source waterkeptthe stream depths constant. Twice aweek, 2 liters (15% ofthe stream volume) were discarded andreplaced with source water.

Colonizationlinitialization ofthe streams is not described in detail, but is stated to have taken place over "3 or4 weeks" with regular additions with waterreplacement keeping the non-control "chronic" treatment streams at 0.01 mg/kg atrazine concentration (10 Ilg/L). After colonization with orwithout the presence of 10 ug/L atrazine, single

2"acute" treatments were applied using commercial atrazine (Aatrex 80 WP) atnominal concentrations based on the active ingredient concentration in the commercial preparation (specific application method and dates not stated). Chronic treatment additions ceased after acute treatmentwas applied.

"A small number" ofanalyses were performed on frozen streamwater samples to estimate the rate ofdisappearance ofthe herbicides from the water (no further details provided). Analysis was performed atthe TAMU Agricultural Analytic Service by anitrogenphosphorus detector method modified from Purkayasthaand Cochrane, J. agric. Food Chem. 21, 1973. No attemptwas made to separately determine effects ofemulsifiers and othernonherbicidal components ofthe commercial atrazine product.

Productivity was measured with open-water oxygen methods, i.e. by estimating the sum ofphotosynthesis and respiration (net community productivity orNCP) via correcting the rate ofchange ofthe dissolved oxygen (DO) concentration for exchange with the atmosphere. Oxygen saturation deficitwas 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 sodiumbisulfite and a crystals/L cobaltous chloride), then circulating the waterin
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 arate of1.6% per degree Celsius. Kwas 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 1hour after sunset; then once perhouruntil midnight; then once the following dawn. (It is unclearwhetherthis 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 enteredthe upstream end. Meters were compared every 0.5 hours to Winklertitrations, which yielded correction factors as coefficients for the raw meterreadings (derived from the averages of two streams' simultaneous readings). Corrections were made to the raw meterdata in a time-varying fashion (linearinterpolation ofcorrection factors overtime). The thiosulfate titrantwas 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 thermometerevery 0.5 hours.

Productivity was calculated with a computerprogram that correctedthe temperature and DO values, computed solubilities from the corrected temperatures, corrected the reaeration coefficient for temperature, and then correctedthe DO change in each time interval for diffusion, ultimately yielding a time series ofNCP (net community productivity) which was integrated overthe day to estimatethe balance ofphotosynthesis

3and respiration (g 02/m\ (No further details provided on the computerprogram used or written.) Photosynthesis (as gross primary productivity, or GPP) was estimated by defining the pre-dawn and post-sunsetNCP 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 blackplastic sheeting for 1 hour at noon, but the dissolution ofbubbles 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 lightrespiration in excess ofdark respiration and thus a negative GPP (which is, ofcourse, impossible), the authors call their estimate ofGPP the "apparent GPP" or AGPP. The principal measure ofinterest
was then dawn-to-dusk AGPP in g 02/m2.

Uptake ofCO2was measured as increases in pH, with the highest pH obtained during the day taken as a rough measure ofcumulative C02 uptake. pH was recorded hourly with
an Analytical Instrument Modell07 pH meter, temperature-corrected and calibrated with buffers before each use. (The authors state that results were similarto the oxygen productivity measurements, but do not provide data.)

Data analysis was primarily factorial analysis ofvariance (ANOYA) on dawn-to-dusk AGPP, NCP, and respiration, and onthe change in these values from the day before acute treatmentto 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 otherherbicides 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.), Nitzschiapalea (Kg.), andRhopalodia gibberula (Ehrbg.). In 1982, the five most dominant algae were: Phormidium minnesotense (Tilden),Achnanthes lanceolata (Breb.),Achnanthes lanceolata v. dubia (Grun.), Amphora ovalis (Kutz.), andNitzschiapalea (Kg.).

Atrazine was calculated to have a half-life in the artificial streams of3.2 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

4algae. "The chi-square median test onthe productivity data and the ANOVA on highest pH confirmedthese patterns" (no p-values ordetails provided). There were no pronounced acute effects ofatrazine treatment on respiration.

Chronic atrazine treatment was often associated with lowerproductivity (see Table 1 and Table 2). In 1981, chronic treatmentAGPP was lowerthan chronic control AGPP seven out ofeighttimes, while in 1982 it was lowereight out ofeight 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) or0.03 (1982) (these correspond to pvalues for the hypothesis thatthe 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 thatthe results show "detectable" inhibition of photosynthesis at 0.0I mg/kg (10 /lg/L). The productivity differences were not causedby respiration, since there was no consistent difference in respiration among the treatment versus control streams.

Although significant acute x chronic interaction effects occurredtwice with AGPP in 1982, there was no pattern consistentwith herbicide resistance being developed in the chronic treatment streams. (No data orp-values provided.)

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Figure 1. Time course ofnet 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 ofrespiration. The reduction ofNCP in the middle ofthe day was caused by darkening the streams with a black covering, but it was not used in the respiration



### 5calculations. The area between the ends ofthe histogram and the dashed line represents AGPP. (study figure 1)



### MS 1 3 1.0 2 

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('oj 4 0 C' A B C 0 E F G 

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Figure 2. Response ofAGPP 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 of0 (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 significantdifferences were detected between treatment and control values. (from study table 2)
DaysAfterAcute TreatmentApplication

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 698Table 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)
DaysAfterAcute TreatmentApplication -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 of Study 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 ofacute 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 atp<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
atp<0.05)

Time weighted averages for acute treatments are equal to nominal, since effects occurred
on Day 0 andthe 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 or4 days (corresponding to twice-weekly waterreplacements described in Methods). Computed

7daily 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 impliedthat 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-watermethod used to estimate gross primary productivity. Commonly acceptedtechniques such as 14C-uptake oreven sealedbottle methods were not applied. It appears (from the many and frequent recalibrations found necessary) thatthe instruments used were not reliable, and additionally much human labor was involved in taking half-hourly measurements. This increases the chance ofhuman error. Aside from that, the GPPresults were clearly not accurate, as negative values were achieved which necessitated the adoption of"apparent" GPP (AGPP) as the metric. Ratherthan 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 thattheir measure ofnet community productivity orNCP was relatively sound, butthatthe respiration estimate
was quite crude). It appears thatthey tried to take alternative GPP measurements by measuring pH as an indirect indication ofCO2 production, butthese data are not provided and the authors simply assertthatthey "confirm" the AGPP patterns. The AGPP data should be interpreted with caution, as they cannotbe considered directly comparable to actual GPP.

8Some comments are similarto comments in the Kosinski 1984 review. Background atrazine in source waterwas not analyzed, and should have been to allow more accurate interpretation ofthe results. No schedule ordates were provided for exactly when sampling occurred, waterwas replaced in the artificial streams, orherbicide was applied. This issue was especiallyproblematic for estimating the day afterchronic treatment at which decreases occurred. Duncan's multiple range test is not arecommended method for multiple comparison due to its tendencyto provide inflated estimates ofsignificance,
so the significance atp-value<O.05 vertical lines may be misleading.

Finally, the authors state thatthe productivities found in their experiment are notably lowerthan those found in natural productive streams. This may be due to faulty measurementmethods, but it may also be due to experimental design (insufficient colonization, etc.).



### Description ofUse in Document (QUAL, QUAN, INV): QUAL

Rationale for Use: The study deficiencies limitthe utility ofthis study. However, the study appears sufficientto contributeto the weight ofevidence regarding the concentration-response relationship ofatrazine and aquatic plants.



### Limitations ofStudy: The study is primarily limited by the use of"apparent" GPP (GPP measured with an apparently faulty method), ratherthan GPP orNPP measured with a modem and commonly accepted method, as the measure ofproductivity. Additionally, the concentrations ofatrazine overtime and the precise schedules ofwhen applications and effects occurred are notprovided and remain uncertain. 

# i/I;//( Primary Reviewer: Sara Hendrix, M.S., StaffScientist,**cambri~e** Environmental Inc 

### II 

# HI?

/ I Secondary Reviewer: Brian Anderson, U.S. EPA

9