### 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 ofherbicides on stream Aufivuchs productivity and nutrient uptake. Arch. Environ. Contam. Toxicol. 17:299- 306. (MRID 47543509).


### 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: November 3, 2008


### Purpose ofStudy: This study examines the potential effects ofatrazine to attached aquatic plants and animals (Aufivuchs) in experimental artificial streams.


### Summary ofStudy Findings:

Methods: Two experiments were conducted, the first examining constant exposure to atrazine, and the second examining pulsed exposure to a mixture offour herbicides. Only the methods relevant to the first experimentwill be described here.

Six replicate streams were used concurrently, each consisting ofa 7.5 em outerdiameter, 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 offresh water, nutrients, and herbicide, and discharge ofoverflow 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.innerwalls 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 of50 em above the water surface.



# III1IIIIII 2087868The atrazine experiment was conducted twice, once at 25°C and once at lOoC, each time testing the effects ofnominal levels of50**~g/L** and 200**~g/L** atrazine. (Average concentrations overtime 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 ofexposure.) 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 ofKN03, 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 ifturnovertime 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 overtwo-day intervals. Daily samples ofthe overflow water were analyzed for nutrient concentrations. Overflow water was collected and aportion 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). Anotherportion 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 ofoverflow sample collection, was processed similarly. Input and output quantities, respectively, wcrc calculated by:

IT =Rs ·TC s 

### +C R .(V T -RsT) 

# o =V .Cp+C c T T 

### 2 where R



### s

### = stock solution pumping rate (Uhr), Vr

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

### = stock concentration (mg/L), C

R

### = composite Rock Creek concentration (mg/L), T

### = pumping interval (hr), Cp 

### = previous artificial stream overflow concentration, and Cc = overflow concentration at end ofpresent 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.

2For seven days pre-treatment, the streams were colonizedbyAufivuchs entrained in the creekwater supply. Larger organisms were excluded by "the presence oftwo reservoirs" (unclear whatthis 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 of0.00057% by volume, and the authors (citing two papers from the literature) do notbelieve any toxic effect is likely from the methanol. Sufficient stock atrazine was added directly to each stream at the start oftreatmentto attain the nominal concentration. The stockwas 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 stockbottles to verify the calculated chemical concentrations. At the end ofeach 20-day experiment, the stream systems were dismantled and the accumulatedAufivuchs inside each Pyrex tube was scraped into aplastic container, homogenized in tap water at aknown volume with a Tekmar Tissumizer, and four replicate aliquots removed from each homogenate for analysis ofchlorophyll a and ash free dry weight (AFDW).

The nutrient samples were analyzed for total P, SRP, N02 

### +N03N, ammoniaN, TKN, and dissolved silicaby the methods ofUSEPA, Methodsfor chemicalanalysis of water andwastes, 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 ofa single mixed reagent, the ascorbic acid was used as a separate reagent and was added directly to the analytical cartridge in orderto increase reagent shelf-life. Distilled waterwas notused in the cartridge and the sample line size was doubled to improve sensitivity.
Chlorophyll a was extracted by adding 

### 3 mL ofa 1% MgC03 suspension and 72 mL acetone to 5 mL offresh homogenized suspension, yielding a 90% acetone solution. The samples in their polyethylene bottles were sonicated in the dark for 1 hourunder refrigeration and were stored at -10°C for up to 3 weeks. Justpriorto 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.

3Aliquots of50 mL homogenized suspension were placed in pre-ashed and tared crucibles and were analyzed for AFDW, following the method ofWeberIUSEPA, Biologicalfield andlaboratory methodsfor measuring the quality of surface waters andeffluents, 1973.

The method for extraction and analysis ofatrazine and the quality control program are described in detail in a separate paperby 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 ofpreparation, using atemperature-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 ttests or by analysis ofvariance.



### Results: Two experiments were conducted, the first examining constant exposure to atrazine, and the second examining pulsed exposure to a mixture offour herbicides. Only the results relevant to the first experiment will be described here.

Atrazine GC analyses were determined to have a detection limit of0.075 

### Ilg/L (uncorrected for percent recovery) and a mean recovery from field spikes of86% (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.OO1, specific test not stated) inhibition ofbiomass accumulation atboth 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 ofuptake 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%

4

### lowerthan 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-treatmentvariation" (no details on precisely which values were significant orwith whattests). In the 24 

### llg/L atrazine concentration streams, uptake rates ofSRP and dissolved silicaremained as high or higherthan 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 afternine days since addition, butthese 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 ofartificial 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 ofcreek water, AEP = air exchange port. Arrows show directions offlow.



### 525 C

o "-

........ 8--I

... {)-...-I

.. 11---H---'-I

... Oc

...... 

# t 10 

### C 

### SRP .I)e
.j).

,0:



# • '"'


# ..**SiO~** 0 .c;**2.~**



### '"'**~u**

Experiment Day Figure 2. Uptake rates ofSRP, Si02 and N02+N03 in each ofthe 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 ofexposure. 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 P04P Si02 8.2 0.15 7.7 4.1 0.23 9.3

6Table 2. Total accumulated AFDW (g) at the end ofeach experiment, including p-value when significantly different from control. (from study table 3)
lO°C 25°C %of %of Treatment Mean StdDev control p Mean StdDev 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 ofeach experiment, including p-value when significantly different from control. (from study table 3)
lOOC 25°C %of %of Treatment Mean StdDev control p Mean StdDev 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 of Study 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.OOI) measured experiment. (Aufwuchs) at end of Declines were biomass 31% study) significant at (ash-free (p<O.OOI) 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

7Effect Magnitude of Nominal Day of Study 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 ofnatural 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 ofbackground concentrations ofatrazine, and the time ofyear over which the experiments took place and discussion ofthat timing relative to agricultural activities.

A few minorpoints ofconfusion affect the study interpretation: first, the definition "turnovertime" 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 citedbut the methods description says that eithertwo-sample 

### ttest or ANOVA may be used, and the exacttests associated with a p-value are neverprovided.
Despite these minor limitations, overall the study appears well-reported, with a wellcontrolled experimental design and clear discussion ofmethods and results. Replication was limited (two replicates at each treatment level), although for biomass endpoints results were still statistically significant.

8

### Description ofUse 

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



### Rationale for Use: Study limitiations limitthe utility ofthe study; however, it contributes to the weight ofevidence regarding the potential for atrazine to affect aquatic communities.



### Limitations ofStudy: This study provides evidence ofatemperature-dependence in the effects ofatrazine on periphyton biomass. The results appearreliable since low variability was found despite the poorreplication, butthey are only applicable to similarly controlled environments, not naturallotic ecosystems. Also, not enough information is provided about the source streamwater.


### Primary Reviewer: SaraHendrix, M.S., StaffScientist, Cambridge Environmental Inc
Secondary Reviewer: 

## ./**p~/1** Brian

# Anderson.~ 

# 1/;/.,/ l /?/ ! 

### /C--- 

# II

9