Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Jurgensen, T.A. and K.D. Hoagland. 1990. Effects ofshort-tenn pulses of atrazine on

attached algal communities in a small stream. Arch. Environ. Contam. Toxicol. 19:617-

623. (MRID 47543504)

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 LaC for atrazine.

Date of Review:

October 16,2008

Purpose of Study:

This study examines the potential effects of atrazine to attached aquatic plants in stream

enclosures.

Summary of Study Findings:

Methods:

The study was conducted in a spring-fed stream, Otter Creek, in western Nebraska near

Lake McConaughy (a map is provided in the study but not reproduced here). The stream

watershed was hilly rangeland with sandy soils, used primarily for cattle grazing.

Background levels of atrazine were not detected in the stream in analyses ofthree

streamwater samples taken at the beginning, middle, and end ofthe 6-week study period.

The stream pH was 8.0, with total alkalinity of 63 mglL as CaC03, and a nitrate-nitrogen

concentration of 0.64 mg/L. At the study site, the stream was 2-3 m wide with an

average depth of 30-40 cm and typical flow velocity ranging from 0.5 to 1 m/s.

Unglazed baked clay square tiles, 7.6 cm long on each side, were used as periphylon

substrates. Eighteen tiles were attached to each treatment tray, consisting of a plexiglass

plate anchored to the stream bottom. Five treatment trays were used at each ofthree

III11IIIII

2087855
replicate sites, with an experimental control (no enclosure), a treatment control (enclosed

but with 0 jlg/L atrazine), and treatments at 2 Ilg/L, 30 Ilg/L, and 100 Ilg/L atrazine.

Initially, the trays were left to colonize for two weeks prior to the first treatment (on June

21, 1985), and then enclosures were attached to the 4 enclosure trays (excluding the

experimental control tray) at each site. Treatment enclosures consisted of plexiglass

boxes (30.5 x 25.2 x 46.6 cm high), open on top and bottom, attached to the trays with

neoprene seals to prevent leakage and weighted to prevent movement with the current.

The height ofthe boxes was sufficient to extend above the water surface and keep the

trays isolated from the surrounding stream. Atrazine (98%, Supelco, Houston, TX) was

dissolved in deionized water and added to enclosures to achieve the desired nominal

concentration, including the control enclosure that received only deionized water. The

enclosures were maintained for 24 hours and then removed. On Day 14, a second 24-

hour atrazine exposure was applied.

Periphyton were sampled 2, 7, and 14 days after the end of each atrazine application

period. For each tray, 2 tiles were removed and scraped with a razor blade, with the

resulting algal suspensions preserved in 6:3: 1 water:ethanol:formalin. These samples

were analyzed for cell counts and total biomass. Cell counts of common and abundant

algal taxa (comprising at least 35 of500 diatom frustules and 10% oftotal algal density,

based on preliminary counts) were conducted using a Palmer cell at 400 x magnification

with an Olympus model FH microscope. Living and nonliving cells were tallied

separately. Permanent slides were also made, using Hyrax mounting medium to facilitate

diatom identifications. Total biomass was determined as ash-free dry weight (AFDW)

modified from American Public Health Association, 1981, Standard methods for the

examination of water and wastewater (APHA 1981).

A 200 mL water sample was taken from two ofthe 100 jlg/L treatment enclosures at the

end of each 24 hour exposure period for atrazine analysis. The samples were extracted

with pesticide-quality methylene chloride (J.T. Bake Chem. Co.) within 8 hours after

collection by shaking twice, for 2 minutes, in a separatory funnel and drawing offthe

methylene chloride. Extracts were stored at 4°C and later concentrated to 10 mL in a

Kudema-Danish evaporator with a 3-ball Snyder column, treated with 10 mL of hexane,

and concentrated to 5 mL (following APHA 1981). Extracts were analyzed for atrazine

using a Tracor model 560 gas chromatograph, a Hall 700A electrolytic conductivity

detector in the nitrogen mode, and a glass column packed with 35 STAP on 80-100

CWHP. Standards were made from 98% pure atrazine (Shell, Pesticides Division). The

method had a detection limit of about 50 jlg/L. Likely actual atrazine concentrations for

the 2 jlg/L and 30 jlg/L nominal treatments were extrapolated from the 100 Ilg/L results.

Statistical analyses were performed on the periphyton biomass and live algal cell density

using 3-way ANOYA with repeated measures to account for the autocorrelation ofstream

samples over time. Sample sizes, especially after the second 24-hour atrazine exposure,

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were smaller than expected due to loss of "several" sample trays "due to storm runoff'

(no further details provided). Because ofthis problem, data were analyzed for collections

made after the first exposure (3 sampling dates) and for collections made after both

exposures (6 sampling dates), rather than after each exposure. Additionally, regressions

were performed on AFDWs, ash weights, total living algal densities, and nonliving

diatom densities.

Results:

Atrazine concentrations measured in the nominal 100 Jlg/L enclosures at the end ofthe

24 hour sampling period ranged from 58 to 93 Jlg/L, with an average of77.5 Jlg/L. The

authors state that, assuming similar atrazine losses from the other treatment level

enclosures, the nominal 2 Jlg/L treatments would have atrazine concentrations of 1.2-1.9

Jlg/L, and the nominal 30 Jlg/L treatments would have atrazine concentrations of 17-28

Jlg/L. (Averages, assuming 77.5% of nominal, would be 1.55 Jlg/L and 22.5 Jlg/L in the

nominal 2 and 30 Jlg/L treatments, respectively.)

Biomass declined in all treatments over time, but no significant differences were found

between the four atrazine treatments (including the treatment control). As shown in

Table 1, 3-way ANOVA found significant effects oftime (p-value 0.05 for the first 3

sample dates, p-value == 0.0004 taking all 6 sample dates) on total biomass, but no

significant effects oftreatment (p-value = 0.73,0.60). The experimental control and

treatment control had significant differences, particularly in the total biomass compared

on all 6 sample dates (p-value of 0.01) (see Table 2). The experimental control, treatment

control, and 100 Jlg/L treatment dynamics oftotal biomass as well as Oscillatoria live

cells and Nitzschia palea live cells are graphed in Figure 1, Figure 2, and Figure 3,

respectively. Total biomass of experimental and treatment controls are also tabulated in

Table 4.

Oscillatoria sp. was the most abundant alga, and Nitzschia palea was the dominant

diatom. Green unicells and the common diatoms Fragilaria pinnata and Navicula

luzonensis were also examined. None ofthe species showed any significant treatment

effects (Table 1), except possibly Fragilaria pinnata on the first three sample dates (pvalue == 0.07).

Sediment (measured as ash remaining after incineration of organic material) increased

over time in all ofthe treatments, in rough correspondence with biomass decline (Table

3). The authors attribute the biomass decline over time on the tiles to sediment

deposition, which they qualitatively argue (citing related work by other authors) is a

much stronger effect on the attached stream community than the atrazine treatments used

in their experiment. These assertions are not quantitatively analyzed. (The authors

present lists ofreferences on maximum concentrations of atrazine residues in surface

waters and on atrazine exposure studies for algae, but they are not reproduced in this

review.)

3
2

-(/)

(/)

<'il

E

o

m

o ot 10 t 20

...... control

..... 0 ug/L

-C-100 ug/L

30

Time (days)

Figure 1. Ash~free dry weight biomass (mg/cm2

) of attached stream communities on tile substrates in

experimental control, 0 Jig/L treatment control, and 100 ",gIL atrazine enclosures. Vertical bars

represent standard errors; arrows on time axis represent onset of 24-hour atrazine treatment

periods.

200

...... control

I ..... OuglL

1. -0-100 ug/L

C\I r I : ·0•• E . . E . '.

(/)

100 ·t \, . = -. Q)

U .f' .... \~

f\1 .

\

0 ....... .»,....0 0

at 20 30

Time (days)

Figure 2. Oscillatoria live cell densities (cells/mm2

) on tile substrates in control, 0 ",gIL treatment

control, and 100 Jig/L atrazine enclosures. Vertical bars represent standard errors; arrows on time

axis represent onset of 24-hour atrazine treatment periods.

4
6

1.......1 ... control

··4.. 0 ug/L

.0- 100 uglL C\l 4 M.: i !

E

11···\.1 E

(/)

= \.

(1)

() 2 f....I

1\1··.. '.

0

of 10 20 30

Time (days)

Figure 3. Nitzschia palea live cell densities (cells/mm2

) on tile substrates in control, 0 J1g/L treatment

control, and 100 J1g/L atrazine enclosures. Vertical bars represent standard errors; arrows on time

axis represent onset of 24-hour atrazine treatment periods.

Table 1. P-values from 3-way ANOVA for comparison of biomass and live cell densities offour

atrazine treatments (0, 2, 30, and 100 J1g/L) with respect to treatment, time, and treatment effects

with time (Trt. x Time) for the first three sampling dates (only the first atrazine treatment) and all

six sample dates (including both atrazine treatments). (study table 1)

First 3 sample dates

Treatment Time

Oscillatoria sp. 0.82 0.97 0.39 0.50 0.03 0.67

Nitzschia palea (Klitz) 0.17 0.1 I 0.92 0.70 0.002 0.85

Green unicells 0.42 0.2 I 0.76 0.44 0.09 0.90

Fragilaria pinnata Ehr. 0.07 0.22 0.90 0.1 I 0.03 0.92

Navicula luzonensis Hust. 0.25 0.62 0.91 0.71 0.81 0.57

Total biomass 0.73 0.05 0.76 0.6 0.0004 0.88

Table 2. P-values from 3-way ANOVA for comparison of biomass and live cell densities of four

experimental control and treatment control (0 J.1g1L) with respect to treatment, time, and treatment

effects with time (Trt. x Time) for the first three sampling dates (only the first atrazine treatment)

and all six sample dates (including both atrazine treatments). (study table 4)

Oscillatoria sp.

Nitzschia palea (Klitz)

0.31

0.05

0.04

0.30

0.63

0.8 I

0.03

0.03

0.21

0.06

0.65

0.60

0.01
Table 3. Ash (mg/cm2

) remaining after organic material was incinerated during ash-free dry weight analysis. Residual ash consisted mostly of

inorganic sediment deposited on the periphyton communities. Percentages are the fraction of total dry weight composed of ash. (study table 2)

2 ug/L 100 ug/L

Sample Community Standard % Standard % Standard % Standard %

date a e Mean Deviation Ash Mean Mean Ash

25-lun 17 days 6.7 1.5 85 25 3.2 95 48 34 95 28 14 94

30-lun 22 days 8.1 2.9 83 23 9.3 95 16 9.3 88 25 26 91

7-Jul 29 days II 0.2 91 16 9.9 95 36 50 92 18 1.6 96

10-Jul 32 days 23 4.1 95 20 1.4 96 18 8.2 93 49 * 97

15-Jul 37 days 45 38 91 19 5.3 96 27 15 97 58

22-Jul 44 days 16 1.6 97 28 29 91 34 2.4 99 89

Table 4. Ash-free dry weight biomass (mg/cm2

) from the experimental controls (no enclosure) and treatment controls (0 ug/L) for the six sample dates.

(study table 3)

Treatment C..antral

Sample

date Mean StandardDeviation Mean Standard

Deviation

25-Jun 1.05 0.52 1.16 0.23

30-lun 1.11 0.33 1.6 0.53

7-Jul 0.44 0.26 1.l5 0.58

10-Jul 0.09 * 1.l7 0.35

15-lul 0.01 * 1.13 0.6

22-lul 0.15 * 0.42 0.21

* No standard deviation due to loss of replicates

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Data Summary Table:

E.1fect Magnitude Nominal Day ofStudy Time Weighted Did Comments

ofEffect Atrazine Effect Average Recovery

relative to Concentration Occurred Atrazine Occurcontrols (flg/L) IConcentration ifso, + Over the Study when (.,ee

Dun"'an comment) I

Total IL- 2 "giL (short- ~jgIL Over all

biomass, ~tenn pulse)_ __~~____ j sample dates,

live eell 30 "gIL (short- Not , I 7-"giL--j p-value for

densities of No effect -,<,nlU'u!,,) ti"hle I~, "g NIA tr,"ment misc. 100 !!g/L (short- PP effect on total

abundant~ tenn pulse) 5.2 !!g/L I biomass was

~hy~n _ -- : 0.60 --------'-

Average exposure concentrations over the study duration are included in the preceding

table.

Reviewer Comments:

The study conditions were apparently not sufficiently controlled, as evidenced by the

disappearance of multiple trays. Perhaps more importantly, the details ofthe events

surrounding the trays being carried off(storm conditions, timing, exactly which trays

were lost, etc.) were not clearly discussed. For example, the number ofreplicates

changed over time, but how or when replicate numbers changed was not reported. The

only information that trays were missing is in Table 3 and Table 4 when standard

deviations cannot be reported. It is not clear how the runoff event leading to the missing

trays affected the study, but it could have had significant impact. Taking the authors'

own assertions about the importance ofsediment effects, the sediment transport of a

storm event could have had a multitude of effects on the streambed periphyton, including

scouring them out, burying them, or carrying away the sediment built up over time.

Overall, the issue ofthe storm that apparently took place during the experiment received

insufficient discussion and may have confounded the results.

The results presented are lacking in certain details. No regression results appear in the

study, despite the description in the methods section. No sediment results are provided

for the experimental controls, and no statistical analysis is used to test correspondence

between sediment and biomass. The authors' points about the importance ofsediment

are thus not well supported by the results presented. Also, the presentation ofthe sample

date timing in the methods section is confusing. For example, the methods section states

that the first sample was taken 2 days after the end ofthe first enclosure period (so 3 days

after the beginning of enclosure), and that the first atrazine treatment began June 21.

However, as shown in Table 3 and Table 4, the first sample date was June 25, not June 24

as would be expected. These inconsistencies confuse time weighted average calculations.
The reasons for and implications ofsignificant differences between the experimental

controls and the treatment controls are not discussed. However, the fact that this was the

single most significant result found for treatment effects (p-value of 0.01 for treatment

effects on total biomass over all sample dates) shows that there must have been a major

impact on the periphyton community simply from imposing 24-hour enclosures in

deionized water. This calls into question whether the atrazine treatment results are

applicable to a more realistic flow-through situation.

Also, the detection limit was 50 ug/L. Therefore, the actual test levels at the nominal 2

ug/L and 30 ug/L nominal test concentrations are unknown. The measured:nominal ratio

observed at the 100 ug/L nominal test concentration was applied to the 2 ug/L and 30

ug/L test levels to estimate a "measured" exposure value.

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

Rationale for Use: Study results may have been confounded by a storm event and by the

high detection limit ofthe analytical methods used in this study. However, the study

contributes to the weight of evidence of potential atrazine effects.

Limitations of Study: This study examined potential impacts ofshort-term (24-hour)

pulses on atrazine on streambed periphyton communities, which did not create noticeable

effects. However, the study is limited by: 1) the occurrence of a storm event during the

study, which decreased the number ofreplicates and could have overshadowed any subtle

atrazine impacts; and 2) the finding ofsignificant differences between the treatment

controls (enclosed with 0 Jlg/L atrazine treatment) and experimental controls (no

enclosure), which points to the artificial nature ofthe atrazine treatment regime. Also,

time weighted average concentrations are highly uncertain, particularly in the 2 ug/L and

30 ug/L nominal levels where atrazine concentrations were not measured. Also, the

detection limit in this study was 50 ug/L; therefore, atrazine may have been present at

background concentrations <50 ug/L.

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

Secondary Reviewer: Brian Anderson j}7/ /1 ..../// ._._

f"! / !~~/ I' Zr'

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