1

Open Literature Review Summary 

Chemical Name: Atrazine 

PC Code: 080803 

ECOTOX Record Number and Citation: 

King, R.S. 2014. Effects of pulsed atrazine exposures on autotrophic biomass, community 

structure and production in field-based stream mesocosms. Baylor University, Center for 

Reservior and Aquatic Systems Research. Waco, TX. 

MRID 49535501 

Purpose of Review (DP Barcode or Litigation): 

Review for inclusion in the CELOC calculation 

Date of Review: 3/15/2016 

Summary of Study and Findings: 

Method: 

This study examined responses of community structure and production for a photoautotrophic 

community established from local native communities of plankton and periphyton, and 

Ceratophyllum demersum. 

The study used flow through mesocosms (60 cm wide, ~20 m long) consisting of riffle (8m long, 

3% slope and ~10 cm depth, turbulent flow velocity 0.5 m/s, ceramic tile substrate), glide (9 m 

long, ~25 cm depth, flow velocity 0.05 m/s, ceramic tile substrate) and pool (1.75 m diameter, 

50 cm depth, “flow negligible except at outflow”, unlined HDPE tanks without sediment) 

sections, and included a grazing enclosure to the glide by adding minnows (Campostoma 

anomalum) to evaluate atrazine effects under both ungrazed and grazing pressures. Water was 

sourced directly from the North Bosque River, through a wetland (2-3 days) before reaching the 

intake for the research facility. Flow to the mesocosms was controlled by a constant head 

regulated tank with 12 regulatory valves at 200 L/min with recirculation. 99% of water was 

recirculated and ~1% of the “polished wetland water” was metered into the headbox mixing 

zones to ensure continuous replacement of associated natural balance of dissolved solids, 

nutrients and other ions. Resulted in a 1.5x replacement of recirculating water every 24 hrs. 

Riffle Design 

Tile sheets cut into strips of ten 25cm2 unglazed ceramic tiles, beginning 10 cm from the mixing 

box to 10 cm above the transition to the glide. Every 12 tiles a double stack of tiles was used to 

create the riffle. 

Glide Design 

Strips of 9 tiles were used. Enclosures for ungrazed portions were upstream of the grazed 

portion, with an additional ungrazed at the downstream section of the glide. Wild caught 
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minnows were added (10 per stream) to the grazing portion of the glide, based on sex and 

biomass to even out the distribution across treatments and replicates. 

Pond Design:Five tip-cuttings of Ceratophyllum demersum were secured to the bottom of a 

shallow pot with cable ties, the lower 20 mm of stems were buried under coarse sand. 12 pots 

were added to each pond. For each sampling event one pot was removed and not replaced. 

Target atrazine concentrations were 60-day averages of 10, 20, and 30 ug/L. Methanol used as 

a solvent (ultimately 0.15 ppb). All treatments and the solvent control were replicated in 

triplicate. Exposures occurred after a 30-day colonization period (February 12 – March 13 

2014). Colonization was aided by placing rocks from the wetland into the headboxes, as well as 

through “standardized collections of benthos from the riffle, glide and pool habitats of the 

North Bosque River (near Valley Mills). Each exposure was a 4-day pulse (~50, 100, and 150 

ug/L respectively) followed by a 7-day period without dosing. Each mesocosm was dosed 3 

times, then were monitored for an additional 26 days. Dosing began March 13 2014. 

Additionally, bromide was used as a tracer (0.05 mg/L), and nutrients, NO3-N (1 mg/L) and PO4-

P (0.15 mg/L), were added and kept at stable concentrations throughout the study to 

“stimulate autotrophic production and estimate daily nutrient uptake”. Analytical 

measurements of atrazine, bromide, nitrogen and phosphorous were provided on a daily basis, 

and continuous reporting for dissolved O2, pH, and conductance. 

Periphyton was monitored in the riffle and glide (grazed and ungrazed) sections of the 

mesocosms. Phytoplankton and Ceratophyllum were monitored in the pool section. All 

biological measures occurred before and after each pulse of atrazine and at intervals 

throughout the “recovery period”, totaling 10 sampling events. 

The study authors stated that “the [source] water coming out of the Lake Waco Wetlands is 

considered typical of relatively pristine streams in the Great Plains region of the USA”, however 

low levels of atrazine were reported in some samples. Water temperature ranges from 5 – 30 

degrees C, conductivity 200-500 uS/cm @ 25deg C. Nutrients are filtered through the wetland, 

Nitrate-N and NH4-N are often near or below detection limits (5-50 ug/L) and Phosphate-P is 

also very low and rarely exceeds 10 ug/L. 

Results: 

Atrazine chemographs were fairly consistent across treatments of the same concentration, and 

across the 3-pulses. In all treatments, atrazine remained present above detection limits (0.05 

ug/L) after the final pulse and through day-60. Measured 60-day average concentrations were 

10.7, 20.9, and 31.0 ug/L. The authors describe that controls contained atrazine at levels above 

the limits of detection but were generally below 0.1 ug/L throughout the study. One exception 

to this was the detection of levels up to 4.2 ug/L during the second pulse phase of the study in 

control stream #3. This occurred between March 28th and April 3rd, showing a clear decline 

curve over a week (4.2, 3.7, 1.0, 0.3, 0.1, 0.08 and 0.06 ug/L) until reaching the detection limit. 

Throughout the study the controls continued to show detections but remained below 0.1 ug/L. 
3

The study reported on several different endpoints reflecting community changes and/or 

responses over time (e.g., DO, AFDM, CHLA, and Community Composition). All of these 

endpoints are discussed for the various portions of the stream mesocosms, with the exception 

of community composition (see Study Limitations below). 

Periphyton (Riffle Portions) 

The riffle periphyton endpoints were reported to be the most sensitive endpoints in the study. 

The authors report that AFDM in the treatment groups was significantly reduced compared to 

the control cosms on day 5. The controls remained significantly higher in AFDM through day 27. 

A decline in control group AFDM was reported between the day 27 and day 34 sampling 

periods, which was attributed to a sloughing event of filamentous algae. This same event 

occurred later in the treatment groups (days 34-48), which the authors report as “due to 

dampened production during the pulse events”. 

The provided photographs of the riffle portions on day 27 of the study (the final day of the 

exposure phase) show clear differences in the coverage of tiles between controls and treatment 

cosms an in a dose response manner. Two and three weeks later (days 41 and 53) it is evident 

in the photographs that sloughing events have occurred in the controls, but differences 

between treatments are less evident. At three weeks (day 48) there are more signs of sloughing 

in treatments and there remains a visible difference between the 30 ppb exposure cosms and 

the remainder of the treatments and controls. By the end of the study, the filamentous algal 

growth on the controls and treatment cosms are rampant and seem to have sloughed off more 

frequently than discussed in the report. There is a slight difference between the 30 ppb cosms 

and the remaining cosms. 

Control AFDM was higher than all treatments from days 5 through 60; during this period the 

difference between the treatment groups and controls is considered biologically significant by 

the reviewer. Statistical significance between the treatment groups and controls was reported 

for the 10 ug/L group on days 5, 11, 16, 22, 27, and 48, the 20 ug/L group on days 5, 22, 27, and 

48, and the 30 ug/L group on days 5, 11, 16, 22, 27, and 48. 

Control CHLA was higher than all treatments. Statistically and biologically significant 

differences were reported for 10 ug/L on days 11, 22, 27, and 48, for 20 ug/L on days 22, 27, 

and 48, and for 30 ug/L on days 5, 11, 22, 27, and 48. Between days 22 and 60, the reported 

reduction in CHLA was biologically significant across all test concentrations as compared to 

controls (Table 2). 
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Table 2. Riffle section Periphyton percent reduction of mean CHLA as compared to controls. 

(bolded statistically significant at p=0.05 based on Generalized Least Squares analysis reported 

authors) 

SAMPLE 

DAY 

10 PPB 20 PPB 30 PPB 

-7 -39 -49 12 

0 -26 -15 8 

5 27 17 43 

11 35 2 59 

16 29 -13 22 

22 42 27 48 

27 42 46 45 

34 16 -44 10 

48 42 30 47 

60 31 41 60 

Periphyton (Glide Portion -Grazed Section) 

The results are similar to those for periphyton in the riffle portion of the study, except that the 

magnitude of effects was lower. The grazing fish suppressed accumulation of algal biomass 

from days 0-27. Despite this, AFDM was higher in the controls than in the treatments during 

the pulses, these were biologically significant for all pulses, with statistical significance for all 

treatments on days 5, and 16. After the pulsed exposures, there was recovery to control levels 

for both AFDM and CHLA in the grazed section of the glide. The rapid recovery was attributed 

to the filamentous algae Cladophora and Hydrodictyon growth. 

Several fish died during the study, however there was no relationship to atrazine dose. 

Periphyton (Glide Portion –Ungrazed Section) 

AFDM and CHLA accumulated rapidly from days 0-16, and was only suppressed during the 

exposure pulses. Significant declines across all treatments and controls occurred from day 48-

60 due in part to shading, senescing and significant accumulation of metaphyton from the 

upstream sloughing events. 

Metaphyton 

Floating algal mats were reported as prevalent in the pools and the ungrazed glide portions of 

the mesocosm. They began removing the metaphyton from pools on day 22, and continued to 

remove them throughout the remainder of the study. By day 27, the mats covered large areas 

of the pools, and the authors report that they were in decreasing density in a dose response 

manner, and that the shading in the controls may have negatively influenced growth and 

productivity of the macrophytes and likely the phytoplankton community. The controls had 

significantly more metaphyton biomass removed than the treatment pools throughout the 

study. 
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The heavy production of the metaphyton would have produced differential light conditions for 

the controls versus the treatments ponds. It is likely that the stable and high nutrient levels 

used in the study design caused these mats to grow rapidly and that the atrazine reduced the 

growth of the metaphyton mats. The end result is a dose response effect on metaphyton with 

significant effects at all concentrations (NOAEC <10.5 ug/L). 

Phytoplankton 

The author’s analysis of community structure indicates that there is little to no difference 

between the controls and treatment pool phytoplankton communities throughout the study. 

Their analysis does indicate that these communities changed throughout the study but it was 

directionally related to the day of sampling rather than atrazine concentration. 

The metaphyton community likely had a confounding effect on algal communities in the 

sections downstream of the riffle section. This is a clear limitation of the study and may have 

obscured treatment related effects. 

The phytoplankton results for CHLA and AFDM were not significantly different from the control. 

These endpoints showed considerable variability across the study. 

Macrophytes 

Reduced biomass and stem length were reported for treatments versus the controls from Day 

16 thorough day 34 however, these were only statistically significant for biomass on day 22 at 

the 20 and 30 ppb levels. 

Dissolved Oxygen 

There are clear and significant differences between control and treatments with regard to 

maximum dissolved oxygen throughout the study. During the pulsed atrazine exposure phases 

this separation was greater than between pulses however, controls remained higher between 

pulses as well. It is unclear as to the cause of the drop in % saturation in the controls during the 

pulses, however the authors attribute it to a decline in Photosynthetically Active Radiation 

(PAR) during rain events. 

Nutrient Removal 

Significant declines in nitrate removal were evident and in a dose response manner during the 

exposure phase, the amplitude of the differences between controls and test concentrations 

declined by the final pulse. This may indicate a shift in the response of the community to the 

atrazine exposures, possibly indicating a more tolerant community to these exposure levels. By 

the end of the study there were no differences between controls or treatment groups with 

nitrate removal, but they were extremely high. The authors were unable to discern if there 

were effects on denitrification or suppression of nitrate uptake during the exposure pulses. 

Also, the recovery cannot be entirely attributed to algal response post exposure, because they 

didn’t account for the denitrification by bacterial communities in the study. 
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Phosphate removal was highly variable and didn’t show clear differences between the controls 

and treatment levels for most of the study. The authors reported significant declines on a few 

days, noting the significant difference between 30 ppb and controls during the third pulse 

exposure period. The author makes an argument that because the phosphate sequestration is 

relatively low as compared to the nitrate sequestration, denitrification by bacteria was a major 

role in removing nitrate from the cosms. 

Authors Conclusions: 

- They believe that these results demonstrate that atrazine had no effect on the community 

structure. 

- There were significant effects on most other endpoints measured. 

- Recovery from the effects occurred for all but metaphyton biomass and DO by the end of 

the study. 

- In streams that already have high nutrient and sediment loads, atrazine effects are 

transient and may not be ecologically significant to stream periphyton, phytoplankton, and 

aquatic macrophytes. 

Reviewers Conclusions: 

- The statistical approaches used to evaluate community structure were not appropriate for 

comparison between treatment levels and controls, see Limitations section below. 

- There were statistically and/or biologically significant effects on some of the measured 

endpoints. 

- The effects on riffle section periphyton biomass, pool section metaphyton biomass, and DO 

did not recover by the end of the study. 

Endpoints for use in the calculation of the CELOC are listed below in the summary table. 
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Data Summary Table: 

Effect Magnitude of Effect 

relative to controls 

Nominal Atrazine Concentration (ug/L) 

Day of Study Effect Occurred

Estimated Maximum 60-day 

average atrazine concentration (ug/L) 

Did Recovery Occur – If so, when? 

Comments 

Metaphyton Biomass

D-27 ~ 80%↓ dry wt;D-60 ~50%↓ dry wt 

10 27-60 10.7 (60-day 

estimated average) 

No Recovery

Statistically significant decrease in metaphyton production starting on day 34 and continued throughout the study. Biologically significant effects started on day 27. 

Metaphyton Biomass

D-27 ~ 80%↓ dry wt;D-60 ~50%↓ dry wt 

20 27-60 20.9 

Metaphyton Biomass

D-27 ~ 80%↓ dry wt;D-60 ~50%↓ dry wt 

30 27-60 31.0 

Riffle Section Periphyton Biomass (AFDM) 

D-05 ~ 30% ↓D-16 ~ 28%↓D-27 ~ 24%↓D-60 ~ 35%↓

10 5, 16, 27 10.7 Recovery 

occurred between and after the pulsed exposure.

Statistically and biologically significant declines in biomassstarted ~day 5 and were associated with the end of each pulsed addition of atrazine.

Riffle Section Periphyton Biomass (AFDM) 

D-05 ~ 29% ↓D-16 ~ 23%↓D-27 ~ 21%↓D-60 ~ 1%↑

20 5, 16, 27 20.9 

Riffle Section Periphyton Biomass (AFDM ) 

D-05 ~ 35% ↓D-16 ~ 31%↓D-27 ~ 22%↓D-60 ~ 8%↑

30 5, 16, 27 31.0 

Riffle Section Periphyton Biomass (CHLA) 

D-05 ~ 27% ↓D-11 ~ 35% ↓D-16 ~ 29%↓D-22 ~ 42%↓

10 5-60 10.7 No 

Recovery

Statistically significant declines in biomass: 10 ppb: days 11, 22,27,48 
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D-27 ~ 42%

↓

D-34 ~ 16%

↓

D-48 ~ 42%

↓

D-60 ~ 31%

↓

20 ppb: days 22, 27, 48 30 ppb: days 5, 11, 22, 27, 48, 60.Although reductionswere not always statistically significant the observed reductions 

were considered biologically significant. 

Riffle Section Periphyton Biomass (CHLA) 

D-05 ~ 17% 

↓

D-11 ~ 2% 

↓

D-16 ~ 13%

↑

D-22 ~ 27%

↓

D-27 ~ 46%

↓

D-34 ~ 44%

↑

D-48 ~ 30%

↓

D-60 ~ 41%

↓

20 5-60 20.9 

Riffle Section Periphyton Biomass (CHLA) 

D-05 ~ 43% 

↓

D-11 ~ 59% 

↓

D-16 ~ 22%

↓

D-22 ~ 48%

↓

D-27 ~ 45%

↓

D-34 ~ 10%

↓

D-48 ~ 47%

↓

D-60 ~ 60%

↓

30 5-60 31.0 

Maximum Daily Dissolved Oxygen 

D-27 ~ 19%

↓

D-60 ~ 14%

↓

10 1-60 10.7 No 

Recovery

All 3 pulses of atrazine resulted in statistically significant but temporary (mainly during pulses) reduction of DO in all treatments relative to the control. After the 3 pulsed exposures statistically and biologically 

Maximum Daily Dissolved Oxygen 

D-27 ~ 26%

↓

D-60 ~ 16%

↓

20 1-60 20.9 

Maximum Daily Dissolved Oxygen

D-27 ~ 29%

↓

D-60 ~ 17%

↓

30 1-60 31.0 
9

significant reductions in DO were reported from day 27 through day 60. 
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Description of Use in Document (QUAL, QUAN, INV): Qualitative 

Rationale for Use: 

This study demonstrates clear effects on periphyton and metaphyton communities after the 

initiation of exposure to atrazine at the tested concentrations of 10, 30, and 100 ug/L. 

Limitations of Study: 

The utility of this study is limited for use in the calculation of the CELOC due to the potential 

mitigating effects of high nitrogen and phosphorous levels used in the study design. High 

nutrient levels would likely be present in most aquatic ecosystems adjacent to and receiving 

runoff from agriculture areas, however phosphorous is typically a limiting factor for algal and 

macrophytes over the long term exposures to atrazine. Additionally, the concentrations tested 

in this study are at the extreme upper end of the measured total nitrogen and total 

phosphorous in the NAQWA water monitoring database. Furthermore, recent research (e.g., 

Baxter et al. 2013) indicates that there are clear interactions with atrazine and phosphorous 

regarding exposure and observed effect, and that concentrations as high as those tested here 

are enough to mask effects of atrazine in some taxa. 

These nutrients are expected to runoff with atrazine however their concentrations would be 

expected to decline rapidly due to biological uptake and movement downstream, not unlike 

atrazine. Therefore the constant nutrient addition in this study does not represent the 

expected natural conditions of streams in the Great Plains. It is unfortunate that the source 

water, as described by the study author as representing “pristine water” was not the control 

baseline for between pulses. Nutrients should have been added as pulses with the atrazine to 

better reflect the conditions of a runoff event such as those that they simulated with the 

atrazine exposures. 

The results for the downstream sections of the cosms (glide and pool) were confounded and 

the ability to detect any treatment related effects may have been reduced. The constant 

addition of high levels of nutrients in this study compromised the utility of many endpoints due 

to the rampant growth of filamentous algae (metaphyton), which sloughed off of the riffle 

portion of the study, often resulting in a scrubbing of the tiles as it went past, then accumulated 

in the ungrazed glide and pool portions of the study causing a difference in light dynamics of 

the pool. The metaphyton likely severely impacted many of the macrophyte and phytoplankton 

endpoints in the pool section that would have been collected later in the study because of the 

decreased light conditions. The metaphyton impacts began around day 22 of the study in the 

controls, which is 5 days short of the last day of exposure, and were more likely to be of greater 

influence in the controls throughout the study. 

Another limitation is that the “pristine” source water clearly had a background level of atrazine. 

It was not reported if other herbicidal chemicals were also present within the water. The 

resident algal community (used to colonize the cosms) may have already been selected for 

more atrazine resistant algae. 
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While there were solvent controls (methanol added) negative controls were not used in this 

study. It is unknown if this would have impacted the results of the study. 

Lastly, the authors did not find a difference in community structure; however, that finding may 

simply be due to the method used to analyze the raw data. 

First, the NMDS ordination was conducted separately for each test group (only 

comparing the similarity among communities within a test group over time). The 

ordination containing all the treatment groups appears to simply be an overlay of the 

separate ordinations (the study authors state that this is this case on pg 30). The 

ordinations show a strong temporal shift over time but do not have meaning in terms of 

the (dis)similarity among all the treatment group communities. A single NMDS 

ordination based on a dissimilarity matrix of all treatment groups is needed to answer 

the question “are there differences among the treatment groups compared to the 

control?” This could be done with all the time points or separately for each time point. 

The ordination would provide a visual way to assess how different the communities 

were. 

Second, the study authors used PERMANOVA to test for statistically significant 

differences among the communities. However, the analysis only asked the question “is 

there a treatment related effect?” (see appendix 17). It does not assess whether or not 

there were treatment related effects at each individual test concentration. 

Primary Reviewer: 

Frank T. Farruggia, Ph.D. Biologist ERB3 

Secondary Reviewer: 

Michael Lowit, Ph.D. Ecologist ERB1 

References: 

Baxter, L.R., P.K. Sibley, K.R. Solomon, M.L. Hanson. 2013. Interactions between atrazine and 

phosphorous in aquatic systems: Effects on phytoplankton and periphyton. Chemosphere 90: 

1069-1076. 

Digitally signed by FRANK FARRUGGIA 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=FRANK 

FARRUGGIA, dnQualifier=0000048865 

Date: 2016.03.24 13:16:15 -04'00'

2016.03.24 13:43:13 

-04'00'