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 Ba rcode 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 st ructure and production for a photoautotrophic community established from local native co mmunities of plankton and periphyton, and Ceratophyllum demersum

.

The study used flow through mesocosms (60 cm wi de, ~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, ce ramic 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 enclosur e 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, th rough 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 20 0 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 a ssociated natural balance of dissolved solids,

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

Riffle Design Tile sheets cut into strips of ten 25cm 2 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 un grazed portions were upstream of the grazed portion, with an additional ungrazed at the do wnstream section of the glide. Wild caught2 minnows were added (10 per stream) to the gr azing 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 aver ages of 10, 20, and 30 ug/L. Methanol used as a solvent (ultimately 0.15 ppb). All treatments and the solvent contro l 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 ex posure 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 tr acer (0.05 mg/L), and nutrients, NO 3 -N (1 mg/L) and PO 4 - P (0.15 mg/L), were added and kept at stab le concentrations throughout the study to “stimulate autotrophic production and estima te daily nutrient uptake”. Analytical measurements of atrazine, bromide, nitrogen an d phosphorous were provided on a daily basis, and continuous reporting for dissolved O 2 , 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 afte r each pulse of atrazine and at intervals throughout the “recovery period”, totaling 10 sampling events.

The study authors stated that “the [source] wa ter 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 NH 4 -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 ac ross 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 th at controls contained atrazine at levels above the limits of detection but were generally belo w 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 28 th and April 3 rd , 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 reachi ng the detection limit. Throughout the study the controls continued to sh ow detections but remained below 0.1 ug/L.3 The study reported on several different endp oints 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 report ed 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 co verage of tiles between controls and treatment cosms an in a dose response manner. Two and th ree weeks later (days 41 and 53) it is evident in the photographs that sloughing events ha ve occurred in the controls, but differences

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

in treatments and there remains a visible diff erence 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 fr om days 5 through 60; during this period the difference between the treatment groups and contro ls is considered biologically significant by the reviewer. Statistical significance between th e 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 da ys 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 biologica lly significant across all test concentrations as compared to controls ( Table 2 ).4 Table 2. Riffle section Periphyton percent redu ction 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 fi sh 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 ther e 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 al l treatments and controls occurred from day 48- 60 due in part to shading, senescing and sign ificant 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 metaphyt on 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 th e phytoplankton community. The controls had significantly more metaphyton biomass removed than the treatment pools throughout the study.5 The heavy production of the metaphyton would ha ve 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 resp onse effect on metaphyton with significant effects at all concentrations (NOAEC <10.5 ug/L).

Phytoplankton The author’s analysis of community structure i ndicates that there is little to no difference between the controls and treatment pool phyto plankton communities thro ughout the study. Their analysis does indicate that these communi ties changed throughout the study but it was directionally related to the day of sampli ng rather than atrazine concentration.

The metaphyton community likely had a confound ing 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 consider able 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 st atistically significant for biomass on day 22 at the 20 and 30 ppb levels.

Dissolved Oxygen There are clear and significant differences betw een 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 th e 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 evid ent and in a dose response manner during the exposure phase, the amplitude of the differen ces 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 to lerant 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.6 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 sequest ration, 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 signific ant 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 biom ass, 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.7Data Summary Table:EffectMagnitude of Effectrelative to controlsNominalAtrazineConcentration(ug/L)Day of StudyEffectOccurredEstimatedMaximum 60-dayaverage atrazineconcentration(ug/L)DidRecoveryOccur– Ifso, when?CommentsMetaphytonBiomassD-27 ~ 80%↓drywt;D-60 ~50%↓dry wt1027-6010.7 (60-dayestimatedaverage)NoRecoveryStatistically significantdecrease in metaphytonproduction starting onday 34and continuedthroughout the study.Biologically significanteffects started on day27.MetaphytonBiomassD-27 ~ 80%↓drywt;D-60 ~50%↓dry wt2027-6020.9MetaphytonBiomassD-27 ~ 80%↓drywt;D-60 ~50%↓dry wt3027-6031.0Riffle SectionPeriphytonBiomass(AFDM)D-05 ~ 30%↓D-16 ~ 28%↓D-27 ~ 24%↓D-60 ~ 35%↓105, 16, 2710.7Recoveryoccurredbetweenand afterthe pulsedexposure.Statistically andbiologically significantdeclines in biomassstarted ~day 5 and wereassociated with the endof each pulsed additionof atrazine.Riffle SectionPeriphytonBiomass(AFDM)D-05 ~ 29%↓D-16 ~ 23%↓D-27 ~ 21%↓D-60 ~ 1%↑205, 16, 2720.9Riffle SectionPeriphytonBiomass (AFDM)D-05 ~ 35%↓D-16 ~ 31%↓D-27 ~ 22%↓D-60 ~ 8%↑305, 16, 2731.0Riffle SectionPeriphytonBiomass (CHLA)D-05 ~ 27%↓D-11 ~ 35%↓D-16 ~ 29%↓D-22 ~ 42%↓105-6010.7NoRecoveryStatistically significantdeclines in biomass:10 ppb:days 11,22,27,488D-27 ~ 42%↓D-34 ~ 16%↓D-48 ~ 42%↓D-60 ~ 31%↓20 ppb: days 22, 27, 4830 ppb: days 5, 11, 22,27, 48, 60.Although reductionswere notalwaysstatistically significanttheobserved reductionswere consideredbiologically significant.Riffle SectionPeriphytonBiomass (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%↓205-6020.9Riffle SectionPeriphytonBiomass (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%↓305-6031.0Maximum DailyDissolvedOxygenD-27 ~ 19%↓D-60 ~ 14%↓101-6010.7NoRecoveryAll 3 pulses of atrazineresulted in statisticallysignificantbuttemporary (mainlyduring pulses) reductionof DO in all treatmentsrelative to the control.After the 3 pulsedexposures statisticallyand biologicallyMaximum DailyDissolvedOxygenD-27 ~ 26%↓D-60 ~ 16%↓201-6020.9Maximum DailyDissolvedOxygenD-27 ~ 29%↓D-60 ~ 17%↓301-6031.09significant reductions inDO were reported fromday 27 through day 60.10 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 test ed 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 phospho rous 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 at razine. 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 data base. Furthermore, recent research (e.g., Baxter et al. 2013) indicates that there are cl ear 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 at razine however their concentrations would be expected to decline rapidly due to biological uptake and movement downstream, not unlike

atrazine. Therefore the constant nutrient ad dition 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 re presenting “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 st udy compromised the utility of many endpoints due to the rampant growth of filamentous algae (m etaphyton), which sloughed off of the riffle portion of the study, often resulting in a scrubbin g of the tiles as it went past, then accumulated in the ungrazed glide and pool portions of the st udy 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 b een collected later in the study because of the decreased light conditions. The metaphyton impa cts 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 wa ter 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 th e cosms) may have already been selected for more atrazine resistant algae.11 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 treatm ent 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 wa y 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 append ix 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 periphy ton. 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

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