1 Open Literature Review Summary

Chemical Name: Atrazine PC Code: 080803

ECOTOX Record Number and Citation: Baxter, L.R., D.L. Moor, P.K. Sibley, K.R. Solomon, M.L. Hanson. 2011. Atrazine does not affect algal biomass or snail populations in microcosm communities at environmentally relevant concentrations. Environmental toxicology and chemistry, vol. 30 (7): 1689-1696.

Purpose of Review (DP Barcode or Litigation):

Review for inclusion in the CELOC calculation

Date of Review: 3/15/16

Summary of Study and Findings:

This study was conducted over 73 d at the University of Guelph Turfgrass Institute Microcosm Facility (Guelph, ON, Canada). Each 12,000-L microcosm is 1.2m deep and 3.9m in diameter,with an actual water depth of approximately 1m. The microcosms were lined with a black foodgrade polyvinyl chloride (PVC) pond liner, contai ned soil covering 44% of the bottom surface (14.5 % OC and 1.8% IC), and were filled with spring water from an adjacent irrigation pond. Water was circulated at 12,000 L/d among the microcosms for seven weeks. Circulation among the microcosms was stopped 3 days prior to treatment.

Atrazine Treatment

Technical grade atrazine (96% a.i.: Syngenta Crop Protection) was added in a random design with nominal concentrations of 0, 1, 10, 30, and 100 ug /L with 3 replicates per treatment level. Acetone was used as a solvent with a final concentration per microcosm at 0.001% acetone, controls also received solvent (no solvent free controls included).

General chemistry included collections of total nitrogen and total phosphorous (d=1,7,14,28,49,70), total hardness and alkalinity (d=1,7,14,28,49,70), pH, conductivity, temperature and DO (Monday-Friday starting on d14). Photosynthetically active radiation (PAR) was measured mid-day (d=0, 8, 17, 38, 44, and 71) at a depth of 60 cm.

Macrophytes

Shoots (5-10 cm length) were added two weeks prior to treatment in 4 L pots. Each cosm received 3 pots with 6 shoots of Myriophyllum spicatum /pot and 3 pots with six shoots of Elodea
canadensis per pot. Total biomass, wet and dry mass, for shoots (both species) and roots ( M. spicatum only).2 Phytoplankton and periphyton

Periphyton substrates were added on the north si de of each microcosm two weeks prior to treatment to allow colonization. A periphytomet er (48 back-to-back microscope slides in a Plexiglas frame) was fixed at the same location in each microcosm and suspended just below the water surface. Additional periphyton measurements were taken using 30 x10 cm strips of PVC pond liner material hung from the edges of the mic rocosms and reaching a depth of 60 cm (or 2/3 of the water depth). Use of periphytometers allowed quantification of periphytic growth at the surface and in the relative absence of snail grazing, while growth on the PVC pond liner was subject to conditions of increased water depth and grazing pressure. Analyses of phytoplankton and periphyton biomass as ash-free dry weight (AFDW) and chlorophyll a were performed on days 1, 4, 7, 14, 21, 28, 49, and 70.

Phytoplankton samples were collected by f iltering 1.5 L of microcosm water through Whatman glass fiber (GF/ C) filters (pre-combusted and pre-weighed for AFDW samples). Periphyton samples were collected by separately scraping each substrate and collecting the resulting slurry on a glass fiber filter as above. Three microsco pe slides were used for each of AFDW and chlorophyll a of periphyton at the surface (six slides total). Polyvinyl chloride liner strips were divided in half and each portion was scraped separately for AFDW and chlorophyll-a analysis. Chlorophyll-a filters were wrapped in aluminum foil and frozen until spectrophotometric analysis. Ash-free dry weight was analyz ed immediately following filtration.

Growth of filamentous algae masses in the microcosms was assessed weekly through a scoring system. Three people separately ranked the amount of filamentous algae in each microcosm on a scale of 1 to 3, where 1=no or low coloni zation, 2=some masses, 3=high colonization. Confidence in the assessment was given a score of 1 (low) to 10 (high).

Snails
Growth and fecundity of snails was tracked using four snail cages in each microcosm. The cages consisted of 35x25x25 cm translucent food-grade polypropylene containers, fitted with 200 micron mesh covering four 6-cm diameter holes to allow for circulation of water through the containers. Lids on the containers were modified so that mesh fabric (2-mm mesh) covered 90% of the surface area to allow light penetration. Photosynthetically active radiation measurements inside and outside the cages indicated no significant shading. Floats were fixed near the top of each container such that the top 10 cm of each cage emerged above the water surface. One day before treatment, 10 adult marsh snails (Stagnicola elodes; 21.4x3.47 mm) were released in one cage per microcosm to track fecundity, and anot her 10 juveniles (6 to 9 mm) were placed in one cage per microcosm for quantification of growth rates. The same procedure was carried out concurrently in separate cages with the snail Physella spp. (adults 10.4x1.35 mm; juveniles <2 mm).

The shell length of caged juvenile snails was measured on days 15, 22, 38, 51, and 73 using calipers and the snails returned to the cages. Egg masses produced in the adult snail cages were removed and counted weekly, and the number of eggs per egg mass was recorded. In each3 microcosm, 50 adult Physella spp. were also fr ee-released to ensure a base population; however, these snails have been found to naturally inha bit the microcosms, so actual populations were expected to be much higher. Substrates for the en umeration of free-living snails were added two weeks prior to treatment. These consisted of 30x30 cm removable mats made from PVC pond liner, suspended at the same location in each mi crocosm. Average size and egg production rates of free-living snails were estimated from these substrates. Free-living snail size measurements were performed weekly starting at day 7 by removing the snail substrates and measuring shell sizes with calipers. The number of snail egg masses on each mat and the number of snail eggs per egg mass were counted and the egg masses were removed. Zooplankton traps as described in Boudreau et al. [ see 33 in Baxter et al. 2011] were also used to quantify the free-living snail population on the bottom of the microcosms. These traps were deployed overnight on days 1, 4, 7, 14, 21, 28, 36, 42, 49 and 65. Snails accumulating in the trap jar and funnel were washed into the sample jar, preserved in 70% ethanol, and enumerated.

Data analysis
Parameters were assessed individually by repe ated-measures analysis of variance (ANOVA) (a=0.05) using the mixed procedure of SAS 9.1 for Windows with the Kenward-Rogers correction. Pretreatment measurements of biomass we re used as baseline covariates for algal parameters and number of surviving snails per cage was used as a time-varying covariate for caged snail size. In each analysis the most appropriate heterogeneous variance structure was det ermined based on the lowest possible Akaike’s Information Criterion value. If a significant interaction was found, the slice command was used to identify the days exhibiting treatment differences and these were further assessed by one-way ANOVA. For size of caged juvenile snails, the treatment response prior to covariate inclusion was investigated by comparing regression parameters for mixed-model growth curves using one-way ANOVA. Filamentous algae scores were assessed by repeated-measures ANOVA on ranks using Sigmaplot 11.0 for Windows (Systat Software). Dunnett’s test (a=0.05) was used for all post -hoc analyses. Nominal concentrations of atrazine were used in all statistical analyses.

Results
Atrazine and general chemistry

Concentrations of atrazine measured 1 h after trea tment were within 10% of nominal values, and dissipation to 36% or less of initial concentrations occurred by day 70 ( Table S1 ). Concentrations in control microcosms were below the limit of detection (0.05 ug/L).4 Table S1 . Mean actual concentration of atrazine with standard deviation (SD) for all measured time-points (1 h to 70 d) by nominal concentration in microcosms ( n =3) after treatment at day 0, measured by enzyme-linked immunosorbant assay (limit of detection = 0.05 μ g/L). ND = nondetect.

Time Contro
l 1 μ g/L (SD) 10 μ g/L (SD) 30 μ g/L (SD) 100 μ g/L (SD)

1 h ND 1.21 (0.091) 11.30 (1.344) 28.43 (4.225) 100.57 (5.661)
1-d ND 1.17 (0.076) 12.88 (0.980) 35.07 (6.543) 117.77 (11.580)
4-d ND 1.03 (0.339) 9.55 (0.934) 24.93 (2.658) 75.70 (2.712)
7-d ND 0.93 (0.130) 8.72 (1.553) 22.07 (2.159) 80.59 (4.662)
14-d ND 0.80 (0.147) 8.40 (1.300) 25.40 (3.293) 88.20 (5.359)
28-d ND 0.55 (0.156) 5.02 (1.028) 17.90 (2.152) 66.93 (10.826)
49-d ND 0.40 (0.188) 5.70 (1.911) 14.80 (4.409) 64.40 (15.492)
70-d ND 0.22 (0.083) 3.50 (1.272) 9.70 (2.458) 36.40 (8.794)

No significant differences in water chemistry parameters including pH, dissolved oxygen, conductivity, temperature, hardness, alkalinity, and total nitrogen and phosphorus were found between treatments and controls, but all changed significantly with time. Although there were no differences between treatments, hypoxic conditions existed (<3 mg/L DO) on at least one day in 13 of the 15 microcosms, and in seven microcosms this occurred on more than 20% of days posttreatment ( Table 3 ). Because water chemistry measurements were taken between 8 and 9 AM after nighttime respiration, the measured values represent minimum values. Photosynthetically active radiation was not different between treatm ents, but was significantly affected by time, with an increase at d 38 and declining values afterward. Measurements of PAR 1m above the microcosms did not decline with time.5 Macrophytes

In the following summary some data that the authors indicated as not being statistically significant (e.g., wet weight of macrophytes and chlorophyll a for phytoplankton) are not discussed. The following summary is intended to identify the results that were reported as significant by the authors.

The authors report statistically significant declines in Myriophyllum spicatum shoot (~ 78%) and root (~ 77%) weights and in Elodea canadensis shoot (~81%) weights for the 100 ug/L treatment as compared to the controls. There were no statis tically significant differences reported for the 1, 10, or 30 ug/L treatment groups. However, the reviewer determined that biologically significant effects are reported for M. spicatum ( Figure 2, Table 4 ) which shows an approximate 46% and 19% reduction in shoot weight compared to the control for the 10 ug/L and 30 ug/L treatment groups, respectively. In addition, the reviewer determined that potentially biologically significant effects to M. spicatum root dry weight were reported at 1, 10, 30 and 100 ug/L ( Figure 2, Table
4 ). There is less certainty in the significance of the reductions in root dry weight as compared to the reductions in shoot weight. This reduced certainty stems from overlapping error bars across the doses ( Figure 2 ). Lastly, effects to E. canadensis shoot weight were biologically significant only at 30 and 100 ug/L, with 17% and 81% reductions respectively.6 Phytoplankton and Periphyton
The authors report no treatment related reductions on periphyton or phytoplankton. It is difficult to interpret the variability for each treatment level from the presented data in Figure 3

.

Snails
Based on the aforementioned potential effects of the high nutrient levels in the microcosms, the study results for snails are in question. The hy pothesis that changes in the autotroph community from increasing atrazine levels cause increases in snail populations on a dose responsive manner cannot truly be tested in this study. If the uncer tainty around high nutrient levels wasn’t present and the results do indeed reflect the pattern of the sail population effects in the presence of atrazine, then there would be some weight to place on this portion of the study. The authors report no chemical related changes in the snail populations.

Table 4: Reviewer estimated mean dry we ights and percent reduction (compared to controls) from Figure 2. Treatment (ug/L) M. spicatum shoot dry weight M. spicatum root dry weight E. canadensis shoot dry weight Control (0) 37 13 52
1 42 (+14%) 7 (-46%) 51 (-2%)
10 20 (-46%) 6 (-54%) 60 (+15%)
30 30 (-19%) 9 (-31%) 43 (-17%)
100 8 (-78%) 3 (-77%) 10 (-81%)78 Endpoints for use in the calculation of the CELO C are listed below in the summary table.

Data Summary Table:

Effect Magnitude
of Effect
relative to
controls Nominal
Atrazine Concentr
ation (ug/L) Day of Study
Effect Occurred Time Weighted Average
Atrazine Concentration from Day 0 to when Effect Occurred (ug/L) Did Recovery Occur

– If so,
when? Comments

Biomass, Macrophytes 46% reduction in

mean M. spicatum root dry weight, biologically significant (reviewer
estimated magnitude
and significance) 1 NA 0.7 No.

Endpoints
measured at study
termination

Biomass, Macrophytes 48% reduction in

mean M. spicatum shoot dry weight

, 54% reduction in
mean M. spicatum root dry weight, both biologically significant (reviewer
estimated magnitude
and significance) 10 Unclear of
initial event, 70-d
used 7.4

Biomass, Macrophytes ~19 % reduction in

mean M. spicatum shoot dry weight

, 31% reduction in
mean M. spicatum root dry weight,
17% 30 Unclear of
initial event, 70-d
used 19.059 reduction in
E. canadensis shoot dry weight, each biologically significant (reviewer
estimated magnitude
and significance) Biomass, Macrophytes reduction in

mean M. spicatum shoot (78 %) root (77%)
and in E. canadensis shoot (~81%) dry weights (reviewer
estimated magnitude), Statistically
and biologically significant (author estimated significance) 100 Unclear of
initial
event, 70-d
used 68.5

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

Rationale for Use:

This study demonstrates clear effects on macroph yte plant communities after the initiation of exposure to atrazine. Statistical and biologically significant effects were noted for macrophyte biomass at the tested concentrations of 1, 10, 30, and 100 ug/L.

Limitations of Study:

Summary data and statistics for many endpoints ca ptured throughout the duration of this study were not presented in the report. This is critically important because the authors report significance information for endpoints but only aver ages across treatments are represented in the figures so within treatment trends could not be verified.

Although there was an acclimation period, it differed for the biotic groups (macrophytes, phytoplankton, periphyton, and snails).

High within-treatment variability was observed, especially but not limited to periphyton and phytoplankton. This was apparent even on day 0. Variability in DO measurements ( Table 3 )10 may be reflective of the high variability in phytoplankton and periphyton response but it is hard to tell. The authors indicate that it wa sn’t a result of block effects. In reality it is difficult to judge the apparent variability in DO measurements because Table 3 just shows > or < 3 mg/L instead of actual concentrations.

These were outdoor microcosms but there was no discussion of weather during the experimental phase or if/how water levels were maintained.

No solvent-free control treatment was included.

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 11:29:43 -04'00'



### 2016.03.24 13:45:02
-04'00'1 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
Date: 2016.03.24 13:16:15 -04'00'



### 2016.03.24 13:43:13
-04'00'1 Open Literature Review Summary

Chemical Name: Atrazine

PC Code: 080803

ECOTOX Record Number and Citation:
Pannard, A., B. Le Rouzic, F. Binet. 2009. Response of phytoplankton community to low-dose atrazine exposure combined with phosphorous fluctuations. Arch. Environ. Contam. Toxicol 57: 50-59.

Purpose of Review (DP Barcode or Litigation):

Review for inclusion in the CELOC calculation

Date of Review: 3/16/16

Summary of Study Findings:
This study investigated the response of microalgal dynamics to long-term exposure (7- weeks) to increasing atrazine doses, which were non-inhibiting during short-term exposure (a two days). The experiment was performed on a controlled phytoplankton community, sampled from the natural envir onment and maintained in semi-continuous culture. The dynamics of the phytoplankton community, exposed to a gradient of atrazine doses in the short or long term, were examined from photosynthetic activity (short-term exposure) or from photosynthetic activity, biomass, and community structure (long-term exposure).

Atrazine (TGAI) was used for short- and long-term exposure experiments. An aqueous atrazine solution consisting of 1.2 ug mL -1 was prepared by dissolving atrazine in distilled water using sonication without any organic solvent.

The natural plankton community was collecte d by sampling 40 liters of water from a freshwater wetland in Brittany (Pleine-Fougeres, France) in autumn 2001. Macrozooplankton community was removed by filtration.

Short-Term Sensitivity of the Phytopl ankton Community to Atrazine
Short-term experiments (48 h of atrazine expos ure) were first performed to determine the non-inhibiting doses to test in the long-ter m atrazine exposure experiment. Five nominal atrazine doses (controls and 0.05, 0.5, 5.0, and 50 ug/L) were tested on the community. Phytoplankton photosynthetic activity was monitored (five replicates per atrazine dose)
from the rate of 14 C incorporation after 1, 16, 22, 40, and 46 h of atrazine exposure in borosilicate glass vessels. These were incubated with 14 C bicarbonates for 1 h in a temperature-controlled incubator, under light a nd temperature conditions similar to those for the initial phytoplankton community. The EC50 and EC10, which correspond to the2 inhibitory doses reducing 50% or 10% of the biological activity, respectively, were calculated by adjusting the model of Bailer and Oris (1993; 1997) to the incorporation rates according to atrazine dose.

Design of the Long-Term Atrazine Exposure Experiments
The initial community was first starved by reducing nutrient inputs and making it phosphorus deficient a few weeks before starti ng the long-term experiment. Phosphorus was then resupplied to obtain medium input (52.6 ug P-PO 4 3- L -1 ) from the day of atrazine input (day 0) onward.

The community was exposed to atrazine for 7 weeks in 500-mL borosilicate Erlenmeyer bottles. Two hundred forty milliliters of th e phytoplankton community was placed in each of the 16 bottles, with four replicates per dose tested (controls and nominal doses: 0.1, 1.0, and 10 ug atrazine/L). Semi-continuous culture conditions were maintained (discontinuous fresh input once a week), and simi lar atrazine doses were added weekly to each medium input to prevent dilution. Given that the atrazine half-life in a water column is between 80 and 150 days (Klassen and Kadoum 1979), the atrazine dose was assumed to remain almost constant throughout the 7 weeks of the experiment, i.e., more than 80% of the initial dose at the lowest half-life calculation (day 48). The phytoplankton biomass was measured by chlorophyll a concentration (Lorenzen 1967) and the biological activity was measured by carbon dioxide incorporation weekly. Taxonomic composition was determined at the beginning of the experiment , then after 1, 4, and 7 weeks of exposure. Fluctuations in the biomass of the coexisting species were evaluated by counting cells and colonies under a microscope using a Nageotte chamber and expressing the concentrations as cell units pe r milliliter for single-cell sp ecies and colony cell units per milliliter for colonial species (Aphanocapsa sp., Oscillatoria sp.).

Dominance within the community was characterized by calculating Simpson’s index D of diversity

Statistical Analysis
The nonparametric two-factor statistical analysis of Friedman was used to test the effects of atrazine doses and time exposures on the rate of carbon dioxide incorporation (for short- and long-term experiment s) and the evolution of biomass. A chi-square analysis was applied to test the homogeneity of communi ty structures at different times and doses. The hypothesis that atrazine has no effect on the evolution of species densities was tested in a single-factor (dose) or two-factor (time and dose) analysis of variance.

Lastly, a Kruskall-Wallis analysis was applied to test the effects of atrazine on Simpson’s index of diversity. In all statistical tests used, the probability level up to which the effect is significant was p<0.05. A factorial correspondence analysis, using software R was performed to see whether atrazine exposure was a predominant factor responsible for the final composition and structure of the phytoplankton assemblage.3 Results Summary:

Short-Term Sensitivity of the Comm unity to Atrazine Exposure
Atrazine-treated samples showed significantly lower biological activity than controls (S = 42.58, 4 df, p<0.001). Communities exposed to 50 ug/L atrazine were inhibited after as little as 1 h with a 30% to 50% decrease in carbon incorporation (Fig. 1a). Results with longer exposure times, 16, 22, 40, and 46 h, confirmed this atrazine toxicity level (Fig. 1a). The calculated EC50 was 60.7 ± 4.0 ug/L.4 Long-Term Atrazine Exposure Biological Activity and Biomass
The changes in primary production over time, based on the amounts of incorporated carbon dioxide, followed the same pattern regardless of atrazine treatment (Fig. 2). According to the author, carbon incorporation after 3 weeks decreased significantly compared to the control followed by potential recovery at six weeks (judged by5 reviewer). For measured chlorophyll a, there were no significant differences observed between controls and treated communities (Fig. 2c). In the first period, increasing growth rates (C absorption rates) were significant following nutrient resupply (still available even 1 week after P-enriched medium input), which induced progressive biomass accumulation (chlorophyll-a abundance). In th e second period, i.e., after 4 weeks, the growth rate decreased with accelerated nutrient impoverishment due to the higher phytoplankton biomass. Whereas biomass pr oduction exceeded loss due to weekly culture removal in the first period, it simply compensated this loss during the second period, as the phytoplankton biomass remained almost stable.

Community Structure and Dominance
The major impact on community structure, eval uated as a comparison of the initial and
final densities of enumerated cells or coloni es, was attributed to the biomass response6 following P resupply ( Table 1 ). Modification of the community structure due to low-dose atrazine exposure was also shown after 7 weeks of exposure, with a clear atrazine dose effect (Fig. 3). The first axis explained more than 61% of the variance of the data set. Going from left to right, the ordination on axis F1 differentiated the control communities from the atrazine exposed communities, at doses of 10, 1, and 0.1 ug/L, although the effect of the 0.1 ug/L dose showed high variability.

It was concluded that community structure was significantly affected by 7 weeks of exposure to a dose of atrazine as low as 10 ug/L or less. In accordance with the correspondence analysis, significant changes in algal density occurred depending on the atrazine dose and taxonomic group ( Table 1 ).7 Further evaluation by the reviewers illustrate the percent reduction in cell densities (species, genus, and community-level) between the controls and treatment groups.

Table 2. Percent Reduction of Treatment Final Cell Densities Compared to Controls

Treatment Skeletonema
bibraianum Chlorolobion

sp. Ankistrodesmus falcatus Chlorella

sp. Oocystis

sp. Unidentified chlorophyte Aphanocapsa

sp. Oscillatoria
limetica Total (Community) Density

0.1 28 73 71 18 69 43 40 79 62

1 -10 39 37 69 60 43 27 40 50

10 22 54 66 73 78 56 33 49 67 Bold values indicate significant difference versus the control. Biological significance without statistical significance described by the authors are italicized.

A shift in community structure was also reflected in changes to dominance and diversity,
as demonstrated by changes in Simpson’s index of diversity (1 – D) (Fig. 5). After 7 weeks of exposure, (1

– D) was significantly higher in the community exposed to atrazine than in the controls (H = 9.2, 3 df, p = 0.027). This higher diversity accounted for a lower dominance in the atrazine treatments. The (1

– D) value was proportional to the atrazine dose, demonstrating a consistent dose-response effect.8 Reviewer Conclusions:

The authors present effects of atrazine on the phytoplankton assemblage, in terms of primary production and community structure fo r all tested concentrations (NOAEC <0.1 ug/L). Their inclusion of phosphorous limitation as a variable in the study adds complexity to interpreting the results, as th e nutrient limitation followed by the addition of phosphorous resulted in dramatic changes in abundance for all tested species independent of atrazine addition. Nonetheless, the data provided in Tables 1 and 2 , and Figures 3 and 5 , suggest that even low doses of atrazine were impacting the abundance of several tested species compared to those in the control community.

The summary table below includes only those endpoints for use in the calculation of the CELOC, thus excludes the results from the short term experiment (Fig 1 and Fig 2).

Data Summary Table for the Long Term Exposure Experiment

Effect Magnitude of Effect
relative to controls Nominal
Atrazine Concentration (ug/L) Day of Study
Effect Occurred Time Weighted Average Atrazine Concentration from Day 0 to
when Effect
Occurred (ug/L) Did Recovery Occur

– If so,
when? Comments

Chlorophyll a No Effect 0.1 NA Not calculable NA Chlorophyll a No Effect 1.0 NA Not calculable NA Chlorophyll a No Effect 10 NA Not calculable NA
Carbon incorporation ~47% ↓ week 2,

~ 18% ↓ week 3

~ 25% ↓ week 4 ~62% ↓ week 5 0.1 7-14 Not calculable Recovery week 6 Difficult to interpret significance with
available
information.9 Carbon incorporation ~47% ↓ week 2,
~12% ↓ week 3
~33% ↓ week 4
~23% ↓ week 5 1.0 7-14 Not calculable Recovery week 6 Difficult to
int erpret significance with
available
information.
Carbon incorporation ~47% ↓ week 2,
~12% ↓ week 3
~17% ↓ week 4
~38% ↓ week 5 10 7-14 Not calculable Recovery week 6 Difficult to interpret significance with
available
information. Cell Density,
Individual Phytoplankton Species andCommunitylevel Chlorolobion sp. (73% ↓ ) Ankistrodesmus
falcatus (71 % ↓ ) Oocystis sp. (69 % ↓ )
Unidentified chlorophyta (43% ↓ ) Aphanocapsa sp. (40
% ↓ )
Oscillatoria limnetica (79% ↓ ) Community (62 % ↓ ) 0.1 49 Not calculable No Recovery Effects only reported in terms
of final

measurement.

Concentrations held constant by weekly additions of compound.

Cell Density,
Individual Phytoplankton Species andCommunitylevel Chlorolobion sp. (39% ↓ ) Ankistrodesmus
falcatus (37 % ↓ ) Oocystis sp. (69 % ↓ ) Chlorella sp. (60% ↓ )
Unidentified chlorophyta (43% ↓ ) Aphanocapsa sp. (27
% ↓ )
Oscillatoria limnetica (40% ↓ ) Community (50 % ↓ ) 1.0 49 Not calculable No Recovery Effects only reported in terms
of final

measurement

Cell Density,
Individual Phytoplankton Species andCommunitylevel Chlorolobion sp. (54% ↓ ) Ankistrodesmus
falcatus (66 % ↓ ) Oocystis sp. (78 % ↓ ) Chlorella sp. (73% ↓ )
Unidentified chlorophyta (56% ↓ ) Aphanocapsa sp. (33
% ↓ )
Oscillatoria limnetica (49% ↓ ) Community (67 % ↓ ) 10 49 Not calculable No Recovery Effects only reported in terms
of final

measurement

Community
Structure Changes in Simpson’s index of diversity and
CCA results (shift in composition) 0.1 49 Not calculable No Recovery An increase in the Simpsons index
as well as a shift
in the composition (changes in the
relative
abundance of the same species) in a dose response
manner indicates Community
Structure Changes in Simpson’s
index of diversity and
CCA results (shift in composition) 1.0 49 Not calculable No Recovery

Community
Structure C hanges in Simpson’s index of diversity and 10 49 Not calculable No Recovery10 CCA results (shift in composition) that the
concentrations
tested affected the community structure leading
to more variable
communities than
the controls and
that the
dominance in the community had
shifted.

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

Rationale for Use:
This study demonstrates clear effects on multiple aspects of plant communities after the initiation of exposure to atrazine. While both the short and long-term experiments demonstrated effects, only the long-term experiments is applicable for use in the CELOC estimation. Effects reported for carbon incorporation suggest biologically significant reductions in primary productivity across all concentrations with recovery by week 6, however chlorophyll a did not reflect the same reductions across treatments. Statistical and biologically significant effects were no ted for biomass (cell density) and different measures of community structure at the tested concentrations of 0.1, 1.0, and 10 ug/L.

Limitations of Study:
The method used in this study refreshed (weekly) each test solution and control with an aliquot of medium plus test material or medium only, respectively. The authors assumed that this maintained a relatively constant concentration at the intended nominal dose concentration based on the study duration and published half-lives of atrazine. They report greater than 80% recovery at the end of the study. Therefore, the reviewer assumed constant exposures at nominal concentrations.

Primary Reviewer:
Frank T. Farruggia, Ph.D. Biologist ERB3

Secondary Reviewer:
Michael Lowit, Ph.D., Ecologist ERB1 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 11:31:35 -04'00'

2016.03.24 13:44:12 -04'00'