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'



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