Open Literature Review Summary

Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation: Moorhead, D.L. and R.J. Kosinski. 1986. Effectofatrazine on the productivity of artificial stream algal communities. Bull. Environ. Contam. Toxico!. 37:330-336.**U~~\P** 

### 4t-5""43513) Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study were also used to parameterize the Comprehensive Aquatic System Model (CASM) and establish the model-associated LOC for atrazine.

Date ofReview: October 30, 2008
Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants (algae and periphyton) in experimental streams (lotic mesocosms).
Summary ofStudy Findings:

Methods: The study is the same as the 1980/MinterSpring experimentdescribed in Kosinski (1984) and Kosinski and Merkle (1984), with only results for atrazine from the control chronic streams (streams with no chronic treatment during colonization) analyzed. (Inconsistencies with these previous studies' methods descriptions will be specifically noted.)

The source ofwater and algae for the study was Minter Spring, Brazos County, Texas. The wateratthis source was nearly anoxic, with a constanttemperature of21°C, and highly mineralized with a specific conductance of54.6 mS/m at 25°C, alkalinity of74 mg/kg CaC03, 404 I.lg/kg nitrate, and 21 I.lg/kg soluble reactive phosphorus. (No details on timing ormethods ofwaterquality measurement; the potential for pre-existing atrazine in the water source is not addressed.) The principal algal genera wereAnabaena, Nitzschia, Rhopalodia, andNavicula. Algal spores and cells in the waterwere usedto colonize the artificial streams (no mention ofthe homogenization procedure described in Kosinski, 1984). Minter Spring water was also used "initially" to replace evaporative



# 1II1IIIIII 2087806water losses, but deionized tap water "was laterused" to avoid excessive buildup of nutrients and minerals (no details on timing ofswitch oron frequency and manner of waterreplacement; this switch was not described in other Kosinski papers).

Sixteen (16) recirculating artificial streams (2.43 m long, 12.5 cm wide, and 6 cm deep, housed on arooftop in Central Texas) were constructed ofplywoodpainted with waterresistant white enamel paint and lined with polyethylene plastic sheeting 0.1 mm thick. Each stream held 12 L water at an average depth of4 em, and the water was recirculated via a waterpump atthe foot ofthe stream passing itto the upperportion ofthe stream through aplastic hose at a flow rate ofabout 5 cm/s (Kosinski, 1984 reportedthatthe velocity was 3 cm/s). A single layer ofstream-worn pebbles (4-8 mm diameter) was placed in the center 1.8 m ofthe streams. Colonizationwas permitted for five weeks (note thatprevious studies described colonization periods of3-4 weeks), beginning September 12, 1980, since initial studies indicatedthat about4 weeks was required for sufficient algal growth. Four concentrations ofatrazine (commercial product, Aatrex 80 WP, CIBA-Geigy Corporation) were created on October 23, 1980, at levels of0, 0.1, 1.0, and 10.0 mg/kg (0, 100, 1,000, and 10,000**~g/L)** atrazine in the streams (no details on application method), with four replicate streams at each level with a single application of herbicide. (Although atrazine concentration measurements are not described, the authors cite Kosinski and Merkle, 1984 and state thatthe expected half-life ofatrazine is 3.2 days in these streams.) One ofthe 10 mg/kg (1 0,000**~g/L)** streams was lost due to leaks, so the results were analyzed with respectto this imbalance in the number ofreplicates. The streams were covered with translucent stream covers.

Waterquality was monitored atregular intervals: conductivities, with an electro-mho meter, every few days; alkalinities, with sulfuric acid titration, every week; diurnal temperature, with an oxygen meter, athalf-hour intervals during one day each week; and diurnal pH, with apH meter, every hour from dawn to dusk during one day each week. Phosphate analyses were performed on water samples taken two days before treatment, the day oftreatment, three days aftertreatment, and one week aftertreatment, which were frozen until analysis. Solublereactive phosphate concentrations were determined by the ascorbic acid spectrophotometric technique with a lower detection limit of5**~g/kg** (American Public Health Association [APHA], Standardmethodsfor the examination of waterandwastewater, 1975). Dissolved oxygen was also measured, as described below.

Net community productivities (NCPs) were estimated with open water oxygen methods by correcting the rate ofchange in dissolved oxygen concentration for exchange with the atmosphere. Dissolved oxygen concentrations were monitoredbetween September22, 1980 and October 30, 1980 on selected days, from sunrise to sunset, athalf-hourly intervals using a dissolved oxygen meter. The meter was calibrated with azide modified standard Winklertitrations (APHA, 1975) at each half-hourreading. NCP was calculated for each stream and daily community netproductivity was summed overthe period from 7:00am to 7:00pm. Additional readings were taken at nightto obtain measures ofnighttime respiration. Equilibrium values ofdissolved oxygen concentration at given

2temperatures were calculated according to Benson and Krause, 

### Oxygen. Limnol Oceanogr25:662-671, 1980.

The oxygen exchange coefficientwas empirically estimated in orderto correct for atmospheric exchange, by placing waterthathadbeen partially deoxygenated with sodium sulfite and cobaltchloride in an uncolonized stream and monitoring oxygen concentrations at one minute intervals. The rates ofoxygen level change were regressed on the mean saturation deficits occurring atthe same time intervals, with the slope yielding the exchange coefficient. All estimates were standardizedto 20°C according to Bansal, 

### WaterRes 7:769-782, 1973. After four separate determinations, a mean oxygen exchange coefficientof0.0568 m/hrwas obtained and used for productivity and respiration calculations.

Fourhorizontally positionedglass microscope slides wereplaced in the upper and lower portions ofall streams atthe beginning ofthe study (note thatthis is inconsistentwith Kosinski, 1984, which states thatthere were only 4 slides per stream atthe pump end), and were randomly selected atweekly intervals during the study forperiphyton sampling. In periphyton sampling, each slide's community was scraped into a vial andpreserved in buffered formalin solution (no details on materials). The preserved algae were suspended in 10 mL distilled water and two I-mL aliquots were taken from the sample, placed in Sedgewick-Raftercells, and five random fields ofview counted afteridentification to genus. The six mostnumerous genera (comprising 95% ofall cells) were statistically analyzed for variations in densities. (The sampling scheme was determinedby the optimal allocation method limited by time constraints, as described in Snedecor, 

### StatisticalMethods, 1967, for the most numerous genus, 

### Chroococcus.)

Statistical methods are not separately described, butDuncan's multiple range test was used for multiple comparisons overtreatmentlevels and some othermethod (most likely ANOVA, butthis is not explicitly stated in mostcases) was used to test for differences between treatments and for differences overtime.

Results: Waterquality results (conductivity and alkalinity) did not show differences due to treatment. Conductivity decreased from 61.0 mS/m (at 25°C) initially to 41.2 mS/m by the end ofthe study, while alkalinity increased from 62.25 mg/kg CaC03 initially to 77.00 mg/kg CaC03, possibly due to abundant limestone in the substrate. Soluble reactive phosphate concentrations were mostly zero or attrace levels, precluding statistical analysis. Changes in pH were observedto agree with patterns ofNCP changes throughoutthe day (no data or statistics provided), and average pH was slightly over 8.0, which the authors attribute to calcareous substrate as well as carbon dioxide removal due to photosynthesis.

3Analyses ofdawn-to-duskNCP for treatmenteffectby date are shown in Table 1. NCP values were low, indicating high respiration relative to oxygen production in the artificial streams. Decline in NCP overtime was significant (p < 0.0001, test not described). There were no significant differences between treatments priorto atrazine application, but afterwards differences were highly significant (p < 0.0001). As Table 1 shows, the highertwo treatment levels became significantly different from the control and the lower treatment level, and neverrecovered, while the lowertreatment level was initially differentfrom the controls, but statistical difference was not observed one week later.
Respiration values, determined solely from night-time observations, showed no significantdifferences between treatments. However, rates decreased overtime (Table 2), which the authors attribute to ambienttemperature changes overthe study period.

Datawere transformed according to Taylor's Power Law (Taylor, Nature 189:732-735, 1961) and then analyzed fortreatment effects with ANOVA. OnlyNitzschia showed significantdifferences between treatments (p < 0.0210), on October 30, 1980 one week aftertreatment, while Duncan's multiple range test showed significant (p < 0.05) differences between the lowesttreatment level, the highesttreatment level, and a group consisting ofcontrol and medium treatment level streams. Cell counts were: Control- 2.7 x 104 cells/em Low Treatment

- 4.5 x 104 cells/em Medium Treatment

- value notprovided, but statedto be similarto control High Treatment

- 1.0 x 104 cells/em where low, medium, and high treatment levels had nominal atrazine concentrations of 0.1, 1.0, and 10 mglkg (100, 1,000, and 10,000 llg/L) at the time ofthe single application in the artificial streams. The authors reported thatthey could not explain the pattern of increase, then decrease inNitzschia cell numbers with atrazine treatment.

4Table 1. Daily cumulative NCP values by date and treatment, atrazine applied October 23. Sample size offourexcept 10,000 uglL level has sample size ofthree. Treatment means and standard errors in g 02/m2/day. Different letters below values indicate significantdifferences (p < 0.05) between treatment means within dates by Duncan's multiple range test. (study table 1)

Atrazine treatmentlevel Date DaysAfter 

## oIlg/L 100 1.000 10,000 Treatment flgiL f1g/L flgiL 22-Sep -31 Mean 0.335 0.093 0.189 0.147 SE 0.064 0.098 0.049 0.144 A A A A I-Oct -22 Mean -0.028 -0.088 -0.100 -0.004 SE 0.103 0.070 0.081 0.063 A A A A 21-0ct -2 Mean -0.042 -0.065 -0.109 -0.030 SE 0.064 0.063 0.048 0.030 A A A A 23-0ct 0 Mean -0.049 -0.410 -0.719 -0.89 SE 0.066 0.038 0.063 0.046 A B C C 26-0ct 3 Mean -0.182 -0.395 -0.596 -0.663 SE 0.059 0.017 0.014 0.034 A B C C 30-0ct 7 Mean -0.214 -0.286 -0.468 -0.502 SE 0.032 0.286 0.011 0.034 A A B B

Table 2. Respiration rate by date and treatment, atrazine applied October 23. Sample size offour except 10,000 ugIL level has sample size ofthree. Treatmentmeans and standard errors in g 02/m2/day. (study table 2)
Atrazine treatment level Date DaysAfter 

## oIlg/L 100 1,000 10,000 Treatment f1g/L f1g/L f1g/L 22-Sep -31 Mean 0.085 0.083 0.092 0.083 SE 0.004 0.003 0.005 0.002 I-Oct -22 Mean 0.076 0.092 0.084 0.079 SE 0.004 0.016 0.002 0.004 21-0ct -2 Mean 0.061 0.061 0.059 0.066 SE 0.002 0.003 0.002 0.001 23-0ct 0 Mean 0.067 0.062 0.065 0.072 SE 0.002 0.002 0.002 0.003 30-0ct 7 Mean 0.050 0.052 0.045 0.052 SE 0.002 0.003 0.002 0.002

5Data Summary Table: Effect Magnitude Nominal Day ofStudy Time Did Comments ofEffect Atrazine Effect Weighted Recovery relative to Concentration Occurred Average Occur

- if controls (/l-g/L) Atrazine so, when (see Concentration comment)? from Day 0 to When Effect Occurred 8 times Magnitudes and lower 100 I-lg/L Day 0 100 I-lg/L yes
- Day 7 recoveries from Table Decline in net I values. community 15 times Differences/no lower 1,000 I-lg/L Day 0 1,000 I-lg/L no differences (recoveries) productivity! 18 times significant at p<0.05. lower 10,000 I-lg/L Day 0 10,000 I-lg/L no
Water quality No treatment effects 100 I-lg/L noted on these (conductivity, alkalinity, soluble no effect through Day 7 not applicable parameters throughout reactive 1,000 I-lg/L course of7-day study. phosphorus, pH) 10,000 ug/L No treatment effects 100 I-lg/L noted throughout Respiration no effect through Day 7 not applicable course of7-day study. 1,000 I-lg/L Sec Table 2. 10,000 Ilg/L 100% Differences with increase 100 I-lg/L Day 7 221-lg/L treatment were

no sig. significant at p<0.021. no- Based on cell counts Change in difference 1,000 I-lg/L Day 7 220 I-lgiL effect described in study text; Nitzschia (atp<0.05) observed unclear ifeffects are abundance" at end of related to atrazine 60% study treatment because no decrease 10,000 I-lg/L Day 7 2195 I-lg/L major shifts in other dominant algal groups were observed. IOOllg/L No significant (p<0.05) Non-Nitzschia differences with algal species no effect through Day 7 not applicable treatment. abundances 1,000Ilg/L 10,000 J..l.g/L

1 This endpoint does not appear in Attachment 2; Attach. 2 erroneously lists "net primary production" of periphyton as not changing. NCP is relatedto AGPP via respiration, and the AGPP endpointappears in the Kosinski and Merkle (1984) data summary table, for a larger set ofartificial streams including those with chronic atrazine treatment during colonization. These NCP data have superior quality and interpretability compared to those AGPP data, and so are listed here despite the overlap.

"This endpoint does not appear in Attachment 2.

6Time weighted averages are computed assuming thatconcentrations were nominal on Day 0, and declined according to 3.2-dayhalf-life subsequently. See linked Excel spreadsheet for calculation.

Nominal TWA (Day 100 21.95315 1000 219.5315 10000 2195.315

Reviewer Comments: Some deficiencies noted in previous publications based on the same data set are not applicable to this study (e.g., use of"apparent" OPP in Kosinski and Merkle (1984), the use ofaggregated species groups in Kosinski (1984), and the confusing factorial experimental design that complicated separation ofchronic versus acute treatment effects). More specific information on dates and scheduling oftreatment and sampling also aids comprehension. As aresult, the data are far more clearly presented and easy to interpret in the present study.

Some importantdetails ofthe experimental design were leftout ofthe study orwere inconsistentwith the previous studies' descriptions. Forexample, the streams are described as recirculating, butthe waterreplacementregime (frequency and quantity) was not outlined as itwas in previous studies. The authors state that after awhile, deionized waterwas used instead ofsource waterfor the replacements; this is not described in the previous studies.

Some problems with the study are as described in the other Kosinski reviews: Background atrazine in source water was not analyzed, and shouldhave been to allow more accurate interpretation ofthe results. Duncan's multiple range test is not a recommended method for multiple comparison due to its tendency to provide inflated estimates ofsignificance, so the significance atp-value<0.05 (forexample, the letters in Table 1) may be misleading. Finally, there was quite high respiration relative to production in these streams, which may indicate thatthe experimental setup was not realistic, at least in terms ofcomparison to productive natural streams. (This issue got more attention in previous studies, butthe effects remain obvious from the data.)

Additional problems with the study include the lack ofproper description ofthe statistical methods used (in several places, p-values are cited or significanteffects affirmed with no test described). Also, the study cites prior Kosinski studies (which were on the same experiment) as evidence thatthe study's observations are consistentwith the literature.

7Description ofUse in Document (QUAL, QUAN, INV): QUAL

Rationale for Use: The study deficiencies limit the utility ofthis study. However, the study appears sufficientto contribute to the weight ofevidence regarding the concentration-response relationship ofatrazine and aquatic plants.

Limitations ofStudy: It is possible that background levels ofatrazine were present in the source water ofthe artificial streams in this study (the issue was not addressed, despite the natural origin ofthe water). The waterreplacement regime for the streams is not clearly described. Atrazine concentration data are not provided, only a generic halflife basedprior studies on this experiment (which was itselfbased on only few samples). The streams were all notably unproductive even without atrazine addition. Although this study's results are far more clear and interpretable than those ofthe two other studies on this experiment, uncertainties about atrazine exposure as well as apotential lack of realism in the artificial stream setup continue to be significant limitations.
Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc


### Secondary Reviewer: BrianAnderson

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