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. Effect ofatrazine on the productivity of

artificial stream algal communities. Bull. Environ. Contam. Toxico!. 37:330-336.

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Purpose of Review:

Determine potential effects of atrazine 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 of Review:

October 30, 2008

Purpose of Study:

This study examines the potential effects of atrazine to aquatic plants (algae and

periphyton) in experimental streams (lotic mesocosms).

Summary of Study Findings:

Methods: The study is the same as the 1980/Minter Spring experiment described 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 of water and algae for the study was Minter Spring, Brazos County, Texas.

The water at this source was nearly anoxic, with a constant temperature of 21°C, and

highly mineralized with a specific conductance of 54.6 mS/m at 25°C, alkalinity of 74

mg/kg CaC03, 404 I.lg/kg nitrate, and 21 I.lg/kg soluble reactive phosphorus. (No details

on timing or methods ofwater quality measurement; the potential for pre-existing

atrazine in the water source is not addressed.) The principal algal genera were Anabaena,

Nitzschia, Rhopalodia, and Navicula. Algal spores and cells in the water were used to

colonize the artificial streams (no mention ofthe homogenization procedure described in

Kosinski, 1984). Minter Spring water was also used "initially" to replace evaporative

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water losses, but deionized tap water "was later used" to avoid excessive buildup of

nutrients and minerals (no details on timing ofswitch or on frequency and manner of

water replacement; 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 a rooftop in Central Texas) were constructed of plywood painted 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 of 4 em, and the water was recirculated

via a water pump at the foot ofthe stream passing it to the upper portion ofthe stream

through a plastic hose at a flow rate of about 5 cm/s (Kosinski, 1984 reported that the

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 that previous studies described colonization periods of 3-4 weeks), beginning

September 12, 1980, since initial studies indicated that about 4 weeks was required for

sufficient algal growth. Four concentrations of atrazine (commercial product, Aatrex 80

WP, CIBA-Geigy Corporation) were created on October 23, 1980, at levels of 0, 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 that the expected half-life of atrazine 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 respect to this imbalance in the number ofreplicates. The

streams were covered with translucent stream covers.

Water quality was monitored at regular intervals: conductivities, with an electro-mho

meter, every few days; alkalinities, with sulfuric acid titration, every week; diurnal

temperature, with an oxygen meter, at half-hour intervals during one day each week; and

diurnal pH, with a pH 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 after treatment, and one week after treatment, which were

frozen until analysis. Soluble reactive phosphate concentrations were determined by the

ascorbic acid spectrophotometric technique with a lower detection limit of 5 ~g/kg

(American Public Health Association [APHA], Standard methodsfor the examination of

water and wastewater, 1975). Dissolved oxygen was also measured, as described below.

Net community productivities (NCPs) were estimated with open water oxygen methods

by correcting the rate of change in dissolved oxygen concentration for exchange with the

atmosphere. Dissolved oxygen concentrations were monitored between September 22,

1980 and October 30, 1980 on selected days, from sunrise to sunset, at half-hourly

intervals using a dissolved oxygen meter. The meter was calibrated with azide modified

standard Winkler titrations (APHA, 1975) at each half-hour reading. NCP was calculated

for each stream and daily community net productivity was summed over the period from

7:00am to 7:00pm. Additional readings were taken at night to obtain measures of nighttime respiration. Equilibrium values of dissolved oxygen concentration at given

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temperatures were calculated according to Benson and Krause, Oxygen. Limnol

Oceanogr 25:662-671, 1980.

The oxygen exchange coefficient was empirically estimated in order to correct for

atmospheric exchange, by placing water that had been partially deoxygenated with

sodium sulfite and cobalt chloride in an uncolonized stream and monitoring oxygen

concentrations at one minute intervals. The rates of oxygen level change were regressed

on the mean saturation deficits occurring at the same time intervals, with the slope

yielding the exchange coefficient. All estimates were standardized to 20°C according to

Bansal, Water Res 7:769-782, 1973. After four separate determinations, a mean oxygen

exchange coefficient of 0.0568 m/hr was obtained and used for productivity and

respiration calculations.

Four horizontally positioned glass microscope slides were placed in the upper and lower

portions of all streams at the beginning ofthe study (note that this is inconsistent with

Kosinski, 1984, which states that there were only 4 slides per stream at the pump end),

and were randomly selected at weekly intervals during the study for periphyton sampling.

In periphyton sampling, each slide's community was scraped into a vial and preserved 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-Rafter cells, and five random fields of view counted after identification to

genus. The six most numerous genera (comprising 95% of all cells) were statistically

analyzed for variations in densities. (The sampling scheme was determined by the

optimal allocation method limited by time constraints, as described in Snedecor,

Statistical Methods, 1967, for the most numerous genus, Chroococcus.)

Statistical methods are not separately described, but Duncan's multiple range test was

used for multiple comparisons over treatment levels and some other method (most likely

ANOVA, but this is not explicitly stated in most cases) was used to test for differences

between treatments and for differences over time.

Results:

Water quality 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 at trace levels, precluding

statistical analysis. Changes in pH were observed to agree with patterns ofNCP changes

throughout the 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.

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Analyses of dawn-to-dusk NCP for treatment effect by date are shown in Table 1. NCP

values were low, indicating high respiration relative to oxygen production in the artificial

streams. Decline in NCP over time was significant (p < 0.0001, test not described).

There were no significant differences between treatments prior to atrazine application,

but afterwards differences were highly significant (p < 0.0001). As Table 1 shows, the

higher two treatment levels became significantly different from the control and the lower

treatment level, and never recovered, while the lower treatment level was initially

different from the controls, but statistical difference was not observed one week later.

Respiration values, determined solely from night-time observations, showed no

significant differences between treatments. However, rates decreased over time (Table

2), which the authors attribute to ambient temperature changes over the study period.

Data were transformed according to Taylor's Power Law (Taylor, Nature 189:732-735,

1961) and then analyzed for treatment effects with ANOVA. Only Nitzschia showed

significant differences between treatments (p < 0.0210), on October 30, 1980 one week

after treatment, while Duncan's multiple range test showed significant (p < 0.05)

differences between the lowest treatment level, the highest treatment level, and a group

consisting of control 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 not provided, but stated to be similar to 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 that they could not explain the pattern of

increase, then decrease in Nitzschia cell numbers with atrazine treatment.

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Table 1. Daily cumulative NCP values by date and treatment, atrazine applied October 23. Sample

size offour except 10,000 uglL level has sample size of three. Treatment means and standard errors

in g 02/m2/day. Different letters below values indicate significant differences (p < 0.05) between

treatment means within dates by Duncan's multiple range test. (study table 1)

Atrazine treatment level

Date Days After 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 of four

except 10,000 ugIL level has sample size of three. Treatment means and standard errors in g

02/m2/day. (study table 2)

Atrazine treatment level

Date Days After 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

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Data Summary Table:

Effect Magnitude Nominal Day of Study Time Did Comments

of Effect 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 (at p<0.05) observed unclear if effects 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 7not 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 related to AGPP via respiration, and the AGPP endpoint appears in the

Kosinski and Merkle (1984) data summary table, for a larger set of artificial 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.

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Time weighted averages are computed assuming that concentrations were nominal on

Day 0, and declined according to 3.2-day half-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 of aggregated species groups in Kosinski (1984), and the confusing factorial

experimental design that complicated separation of chronic versus acute treatment

effects). More specific information on dates and scheduling oftreatment and sampling

also aids comprehension. As a result, the data are far more clearly presented and easy to

interpret in the present study.

Some important details ofthe experimental design were left out ofthe study or were

inconsistent with the previous studies' descriptions. For example, the streams are

described as recirculating, but the water replacement regime (frequency and quantity)

was not outlined as it was in previous studies. The authors state that after awhile,

deionized water was used instead ofsource water for 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 should have 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 at p-value<0.05 (for example, the letters in

Table 1) may be misleading. Finally, there was quite high respiration relative to

production in these streams, which may indicate that the experimental setup was not

realistic, at least in terms of comparison to productive natural streams. (This issue got

more attention in previous studies, but the effects remain obvious from the data.)

Additional problems with the study include the lack of proper description ofthe statistical

methods used (in several places, p-values are cited or significant effects affirmed with no

test described). Also, the study cites prior Kosinski studies (which were on the same

experiment) as evidence that the study's observations are consistent with the literature.

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

Rationale for Use: The study deficiencies limit the utility ofthis study. However, the

study appears sufficient to contribute to the weight of evidence regarding the

concentration-response relationship of atrazine and aquatic plants.

Limitations of Study: It is possible that background levels of atrazine were present in

the source water ofthe artificial streams in this study (the issue was not addressed,

despite the natural origin ofthe water). The water replacement regime for the streams is

not clearly described. Atrazine concentration data are not provided, only a generic halflife based prior studies on this experiment (which was itself based 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 a potential lack of

realism in the artificial stream setup continue to be significant limitations.

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer: Brian Anderson II11-- L/;!,IIi

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