Open Literature Review Summary

Chemical Name: 

### Atrazine CAS No: 

### 1912-24-9

ECOTOX Record Number and Citation: 

### Lampert, W., W. Fleckner, E. Pott, U. Schober, and K.U. Storkel. 1989. Herbicide effects on planktonic systems ofdifferent complexity. Hydrobiologia. 188/189:415-424. ('

### \lVl~r{)**Lt"t~43** 

## S-ft-") 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-associatedLOC for atrazine.

Date ofReview: 

### November 19,2008
Purpose ofStudy: 

### This study examines the potential effects ofatrazine to aquatic plants and animals in laboratory microcosms and in experimental enclosures in anatural lake.
Summary ofStudy Findings:

Methods: 

### A set ofexperiments was conductedranging from directtoxicity tests of atrazine onDaphnia

### to full community assessments that allowed for interpretation of indirect effects. Methods andresults presentedhere will focus primarily on the tests that allowed for atleast atwo-step food chain.

Atrazine used in all experiments carne from Ciba Geigy, Basel (it is not statedwhether it was reagentgrade orin acommercial product formulation). A solventwas notused. The authors state, "All chemical and biological analyses were carried out according to routine protocols" (the source ofthe protocols used is not stated). Particulate organic carbon (POC) was measured using an infrared carbon analyzer described in a 1981 German journal article (Krambeck et aI., GITFachz. Labor. 25).
Laboratorytests were carried outwith an obligatelypathenogenetic strain ofDaphnia pulicaria 

### Forbes and withD. magna 

### Straus, whose stocks had beenkept for several years at 20°C and continuous dim light. The food for the daphnids in all experiments was 

# III1IIIIII 2087866Scenedusmus acutus Meyen green algae, grown in CHU 12 media in chemostats (described in a 1976 article by the lead author). 0.45-flm membrane filtered lake water from eitherLake Constance orapond in the FrankfurtBotanical Gardens was the test water, kept at 20°C for all experiments. Acute lethalitytests ofatrazine on daphnids were conducted, as well as sublethal tests for effects on growth and reproduction and on feeding inhibition.

A two-step "food chain" system in the laboratory assessed the effects ofatrazine on algae andDaphnia. See Figure 1 for a diagram ofthe set-up. Scenedesmus was grown intwo different chemostats, one with and one without atrazine, and the algae samples were diluted with filtered pond water (with orwithout atrazine) before addition to the microcosms containingD. magna females. The experimentbegan with 10 neonateD. magna females in each microcosm (2-L glass beaker). Fourreplicate microcosms were created at each treatmentregime: atrazine in algae chemostatonly; atrazine in dilution water only; atrazine in both algae chemostat and dilution water; and no atrazine exposure (controls). Experiments were run for atrazine concentrations of0.3, 0.1,0.05 (two experiments), and 0.01 mg/L (300,100,50, and 10 flg/L). Each experimentran for 3 weeks, and afterwards the number ofanimals perbeaker, dry weight ofthe population, and the number ofeggs were measured. (Atrazine concentrations were apparently not monitored.)

Enclosure experiments with natural communities were carried out in plastic bags mounted in an aluminum frame floating in Schohsee, amoderately eutrophic lake in northern Germany (then West Germany). The lake has a small catchment areawhere no com ortreated crops are raised, and so no priorexposure to atrazine was expected to affect the community responses. The enclosures had about 1 m diameter and were about 2.7 m long, and held 1.7 m3 ofwater. Experiments were carried out in different seasons and with different initial atrazine conccntrations (0.1, 1, 10, and 100 flg/L). In each experiment, four bags were filled with lake waterthathad been filtered through a 100 flm meshto screen out large zooplankton, and the bags were then inoculated with equal amounts ofzooplankton collectedwith aplankton net. Priorexperiments had shown that all four bags developed "very similar" communities when leftuntreated (prior experiments not further described). Two bags received atrazine, and two remain as controls. Experiments lasted "typically" "no longerthan 3 weeks" because ofthe growth ofperiphyton on the bags (exact durations ofthe experiments not stated). SamplesJor chemical andbiological analysis, including zooplankton sampledby vertical 1OO-flm net haul, were taken "atregular intervals" (about every few days; see results figures). Atrazine concentrations were measured by gas chromatography (a 1989 dissertation from Univ. Kiel is citedbutno additional details are provided).

Results: Methods and results presentedhere will focus primarily on the tests that allowcd for at least atwo-step food chain. Direct atrazine toxicity tests found thatDaphnia has a 48-hr LDso ofabout 10 mg/L (10,000 flg/L), with sublethal effects observed at about I

2order ofmagnitude lower atrazine concentrations. At 2 mg/L (2,000 /lg/L) atrazine, 

### Daphniahad significantly (p<0.05; testnot stated) reducedreproduction and growth.
Significant (p-value < 0.01) reductions in final daphnidbiomass in the two-step laboratory food chain system were found at 0.3 and 0.1 mg/L (300 and 100 /lg/L) atrazine, and at 0.05 mg/L (50 /lg/L) atrazine in one ofthe two experiments atthis concentration (p-value < 0.01 for combined effect, p-value < 0.05 for algae effect only). These reductions were seen only in the treatments where algae were affected by atrazine treatment. An apparent contradictory effect occurred, where microcosms with

### Daphnia being directly butnot indirectly exposedto atrazine (i.e. atrazine was in the dilution water, butnot in the algae growth chamber) actually had increasedbiomass relative to controls. The authors explain this effect as being due to initial but subtle relative decreases in the development ofthe directly-exposed daphnid population, which allowed these populations to avoid the

### Daphniabloom (and subsequent crash) that affects the control populations; they state that the duration ofthe experiments should have been either shorter ormuch longerin orderto avoid this artifact. See Figure 2 for a figure showing theresults ofthe laboratory food chain experiments.

In the enclosure experiments, similarresults were found for all atrazine concentrations between 1 /lg/L and 100 /lg/L. Atrazine was persistent in these experiments, with day 18 concentrations being within 90% to 95% ofinitial values. In these experiments, primary production and chlorophyll a decreased in the treatments after about 7 days, and as algae died, POC grew and oxygen decreased. (See Figure 3 and Figure 4.) Concurrently, zooplankton abundance declined and composition changed, with cladocerans declining more than copepods (see Figure 5). (Figures are for the 1 /lg/L atrazine experiment only; the authors state that 10 /lg/L and 100 /lg/L atrazine experiments "were qualitatively identical to those at 1 /lg/L, but were even stronger" and do notprovide figures ordata.) The authors also note thatbacterial and blue-green filaments occurred atthe time of oxygen decline, and attribute the increase in particulate organic carbon to bacterial production and detritus formation. They also attribute the excessive cladoceran decline relative to copepods to loss oftheir algal food base and increased abundance offlakes and filaments that interfere with their food collection, whereas the copepods still had abundant small zooplankton prey.

Tests with 0.1 /lg/L atrazine were performed twice in 1982, once in June/July and once in September, and once in August and September 1984. In the latterexperiment, the authors state that no differences between treatments and controls were seen, so results are notpresented. The 1982 experiments have results shown for the Warm (W; June/July) and Cold (C; September) experiments. The Cold experiment, bags were left in the lake after experimenttermination for another 17 days and sampled again on Day 42. Atrazine in the treatmentbags was no longer detectable after 10 days. In the 0.1 Ilg/L atrazine experiments, algal biomass (chlorophyll a) did not differbetween treatments and controls in the Warm study, buttransientrelative decreases with treatment were noted in the Cold study that ultimately led to a large spike (algal bloom) noted on Day 42 (see Figure 7).

3Primary production (photosynthetic rate) was more sensitive to treatment in both the Warm and Cold experiments, but like biomass it appeared to recover by the end ofthe experiments. Oxygen was reduced in the treatments and did not fully recover, except partially after42 days in the Cold experiment (see Figure 8). (The authors state "recovery ofthe oxygen concentration occurred aftertwo andthree weeks, respectively"; it is not clearwhat "respectively" refers to, andthe statement seems in contradiction with the figure provided.) 

### Daphnia was nearly eliminated in the treatment bags, butwas seen to rebound after42 days in the Cold experiment (see Figure 9).



# 1 l f·L.1 

### PI ["Mer· l

.. 

### P · I

.

, I 

# T

'j 

## MP 

## l • 

# [J

# LJ [J

# IJ c J,.

- -! ,.t\,

### IR



# l!J**[~** W ,....j "

..,**I'~'l**

## .~_ 

## l.!.,J l!J 

## A 

# w

,

### ....

## _-.-
...........----, I

Figure 1. Schematic ofthe "food chain" laboratory experimental set-up. 

### s: chemostat containing Scenedesmus; M: algal nutrient medium; W: filtered pond water; PP: peristaltic pump; PI: piston pump; MP: multichannel peristaltic pump; 0: overflow. Asteriskdenotes presence ofatrazine. Treatments labeled by letters. C: controls; W: water effect; A: algae effect; T: combined effect.

440

Q) 

# 3°f 

# () i .;,:; to , Q) .1- .0 

## I i 

## I "- 0) 

## I**.,~"** E 20 I 

### , i**~.** .c 

## I C1 (l) 

# .1 ", 10 I

## ~: ;.>-

# .~. .... 

### sr 0

## ~~ 0 

## - •.• j
T .A wC T A\11 C 0.3 0.1 ;Awe 0.05 TilWe 0.05 0.01 Concentration of atrazlne (mg/U Figure 2. Biomass ofDaphnia populations in the "food chain" laboratory experiments after 3 weeks with different atrazine concentrations. Letters denote treatments. C: controls; W: water effect; A: algae effect; T: combined effect. Vertical bars show the standard deviation (n=4). Asteriskindicates that the mean is significantly different from the control mean according to Tukey's test. Large asterisk: p-value < 0.01; small asterisk: p-value < 0.05.

;::: OJ 2 :J.,
l1J

>. 0. 0 

## \-. 0

# ,";~~I J:: u _LI ! 0.8

## ~I '

....

# ~I OJ E
u 0.4

# -~I 0 

## . I

a.. !
0 0 5 10 15 Days Figure 3. Enclosure experimentwith 1 J.tg/L atrazine. Time series ofchlorophyll a and particulate organic carbon (POC). Open circles: control bags; closed/solid circles: treatment bags.

5

### F 

### ;e 100
c .C2 ':ij

'-

(/i 50
c OJ 0) >. x 

### o 

## o o 5 10 Days 15
Figure4. Enclosure experiment with 1 Ilg/L atrazine. Time series ofoxygen saturation. Open circles: controlbags; closed/solid circles: treatment bags.

15

15 10

10 5

5 DA

80 

### o

o 

## -01

300

# ~I 200 100

### o~r: I I 400 

### NAU 1000

::J n::l 100 ..c +-' (1) 10 t:: "'- 0 10 15 C/)



### -- CO 

### Cy :J -0 10001-

# ~~ > 8S" -0 c: 100

101- I I I , J 0 5 10 15 

### Day s Figure 5. Enclosure experiment with 1 /-lgiL atrazine. Time series ofzooplankton. NAU: nauplii; DA: Daphnia; CY: Cyclops; BO: Bosmina. Open circles: control bags; closed/solid circles: treatment bags.

6Figure 6. Enclosure experimentwith 0.1 /-lg/L atrazine. W: Warm experiment; C: Cold experiment. Time series ofchlorophyll a. Open circles: control bags; closed/solid circles: treatment bags.

25 20 5 ! I ! I ! l' I !

I I! I I '..Ll.!! I, ! I ! I ! I ! \ .!:

# 1O~

':' 51- E u Cl E 0 ro 10 >-. '=:' +-'

# J (l) .£: C >- u;, 0 0 I" f- a. 

# ot
'! ' 0 10 15 Days Figure 7. Enclosure experimentwith 0.1 /-lglL atrazine. W: Warm experiment; C: Cold experiment. Time series ofprimary production. Open circles: control bags; closed/solid circles: treatment bags.



### 7

## w

100 o

# 50~ C

OLU \.;[ LU. : U l LL-L.;..l1L:.· I I 'I II- 1 o 5 10 15 20 25 ' 

### 42 Days Figure 8. Enclosure experimentwith 0.1 /-lg/L atrazine. W: Warm experiment; C: Cold experiment. Time series ofoxygen saturation. Open circles: control bags; closed/solid circles: treatment bags.

## 1000~~ 100 

# \,
:3 

### '" 10· 

# \ ..c: ; 

### W co 1f· "- .5!2

" , • ! I II 1 ' , I

. I I.LLL'-LLLLLl..U-/f-J Ol ::l <:I 5000 <:I .J> .S 1000

Ol 

# \ c 100 

## ! .c 

# \cOl Cl 10 !

1 

### C

II I!! t! t!""'!!'! I!' I!!!!' ';-....J a 5 10 15 20 25 ' 42 Days Figure 9. Enclosure experimentwith 0.1 /-lg/L atrazine. W: Warm experiment; C: Cold experiment. Time series ofDaphnia abundance. Open circles: control bags; closed/solid circles: treatment bags.

8Data Summary Table: Effect Magnitude Nominal Dayof Time Weighted Did Comments ofEfJect Atrazine Study AverageAtrazine Recovery relative to Concentra EfJect Concentration Occur

- 

### if controls tion Occurred from Day 0 to so, when (flgIL) When EfJect (see Occurred comment)?
>95% 300 Ilg/L 300 Ilg/L Results oflaboratory "food Decline in chain" experiments with Daphnia Day 21 combined algae and water biomass 90% 100 Ilg/L (end of 100 Ilg/L No (end of effects. P-values for effects are resulting from study) study) all <0.01. Magnitudes effects on food (algae). 60% 50 Ilg/L 50 Ilg/L estimated from Fig.2; the leftmost 0.05 mg/L plot is used. No effect 10 Ilg/L 10 Ilg/L NR 100 Ilg/L NR not calculable NR Decline in NR 10 Ilg/L NR not calculable NR Results ofenclosure Daphnia experiments. Magnitudes biomass I >90% I Ilg/L Day II 0.97 Ilg/L No estimated from Figs. 5 and 9 >90% 0.1 Ilg/L Day 18 0.03 Ilg/L No ("C").
Decline in NR 100 Ilg/L NR not calculable NR Results ofenclosure NR 10 f,lg/L NR not calculable NR experiments. Magnitudes primary est.imated from Fig. 7 ("C"). production NR 1J.1g/L NR not calculable NR (photosynthet Effect at 0.1 ug/L was not ic rate) 60% 0.1 J.1g/L Day 18 0.03 J.1gIL Yes
- Day considered to be clearly related 25 to atrazine treatment. NR 100 Ilg/L NR not calculable NR NR 10 Ilg/L NR not calculable NR Results ofenclosure Decline in 50% 1 J.1g/L Day 11 0.97 J.1g/L No experiments. Magnitudes chlorophyll a estimated from Figs. 3 and 6 No clear 0.1 J.1g/L NA NA NA ("C"). effect NR 100 f,lg/L NR not calculable NR Results ofenclosureexperiments. NR 10 f,lg/L NR not calculable NR Results presented in Figs. 4and 8 ("C"). There is uncertainty in the 50% 1J.1g/L Day 11 0.97 J.1gIL No results at 0.1 ug/L becausethe oxygen levels may have been affected by daphnid mortality and Decline in corresponding decomposition. oxygen However, there was an apparent saturation2 decrease in photosynthesis at 40% 0.1 J.1g/L Day 18 0.03 J.1g/L No (partial approximately the time of at Day 42) observed reductions in oxygen levels. Therefore, these data suggestthat atrazine may have impacted photosynthesis and corresponding oxygen levels at0.1 ug/L. Note: NR=Not Reported; NA = Not applIcable; Balded values are those data pomts specIfied m the "Cosm DataPoints and Exposure Profiles" spreadsheet.

I Ibid. Note that three other zooplankton declines appear in Fig. 5; onlyDaphnia is included here, because it is the only zooplankton that was also examined at 0.1 Ilg/L, and ithas the most extreme responses ofall zooplankton studied. 2Ibid.

9Since the laboratory "food chain" experiments used controlled continuous inputs of treated water and growth medium, it was assumed thatno significant declines in atrazine concentration occurred overtime. Forthe enclosure experiments, time weighted averages were calculatedbased on the information in the study text: I) 90% ofthe atrazine from the 1 /lg/L treatmentbags remained on Day 18; and 2) No atrazine could be detected in the 0.1 /lg/L treatmentbags on Day 10. The detection limit was notreported; therefore, a conservative assumption of0 ug/L was used to calculate time-weighted average concentrations at time points when no atrazine was detected. Chemographs were computed assuming a linear decline in atrazine concentration overthe time period ofthe experiment, and time-weighted averages were then simple cumulative average concentrations. Calculations are shown below.

0.01 ug/L1day 0.005556 ug/L1day 0.1 ug/L 1 ug/L Cone Day TWA Cone Day TWA 0.10 0 0.10 1.00 0 1.00 0.09 1 0.10 0.99 1 1.00 0.08 2 0.09 0.99 2 0.99 0.07 3 0.09 0.98 3 0.99 0.06 4 0.08 0.98 4 0.99 0.05 5 0.08 0.97 5 0.99 0.04 6 0.07 0.97 6 0.98 0.03 7 0.07 0.96 7 0.98 0.02 8 0.06 0.96 8 0.98 0.01 9 0.06 0.95 9 0.98 0 10 0.05 0.94 10 0.97 0 11 0.05 0.94 11 0.97 0 12 0.04 0.93 12 0.97 0 13 0.04 0.93 13 0.96 0 14 0.04 0.92 14 0.96 0 15 0.03 0.92 15 0.96 0 16 0.03 0.91 16 0.96 0 17 0.03 0.91 17 0.95 0 18 0.03 0.90 18 0.95

Reviewer Comments: Interpretation ofthis study was hampered by inadequate descriptions ofthe protocols and methods used for analysis, as well as incomplete presentation ofthe results ofthe enclosure experiments. The lack offull descriptions ofthe protocols is particularly problematic becausethis study tookplace in Germany, and so differences with standard American procedures may affectunderstanding ofthe results. Exact dates and durations ofthe enclosure experiments are notprovided. The data summary table is incomplete because results for 10 /lg/L and 100 /lg/L atrazine experiments were notpresented. The fact that wann versus cold experiments were conducted at 0.1 /lg/L atrazine levels butnot

10for other concentrations also confuses interpretation ofthe results, as inter-treatment level comparisons may notbe appropriate.

The laboratory "food chain" experimentbarely qualifies as an ecological study, since only one zooplankton species and one algal species were utilized. Indeed, the authors use this experiment as an example ofthe effects that canbe observed at alevel only slightly more complex than a directtoxicity bioassay. Results ofthe laboratory experimentthus cannotbe generalizedto natural ecosystems; however, the laboratory conditions allowed areasonable level ofreplication (fourreplicates at each treatment) and well controlled conditions.

The enclosure experiments appeared well controlled, but only two replicates were used at each treatment level, so no statistical tests could be applied. Additionally, the bag enclosures may have generated artifacts inthe observations due to inhibited mixing and inability ofcommunities to recolonize, which limittheir applicability to natural ponds.

Overall, the study is successful in its goal ofshowing that indirect effects may be more importantthan direct effects in a complex community, butthe experiments do notprovide arealistic simulation ofenvironmental conditions.
Description ofUse in Document (QUAL, QUAN, INV): QUAL

Rationale for Use: The utility ofthis study is limitedby the deficiencies described in this review, and its results should be interpreted with caution. However, the study does contribute to the weight ofevidence regarding potential effects ofatrazine to aquatic communities.

Limitations ofStudy: The highly simplified, artificial structure ofthe laboratory "food chain" experiments limits their applicability to real-world aquatic systems. Results for higher-concentration enclosure experiments were notprovided, and the use of"warm" VS. "cold" lowest-concentration enclosure experiments interferes with comparison ofeffects at different treatment levels. Finally, the study does not describe orprovide sources for nearly all analytical methods and sampling protocols used.
Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc


### SecondaryReviewer: Brian Anderson, U.S. EPA

# #**~_**

] 1 /) '/ I 

## t; (t:... / -, "J/ i