Bioresponse of Nontarget OrganismsResulting from the Use of Chloropicrin

To Control Laminated Root Rot in a Northwest Conifer Forest:

Part 2. Evaluation of Bioresponses

by

Elaine R. Ingham', Walter G. Thies', Daniel L. Luoma 2 , Andrew R. Moldenke3 and Michael A. CasteIland

Author affiliation: 'Department of Botany and Plant Pathology, Oregon State University, 'Pacific NorthwestResearch Station, USDA Forest Service; Department of Entomology, Oregon State University, OR.

Known impacts of chloropicrin

Most research hasconcentrated on the effects ofcombinations of methyl bromideand chloropicrin fumigants ondisease-causing organisms. Littleresearch has been reported on theresponse of saprophytic fungi tochloropicrin. Some evidence existsfor the escape of some species offungi during fumigation effortsthus positioning the survivors forrapid recolonization of theavailable substrate. Theoccurrence of Trichoderma spp. inroots of fumigated Douglas-firstumps was noted during theevaluation of several fumigants forthe control of laminated root rot(Thies and Nelson 1982).Following fumigation, increasednumbers of Trichoderma spp. werefound in some soils (Mughogho1968). Combinations of methylbromide and chloropicrin control avariety of soil-borne diseases inforest tree nurseries (Peterson andSmith 1975) and can reducepopulations of various other fungi.These include VA mycorrhizalfungi (Jones and Hendrix 1987,McGraw and Hendrix 1986),ectomycorrhizal fungi (Trappe etal. 1984), as well as rootdisease-causing species of Pythium,Rhizoctonia, Phoma (Sumner et.al. 1985), Helicobasidum momta,(Sakuwa et al. 1984), Verticilliumalbo-atrum, and Sclerotium(Himelrick 1986).

Nematode populations arereduced by chloropicrin alone andin combination with methylbromide or dazomet. Researchhas concentrated on thecommercially important parasitesof plant roots and no informationwas found on free-living fungal orbacterial feeding nematodes.Fumigation reduced the nematoderoot pathogens Meloidogyne spp.and Paratrichodorus minor(Sumner et al. 1985) andchloropicrin in combination withethylene dibromide reducedBelonolimus longicaudatus,Meloidogyne incognita, andHoplolaimus galeatus (Rhoades1983).

As a measure of speciesrichness, soil arthropods in coniferforests of the Pacific Northwestaverage nearly 200 species/m2(personal communications AndrewR. Moldenke, Oregon StateUniversity, Corvallis, OR). Thussoil arthropods may be animportant and sensitive indicatorto evaluate potential ecosystemeffects of chloropicrin onnontarget soil organisms. Soilarthropods are sensitive indicatorsfor soil moisture, successionalstages, plant communities, andmycorrhizal biomass (Cromack etal. 1988, Moldenke and Fichter1988). All arthropod species arepresumed to be sensitive tochloropicrin, although differingfeeding preference, microhabitat

choices, and position in the foodweb will expose the diversity ofspecies to different concentrationsof fumigant.

Objectives

One objective of this study wasto determine changes in diversityof specific nontarget organismcomponents of a coastal ecosystemwhich occur as a result ofapplication of chloropicrin tostumps to control laminated rootrot. A major challenge was toevaluate the impact of chloropicrinon a range of organism groups. Asample scheme capable ofdistinguishing at least two levels ofspatial variability was designed.Background variability of theorganisms resulting fromheterogeneity in soil micrositesand seasonal shifts is a commondifficulty in studies of soilorganisms. Sampling intensity hadto be great enough that treatmenteffects could be assessed, withoutincreasing the work load beyondthat of a limited budget.

The approach described herecan be applied to any ecosystem,and to a number of situationswhere effects of pesticideapplication need to be assessed.With this approach, the effect ofpesticide on plants, soil detritalfoodweb organisms, mycorrhizalcolonization of dominant plants,and survival of sensitive bioassay

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NONTARGET BIORESPONSE TO CHLOROPICRIN: 2. EVALUATION

plants can be interpreted in abiologically meaningful manner ata number of spatial scales. Forexample, the pesticide may reduceroot rot, but destroy thoseorganisms responsible for nutrientcycling, or could allow anotherpathogen to become a problem,exchanging one pathogen problemfor another. Root-feedingnematodes might be advantaged,resulting in seedling death.Alternatively, greater mycorrhizalcolonization of roots might occur,and benefit the survival of desiredplant species. Before we continuewith the use of pesticides, weshould attempt to understandbeneficial or detrimentalinteractions which may beproduced. Our approach allows usto assess these possible beneficialor detrimental effects.

Experimental site

When the experimental sitewas cut early in the fall of 1988,see Part 1. of this study, p. 81) soilconsisted of a moss (bryophyte)layer overlying poorly developedlitter, and fermentation layers, witha 1-5 cm depth humus soil horizonresulting from the rapiddecomposition rates in thesesystems. When soil developmentis limited, nutrient cycling isusually tightly coupled to organismdynamics (Read and Birch, 1988;Perry et al. 1989). When litterfalls to the forest floor, it is rapidlydecomposed, and the nutrientsconverted to microbial biomass.These nutrients are then releasedby arthropod, nematode andprotozoan grazing of decomposers,resulting in high soil fertility andmaximum nutrient availability toplants during times of rapid plantutilization (Coleman 1985).However, with clearcutting, muchof the thin soil layer was destroyedand mineral soil revealed.

The location of each infectedstump in the clearcut stand and

three mature trees in adjacent un-cut stands were mapped. Circulartreatment plots 0.4 ha in size wereestablished in the clearcut areaand blocked into groups of fourbased on similar inoculum rating(see part 1 for explanation). Eachof the four plots in each blockreceived one of the followingtreatments: all stumps treated with100% chloropicrin, only infectedstumps treated with 100%chloropicrin, all stumps treatedwith 20% chloropicrin, and acontrol plot with no applicationchloropicrin. The 100% labeldosage was 3.3 ml of chloropicrinper kilogram of stump and rootbiomass.

Choice of bio-response parameters

Bio-response assessment wasinitiated in the spring of 1989.Five major component groups oforganisms were assessed; theabove ground plant community,detrital foodweb organisms,mycorrhizal colonization ofDouglas-fir roots, and the responseof chloropicrin-sensitive plants.These assessments will continueuntil the fall of 1991.

Soil foodweb organismsrespond more rapidly than plantsto environmental change anddisturbance. Responses ofbacteria and fungi often reflectday-by-day fluctuations intemperature, moisture, grazing,and nutrient availability, and thusare not useful as measures ofecosystem response to disturbance.However, by examining changes inactivity, in ratios of fungal tobacterial biomass, or importantpopulations of microorganisms,longer term impacts on the systemcan be assessed.

Protozoa, nematodes andmicroarthropods are intimatelyinvolved in nutrient cycling(Coleman 1985) and thus are goodindicators of ecosystem health

(Ingham et al. 1985; Ingham andHorton, 1987). Mycorrhizal fungiare extremely important in survivalof Douglas-fir; in field sites,Douglas-fir is not found withoutthe symbiotic fungus (Trappe et al.1984). Thus, monitoringmycorrhizal fungi can be extremelyimportant in determining ifpesticides have an effect.

Tomatoes and alfalfa wereplanted in the field sites. Tomatois extremely sensitive tochloropicrin (Rhoades 1983), andalfalfa is symbiotic with N-fixingrhizobium. Release ofchloropicrin in field soils could bemonitored with tomato, andnatural populations of rhizobiacould be assessed by examining theroots of the alfalfa.

Spatial scales

Within the 0.4 ha circular plots,responses were monitored onseveral spatial scales, as well asover time.

Aboveground higher plantresponses were assessed eachspring and fall by monitoringpercent cover of each speciespresent,

in the entire plot,

in six 2 m X 3 m plotsarranged sequentially at 1, 2 and 3meters from four individual stumpsat the edge of the plot, and

(3) in six 1 meter square plotslocated between two stumps within2 meters of each other.

Detrital foodweb responses(numbers and activity of bacteria,active and total fungal biomass,and numbers and communitystructure of protozoa, nematodes,and microarthropods) weremonitored over time. Sampleswere taken mid-spring, early

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summer, late summer, and mid-fall each year.

To determine if soil organismnumbers, activity or communitystructure changed on a whole plotspatial scale, twelve random pointswere sampled in each of five plotsper treatment on the four sampledates each year. The same fourrandom sets of coordinates weredesignated in each third of the plotto maximize coverage of the area.

To determine if a localizedeffect of chloropicrin treatmentoccurred, soil samples were takenat 4 distances (0.5, 1.0, 1.5, and 2.0m) along three equally spacedradii extending from tree stumps(five separate tree stumps pertreatment). While soil samples formicroarthropods were taken fromeach point, samples for the otherorganisms were bulked bydistance. Since it was chloropicrintreatment, not soil heterogeneity,that was being assessed, averagingthe differences resulting from soilmicrosite heterogeneity wasacceptable.

On each sample day, the actualpoint from which soil was removedwas repositioned from the originalmarker by pre-determineddistances. A 7.5 cm diameter, 7.5cm deep sample of soil wasremoved from each point formicroarthropod estimates. Anapproximately 2 cm diameter, 5cm deep soil sample was takenfrom each point, but soils fromeach of the three quadrants (i.e., 4soil samples) were bulked forbacteria, fungi, nematodes, andprotozoa assessments.

Active bacteria, active fungi,and total fungal biomass wereassessed by the FDA method ofIngham and Klein (1984). Totalbacterial numbers were assessedby FITC staining (Babiuk andPaul, 1970). Protozoa weredetermined by MPN and direct

microscopic viewing (Darbyshire,et al. 1974). Nematodes wereassessed by Baerman extractionand microscopic observation(Anderson and Coleman, 1977).Microarthropod numbers wereassessed by high efficiencyTullgren extraction andobservation (Merchant andCrossley 1970).

Chloropicrin-sensitive bioassayplants (tomatoes and alfalfa) wereplanted each year in the spring at1 and 2 meters distance fromstumps of five trees of eachtreatment type and were randomlyplaced in another five plots of eachtreatment. Survival was assessedon each sample date. In thesecond year, a ring of alfalfa wasplanted at 1 meter distance fromeach stump. All alfalfa plantswere examined for N-fixingbacteria nodules on their roots atthe end of the second year.

RESULTS

Chloropicrin application wasnot the only environmentalvariable to which these sites wereresponding. Two extremelyimportant correlated variableswere (1) removal of canopy coverand (2) compaction of the soil byheavy machinery. All theexperimental sites were exposed toboth, either of which may haveecosystem effects equal to orgreater than the chloropicrintreatments. The three types ofplant plots, the two types ofdetrital foodweb organism plots,placement of chloropicrin-senstitive plants, and placement ofDouglas-fir seedlings to assessmycorrhizal colonization allowedassessment of bio-responses tothese environmental variables.

In addition to compaction andcanopy removal, we observed agradient of organism numbers andcommunity structure from north tosouth and east to west in this

stand. The blocking initiated atthe beginning of the study basedon Phellinus inoculum density instumps reflected changes in soilorganism community structure,and was related to surface soilcharacteristics. We use theseblock effects as covariates instatistical analyses.

Pesticide application did notimpact establishment, growth orsurvival of any plant species in thefirst year after application ofchloropicrin.

In the first year, chloropicrinimpacted soil bacteria, fungi,protozoa, and nematodes only in afew isolated points near stumps:

area 8, point 2, 20%chloropicrin treatment,

plot 2, 20% treatment, 2.0distance,plot 602, 100% treatment, 2.0distance, and

plot 736, 100% treatment, 2.0distance.

When these single points wereaveraged with all five replicatesfrom a treatment, variance wassignificantly increased, but nosignificant treatment effect wasobserved.

In these isolated cases,reductions in numbers oforganisms were considerable, fromaround 101 , or 10 million totalnumber of bacteria per gram soilto less than 100,000 per gram soilin impacted points. Funginormally measured about 600 m ofhyphae per g with between 10 and50% of those hyphae active,dependent on season. In impactedareas, less than 5 m of hyphae perg were found, with no activehyphae present. Protozoa tendedto number around 10,000 per gsoil, but in impacted soils, wereless than 10 per g. We have notfinished assessing protozoancommunity structure, but no

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NONTARGET BIORESPONSE TO CHLOROPICRIN: 2. EVALUATION

immediately obvious changes weredetected in the first year. Incontrol soils, nematodes tended tonumber around 100 per 5 g soil,but in impacted soil, less than 10per 5 g soil were found. However,no significant changes in nematodecommunity structure wereobserved in the first year.

Microarthropod communitystructure has been assessed for thefirst year, and it is clear thatthough individual species respondboth positively and negatively to achloropicrin-fumigatedenvironment, these responses werenot of great enough magnitude toradically alter total densities orbiomass either around individualtrees, or in whole plots. However,in tree-centered samples, guilddiversity was decreased in the 20%treatments, and comparisons ofindividual species showed that 15of the commonest speciesdecreased with increasing dosage,8 increased with dosage, 2 werehighest in 20%, and 3 were lowestin 20% chloropicrin treatments.The commonest species ofspringtail was unaffected bychloropicrin, whereas mostoribatid species were affected,although increases and decreasestended to cancel out overalleffects. Coupling these analyseswith covariate information(removal of canopy cover,compaction, plant diversity, etc.),should lead to identification of asmall set of indicator taxa that canbe used for more efficientmonitoring of similar situations.

In the second year, the areasimpacted in the first yearexpanded, and four to five newpoints of impact were observed.The position of newly impactedpoints appear random. Completeanalysis of variance has not beenperformed at this time, becausenot all samples have beencompletely analysed.

There was no differencebetween survival of tomato plantsin year one in any treatment, butin year two, more tomato plantsdied in the 100% treatments thanin other treatments. There was nosignificant difference in nodulationof alfalfa roots in any plot in anyyear.

CONCLUSIONS

In the first year afterchloropicrin application, reductionsin organism numbers in a fewisolated points and changes inindividual species ofmicroarthropods were significanton the spatial scale of a singlestump. These impacts could beimportant to a new seedling tryingto obtain nutrients from the soil inan impacted area. On an largerspatial scale, such as the 0.4 haplots, and certainly on anecosystem-level, the impact wasnot significant, based on plantresponse (no impact on plantcommunity structure or onchloropicrin-sensitive bioassayplants) or on numbers of bacteria,fungi, protozoa, nematodes ormicroarthropods.

Will the impact be detrimentalor beneficial, in the long term? Inthe second year, numbers of plant-feeding nematodes have increasedfrom barely detectable numberspresent in soil to comprising up to50% of the nematode populationin some samples in the secondyear. This could be detrimental toDouglas-fir seedlings, but this is astand-wide effect, not an effect ofchloropicrin application. In fact,those points impacted by thechloropicrin have below-detectionlevel numbers of nematodes, andso, chloropicrin application couldbe beneficial for Douglas-firseedlings growing in those areas,because the plant-feedingnematodes have been negativelyimpacted in those places.

The increase in plant-feedingnematodes has coincided withincrease in exotic weed species onthese sites. Plant-feedingnematodes may be attacking theroots of these weedy species andthus be reducing competitionbetween the weed species andDouglas-fir seedlings. In areasimpacted by chloropicrin, it maybe that the weeds don't sufferlimitation by root-destroyingnematodes and the Douglas-firseedlings will experience increasedcompetition. We will be able toassess this interaction by continuedexamination of the soil and theplant communities over time.

All of our information supportsthe conclusion that very littlechloropicrin escaped from roots inthe first year. Only at a fewpoints, not significant on anecosystem scale, were effectsdetected. In the second year,responses of soil organisms andchloropicrin-sensitive plantsprovided information that morechloropicrin escaped from roots,but still not enough to result in aneffect on the plant community.Early warning that potential effectsmay occur is being provided by thesoil organisms, but we don't havethe database to tell us if thismeans an overall detrimentaleffect on the ecosystem, or anoverall beneficial effect on theecosystem.

We know that the fungus thatcauses laminated root rot is beingkilled in these stumps (Thies andNelson, 1985). Within the stump,other organisms are being killed,and it is likely that wooddecomposition is being slowed. Isthat positive or negative? Wedon't know. What is the balancesheet going to indicate in tenyears? Will the detrimental effectsoutweigh the positive?

Whatever happens in thisparticular ecosystem, however, thetype of sampling being done,

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Ingham, et. al.

.limited as it is, allows biologicallymeaningful conclusions to be madeabout the impact of this pesticidein this ecosystem. This approachof assessing bio-responses gives usthe ability to make predictionsabout the possible trajectories thisecosystem may take. We have themeans to make useful predictions,and by looking at this suite oforganism responses, we canindicate problem areas before asituation may result in irreversibleloss of a particular habitat orspecies.

So, is chloropicrin usedetrimental? If the points whereorganism numbers have beennegatively impacted don't spreadfarther than they have in thesecond year, and if the pathogensdon't cause a problem as impactedareas are re-colonized, theliklihood is that the pesticideshould continue to be registeredfor this use, with the clearexplanation that higher doses,applied in a different manner,might be detrimental. However,impacted areas are likely tocontinue to expand, because not allthe pesticide has volatilized fromthe stumps. How far will theaffected areas expand? Willpathogens colonize the center ofthese impacted areas and causeproblems? Will we select forworse disease problems by usingthis pesticide? Once all thechloropicrin is out of the stumps,how long before affected areas willbe re-colonized by the normalorganisms? Or will these areas bepushed into completely differentecosystem trajectories, similar towhat has occurred in some plots insouthern Oregon with completelydifferent disturbances (Borchers

1 and Perry, 1989)? Answers tothese questions are not available.

t

1 But continued monitoring in this

`,,,,,

system will allow these questionsto be answered for this system.Extrapolations can be made toother systems with theunderstanding that differences

between another system and thisone must be understood inpredicting possible impacts.

COOPERATION

The following organizations arecooperating in support of thisstudy. Simpson Timber Co.; GreatLakes Chemical Co.; NationalAgricultural Pesticide ImpactAssessment Program (NAPIAP),US Department of Agriculture;Pacific Northwest ResearchStation, US Department ofAgriculture, Forest Service; andthe departments of Forest Science,Botany and Plant Pathology, andEntomology, Oregon StateUniversity.

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