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  The Impacts Of Ultra
Yiolet B Radiation On
 Biological Systems:
  A Study Relate
 Stratospheric Ozone
       Depletion
         Submitted To:

    The Stratospheric Impact Research
    and Assessment Program (SIKA)
  The U.S. Environmental Protection Agency
      Washington, D.G. 2O6O4
        Volume I

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                         DISCLAIMER






     THIS REPORT HAS NOT BEEN REVIEWED FOR APPROVAL BY THE



AGENCY AND HENCE ITS CONTENTS DO NOT REPRESENT THE VIEWS AND



POLICIES OF THE U.S. ENVIRONMENTAL PROTECTION AGENCY, NOR DOES



MENTION OF TRADE NAMES OR COMMERCIAL PRODUCTS CONSTITUTE ENDORSEMENT



OR RECOMMENDATION FOR USE.

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                                     CONTENTS

                                                                        SIRA FILE #

 VOLUME  I

 1.    Study Of Increase In Skin Cancer As A Function
      Of Time And Age And Changing Stratospheric Ozone:
      Need For Careful Measure Of The Ultraviolet Dose    .............   # 132.11

 2.    The Influence Of Age, Year Of Birth, And Date On
      Mortality From Malignant Melanoma In The Populations
      Of England & Wales, Canada And  The White Population
      Of The United States    				   # 132.31

 3.    Non Melanoma Skin Cancer Surveys In The United States
      -  An Environmental Epidemiologic Project   ......................   # 142.11

 4.
5.   Biological Effects Of Ultraviolet Radiation On Plant
     Growth And Function	,.	   # 142.21

6.   Effects Of UV-B Radiation On Selected Leaf Pathogenic
     Fungi And On Disease Severity	   # 142.21g

7.   The Effect Of Ultraviolet (UV-B) Radiation On Englemarm
     Spruce And Lodgepole Pine Seedlings	„	   // 142.22

VOLUME II

8.   UV-B Biological And Climate Effects Research	   # 142.23

9.   Ultraviolet Effects Of Physiological Activities Of
     Blud-Green Algage	'...   # 142.24

10.  Impact Of Solar UV-B Radiation On Qfops And Crop
     Canopies	„....„*..ซ	   # 142.25

11.  High Altitude Studies Of Natural, Supplemental
     And Deletion Of UV~B On Vegetables And Wheat   .................   # 142.26

VOLUME III

12.  UV-B Radiation Effects On Photosynthesis And
     Plant Growth	   tf 142.27

13.  Influence Of Broad Band UV-B On Physiology And
     Behavior Of Beneficial And Harmful Insects	   // 142.28

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                                                                        SIRA FILE

14.  A Study Of The Effects Of Increased UV-B
     Irradiation On Environmental Dissipation Of
     Agricultural Chemicals   	.......	    # 142 v. 29

15.  Biological Effects Of Ultraviolet Radiation
     On Plant Growth And Development In Florist And
     Nursery Crops	    .# 142.210

16.  Biological Effects Of Ultraviolet Radiation On Cattle:
     Bovine Ocular Squamous Cell Carcinoma	    # 142.211

17.  Radiation Sources And Related Environmental Control
     For Biological And Climatic Effects UV Research (EAGER)   	    # 142.212

18.  Instrumentation For Measuring Irradiance In The
     UV-B Region	    # 142.213

19o  Annual Report To EPA, Bacer Program For
     Fiscal Year 1978		.......	    # 142.34

20.  Penetration Of UV-B Into Natural Waters	    # 142.36'

21.  Higher Plant Responses To Elevated Ultraviolet:
     Irradiance	    // 142.4.1

22.  Assessment Of The Impact Of Increased Solar
     Ultraviolet Radiation Upon Marine Ecosystems	    // 142.42

23.  UV-B Instrumentation Development   ............................    # 142.51

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                    STUDY OF INCREASE IN SKIN CANCER

        AS A FUNCTION OF TIME AND AGE AND CHANGING STRATOSPHERIC OZONE:

             NEED FOR CAREFUL MEASURE OF THE ULTRAVIOLET DOSE
                 By  June Morita and Elizabeth L.'Scott
                          Statistical Laboratory
                     University of California, Berkeley
     The work which we are reporting is part of a large cooperative study

of the Panel to Review Statistics on Skin Cancer of the Committee on

National Statistics of the National Research Council, of an informal

Skin Cancer Workshop at the University of California, Berkeley, and of

members of the staff of the National Center for Health Statistics, and

of the National Cancer Institute.  We have been' studying both melanoma

and nonmelanoina skin cancer as well as actinic skin damage.  These are

responses to ultraviolet radiation, perhaps associated in some cases with

certain chemicals.  They are rather different but each is important and

the studies support each other.  We are estimating the increase in skin

cancer for a specified scenario of the change in ultraviolet radiation, as

a function of time, age,  sex,  location, and other predictor variables.

     Note that a scenario about change in ultraviolet radiation implies that

an individual at a given  locality will receive different doses as time

changes.   Actually the present dose is not constant;  there is  diurnal

change,  seasonal change,  year  to year change (influenced by changes in  the

pollution of the earth's  atmosphere and stratosphere  as  well as changes on

the sun),  and also local  changes within our atmosphere.   The individuals at

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                                       -2-
 a given locality  have  different life styles, different  sensitivity  spectrum

 of the  skin,  and  so  forth, all of which affect  the  dose received.
                  C#i1iซ'ซป
      The effect of,new pollutants entering  the  stratosphere will be to change

 the proportion of ozone and thus the amount of  ultraviolet radiation that

 passes  through to the  earth's surface.  Because the vertical mixing in the

 atmosphere  is very slow and because cancer has  a long latent period, any

 change  in ultraviolet  radiation will have a long-time effect on skin cancer.

 We will need  to consider ultraviolet flux as a  function of time as well as

 of locality,  life style, and other variables.

      Observational data about skin cancer are sparse and inaccurate, due

 partly  to the gross under-reporting of nonmelanoma skin cancer.  We need

 to  use  many different  sources of data from independent  studies, employing

 individual sets to obtain reinforced conclusions.

      Our  method of study is to estimate the increase in incidence,  preva-

 lence or  mortality, as the case may be, for each age and sex category,

 as we consider one locality after another with increasing flux.  In addition

 to  our  difficulties with the uncertainties in the  skin cancer data,  we  also

have difficulties with uncertainties in the dose of ultraviolet radiation

received  by an individual at a specified locality.   In the first place,

we do not have measurements of the ultraviolet flux received at each

locality  during any short period of time.   In the  second place, we  do not

have measurements of the variability in dosage from one person to another

at the  same locality or from one time to another for the same person.  We

need to know the average dosage at  each locality for each short time period,

and we also need to know how this  dose  varies according to the life  style

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                                       -3-
  of  the  individual,'according  to his skin sensitivity7  his  year  of  birth,

  and so  forth  and  so forth.  This information is not availablej  we  have

  made use of estimates.

      We estimate  the average  ultraviolet flux dosage, for each, locality

  with the help of  extensive computations made for us by S.V, Venkateswaran

  and his colleagues,  D.E. St.  John and N.  Sundaranaraman.

  As  shown in the schematic drawing in '.Figure 1",      .   • '         -

.  the ultraviolet radiation received by an individual ฑs the product of, the

  solar irradiance  in  the ultraviolet times the transmittanee.through the

  stratospheric ozone  layer and the earth's atmosrphere (as computed for us

 by Venkateswaran using ozone measures)  times the transmittanee through

  the  layer of clouds and murk at the' earth's surface times the transmittanee

 through the  individual's clothing and  other protection and finally into

 his  skin.   We have labelled this product ''sensed -flux,"  Each of the factors

 entering the product sensed flux  is uncertain,  particularly the estimated

. sensitivity of the skin as a function of wavelength.   Following  Setlow,  v?e

 have used  the relative sensitivity,  as  a function  of wavelength, in terms

 of damage  to DNA by radiation.  It  is important  to have a  check  on  the
               calculated values, of.     .              . .       .."'''
 accuracy of the/flux of ultraviolet  radiation at the'earth's surface and

 also the amount  entering the individual's skin.  Ultraviolation  radiation

 meters, in particular, the Berger meter,  provide a  partial check.  We are

 now engaged in a comparison  of the results we deduced from  the Venkateswaran

 computations, with those, obtained by. the  Berger meter.

      A  further complication  is that  the ultraviolet radiation'dose  varies

markedly from  one  individual to another  in the same locality, depending

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                                OZONE
                                LAYER
                                :f<^/\\
FIGURE "L.  Schematic dirawing of path of ultraviolet: radiation through the


           stratosphere and atmosphere of the earth.

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                                      -4-
 on life style,  protective  clothing, and  so  forth..   This  means  that  some


 individuals  will  be  subject to higher risk  than others,   It also  intro-


 duces  a statistical  complication.  The standard methods  of estimating the


 relationship between skin  cancer raLes and  ultraviolet flux will  be biased,


 underestimating the  slope.   To correct for the bias du6  to the variation


 in ultraviolet  dose  from one individual to another at the same locality,


 we need data on the variability.  Direct measurements are hot yet available,


 but estimates can be made  from the surveys of Urbach for  the two  localities.


 Philadelphia, Pennsylvania, and Galway, Ireland.  Further information is


 available from  the exposure data of the Health and Nutrition Examination


 Survey  of the National Center for Health Statistics.  However, these are


 only estimates  a.nd they are not scaled.  We need actual measurements from


 personal dosimeters on individuals in different localities,  some in the


 South and some  in the North, in different age and sex categories,  with


 various skin sensitivities,, various occupations, and so forth.


     We want to make a strong plea for personal dosimeter data, collected


 on a regular basis on each of many individuals for.successive short  periods


 of time, preferably daily,  so as to obtain estimates'of the  dose under

                               v       .                  •
 specified employment a.nd recreation regimes, and. as a function of  micro-

 climate,, age, sex, etc.   We have already  stated the'importance  of  verifying


 the estimated avex'age flux  at each, locality, and the importance of estimating


how this flux dosage varies in time and varies from one individual to


another at the same locality and time.  We also should  emphasize the dose-


rate problem.  There is  clinical evidence that persons  who expose  themselves


 to a large dose of ultraviolet radiation,  even for a relatively short

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                                       -5-
 period  of.  time,  are much more  likely  to  develop  actinic  skin damage and




 even malignant melanoma than persons  who receive the  same  total dose in




 small sub-doses  distributed over a long  period of time,  This, effect is




very striking, for example, in Scandinavians who spend a, short vacation




 in the Mediterranean area, but it is  also noticeable  in  office workers




who enjoy an outdoor weekend.  Personal  dosimeter observations are needed




to establish, the dose-rate relationships for different types of  individuals,




and to compaje the estimated relationships with those predicated from




studies of laboratory mice.




     Our study comparing the ultraviolet flux estimates based  on the compu-




tations of Venka.te3wa.rain a.nd the. observations provided by the Berger meter




is still preliminary-,   Therefore.,  the report'we are. presenting today is




an interim  report.




     First, we need to be .clear.!about what, we are comparing.   On the one




hand, we have sensed flux, an  estimate of what affects the cells of  the skins-




computed as the product of many factors, as described above.   This is to be




compared with the measurements from the  Berger meter t as provided to us by




W. N. Haas and his colleague G. Cotton at the Air Resources Laboratories,




National Oceanic and Atmospheric Administration.




     Speaking roughly, the Robertson-Berger meter integrates over the radi-




ation actually observed at a specified locality during each' time    • ;•  . .




interval.  The integration is weighted so that the result approximates what




the cells of the skin would receive if the sensitivity spectrum of the  skin




were a standard erythema (sunburn) spectrum.  For each place where a Berger




meter is located, we have observations for each half-hour over the recorded




life of the meter there, usually from January 1, 1974 through  October 30, 1976,




with .later observations to become available shortly.  The sensed flux and the

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                                  -6-
 Berger  meter  flux are not  strictly  comparable.   The  sensed flux is a

 product of  extensive  theoretical  computations based  on  long-range ozone

 observations,  adjusted for altitude and for  long-range  cloudiness, .for  skin

 absorption  and for the DNA-damage spectrum.  It  is available  for each of

 74 localities  in  the  United States  on a per  month basis.   The Berger

 meter flux  is  strictly observational, subject to the vagaries of real clouds

 and other absorbing material at its real altitude, but with the  observations

 weighted in an approximate fashion  designed  to correspond  to  the erythema

 spectrum rather than-DNA-damage spectrum.  Nevertheless, it is of interest

 to see how well the two sets agree  and to examine any systematic differences.

 In particular, are there systematic differences in the higher flux (southern)
    . *
 localities  that are different from  those in  the lower flux (nortbern)

 localities?

     The short time-period observations from the Berger meter were combined

 into daily observations which were then combined to obtain averages  over the  '•

month when the meter was in operation.   Missing observations were  interpo-

 lated or were  supplied from the auxilliary record?" at each locality.  For

purposes of comparison with sensed flux,  averages were taken over  the years

 the meter was  in operation.  The results  are illustrated in Figure 2 for the

meter in Minneapolis.  The triangles connected by solid-lines are the Berger •

meter readings for a particular month.   The crosses connected by dashed' lines

are monthly averages over the typically three years the meter was in operation

drawn again for each year to illustrate the variability from year to year

contrasted with the average over only  three years.   For example, the spring

of 1976 tended to have higher flux than the three-year average.

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                                                       Minneapolis
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                           MONTH
                                                     1/76
7/76
1/77
                                              A	Berger Meter Flux
                                              X	AvQ.(Mean)For Each Month
FIGURE 2.  Comparison of Berger meter  readings for each month  (triangles

           connected by solid lines) and  monthly averages over  three years

           (crosses  connected by dashed lines).                    >•   . .   .

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                                      -7-
       These Berger  meter monthly  averages  are  drawn  again  on  Figure  3


  along with the  monthly sensed  flux values (as circles  connected  by  solid


  lines).   Since  the units are arbitrary  for .both  the Berger meter values and


  the  sensed flux (because the scale of the action spectrum is unknown in


  both cases,  and is arbitrarily set equal  to unity at one  wavelength), we


  have adjusted the  plot in Figure 3 so that distances from minimum to maximum


  agree.


       We have studied the relation between the monthly Berger meter measure-


  ments and  the corresponding monthly sensed flux  in  those  localities where


<•  both are available to us:  Albuquerque, El Paso, Des Moines, Oakland,


  Minneapolis, PHiladelphia and Ft. Worth (except  that we made an error in


  computation  on  the Ft. Worth data).  The  localities group nicely into pairs


  by climate:  clear  (Albuquerque and El Paso), mixed  (Des Moines and Oakland),


  and  cloudy  (Minneapolis and Philadelphia).


       In Figure  4 is shown a direct comparison between the sensed flux and


  the  Berger meter flux for each month of the year 1975 for the two locations,


  El Paso and Albuquerque.   We have used different symbols for the six months


  January to June and the remaining six months July to December.   For the first

                                v
  year  in which the Berger meters were in operation the relationship between


  Berger meter and latitude measures was systematically different for the first


 half of the year and the second half.   This effect persists  when comparing


  sense flux with Berger meter in each of the years 1974, 1975, 1976.   For the


  clear stations  (which are the southern stations in our  comparisons), the


  relation for the first half of  the year tends  to be higher than that of the


  second.  This is also true  for  the cloudy stations (which are further north),

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                                             O	Monthly Sensed Flux

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N   FIGURE 3.  Comparison of Berger meter monthly averages  (crosses^connected

       . ..      hy dashed lines) with the monthly sensed  flux values (circles

              connected hy solid lines).

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                      MONTHLY  RESIDUALS
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                    MONTHLY RESIDUALS
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/75
 MONTH
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FIGURE 5.  Direct comparison between the sensed flux and the Berger meter

          flux for each month of the year 1975 for the two locations,,

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                                    ~7a-
as shown in Figure 6.  On the other hand, the early months tend to be lower




for the two mixed climate stations which we have studied, Oakland and




Des Moines.  That is, if one were to trace the points in the scatter plot




for El Paso (Figure A), starting with January, then to February, and so




forth to December, one would find oneself tracing an ellipse.   Thus it seems




that for the first half of the year the observed Berger meter flux values




are consistently higher than the corresponding calculated sensed flux values,




while for the second half of the year,  the Berger meter values are consistently




lower than the corresponding sensed flux values.   Seasonal deviations were



already noticed by Urbach in the 1974 data.

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                     MONTHLY RESIDUALS
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                                  MONTH
   FIGURE 6.  Direct comparison between the sensed flux and  the Berger meter

            . flux for each month of the year 1975 for the two locations,,

                      s and Phi

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                                   -8-
 Both of these stations happen to be at  middle  latitude.   We should point




 out that the cloudiness we are using refers  to the  middle of the day in




 July,  the period of  highest ultraviolet radiation intensity.   In spite of




 the suggestion of the  systematic differences between  the  two sets of obser-




 vations,  which could be explained by inadequacies in  the  factor  correcting




'for cloudiness in the  computations  of the  sensed  flux, the relationship




 between the  two measures is very reasonable and nearly linear.  (We could




 also explain this discrepancy by prolonged periods  of unusual cloudiness in




 the years  under study.)




     We have started to  make a more  detailed examination  of  the  residuals.




 The results  are shown  in Figures 7,  8 and 9 for the eight  localities  under




 study,  again paired  according to climate.  In  the plots,  the  least-square




 line relating  the Berger meter flux measures to the sensed flux  has been




 eliminated and  only  the  residuals from  the line are plotted.  Our  original




 plan had been  to  compare the  residuals with cloudiness data for  each month




 shown.  We have not  extracted  the cloudiness data so that part of  the study




 is  unfinished.  The  plots of  the residuals do give an impression of smooth




 changes with time and  it  is of interest to determine whether  these changes




 reflect long periods of  unusual weather.




     . The last  three  Figures again show the residuals from a straight-line




 relationship between the  two measures of flux,  but the residuals have -.been'.,'.




 smoothed by Tukey's 3R or Running Medians-of  three\ .repeated .until.convergence.




 The.suggestion of systematic changes does persist  but  the  evaluation of'the




 contribution of unusual weather has  still not been done.




     We are continuing our studies comparing Berger  meter  flux with the sense




 flux.  As stated above, we are searching for  systematic  differences between

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                        MONTHLY FLUX VALUES 1975
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                    SENSED FLUX X IO"17
                                                             .70
                                                     .80     .90
  FIGURE  7.  Monthly residuals from the line relating the Berger meter flux


             measures to the sensed flux for El Paso and Albuquerque".

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                        MONTHLY  FLUX VALUES 1975
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                         • ''-A
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   FIGURE 9.   Monthly residuals from  the  line relating the Berger meter  flux


              measures to the sensed  flux for Minneapolis and Philadelphia.

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  FIGURE 10.  Running  medians of monthly residuals for El  Paso and Albuquerque.

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 FIGURE 11. Running medians of monthly residuals for Oakland and Des Moines.

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    -Fl'tanfe. 12. Running medians of monthly  residuals  for Minneapolis and



                Philadelphia.

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                                  -9-
 the two  systems which  cannot  be  explained  by  prolonged  periods  of.  unusual




 weather.  When observations are  available  from  the  Berger meters over  a,




 long period,  the effects of irregularities of weather will be minimal.  •




 At  the present time, with  less than  three  years of  data at hand, we can




 only estimate the effects  of  unusual weather  and we need further meteorolog-




 ical data to do even this.  In order to check the adequacy of the  cloudiness




 corrections we have been using, we need many more than  three years of  com-




 parison.  We suspect that  our cloudiness corrections are too simplistic.




 The  means of studying  the  adequacy of cloudiness corrections lies in the




 direct comparison of Berger meter readings with the sensed flux data, com-




 bined with meteorological  observations on  cloud cover,  and extended over a




 reasonably long period of  time.




     We have been troubled by the cloud corrections we, and everyone else




who makes any correction at all, have been using because we noted systematic




 grouping of the residuals  in age-adjusted mortality from malignant melanoma




 considered as a function of flux.  Localities with low cloudiness tend to




have higher than predicted mortality; localities with more cloudiness tend




to have lower than predicted mortality even when corrections for microclimate




are included.  Even though residuals in our more recent studies  do not show




any such pronounced effects,  we realize that we  should employ better  means




for considering the attenuation by high and low  clouds and  other absorbing




materials.   A careful study of the Berger meter  readings over a  period of




years, combined with a  corresponding series of meteorological observations,




should provide the  information we need.

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                     LEGENDS FOR FIGURES





FIGURE  1.  Schematic drawing of path  of ultraviolet radiation  through  the


           stratosphere and atmosphere of  the earth.





FIGURE  2.  Comparison of Berger meter readings for each month  (triangles


           connected by solid lines) and monthly averages over three years


           (crosses connected by dashed lines).





FIGURE  3.• Comparison of Berger meter monthly averages (crosses connected


    .ซ.     hy dashed lines) with the monthly sensed flux values (circles


           connected by solid lines).





FIGURE  4.  Direct comparison between the sensed flux and the Berger


           meter flux for each month of the year 1975 for the two locations,


           El Paso and Albuquerque.
FIGURE 5.  Direct comparison between the sensed flux and the Berger meter
                              v

           flux for each month of the year 1975 for the two locations,


           Oakland and Des Moines.
FIGURE 6.  Direct comparison between the sensed flux and the Berger meter


         .  flux for each month of the year 1975 for the two locations,


           Minneapolis and Philadelphia.





FIGURE 7.  Monthly residuals from the line relating the Berger meter flux


           measures to the sensed flux for El Paso and  Albuquerque.

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FIGURE 8.  Monthly residuals from the line relating the Berger meter flux




           measures to tVie sensed flux for Oakland and Des Moines.









FIGURE 9.  Monthly residuals from the line relating the Berger meter flux




           measures to the sensed flux for Minneapolis and Philadelphia.









FIGURE 10. Running medians of monthly residuals for El Paso and Albuquerque.









FIGURE 11. Running medians of monthly residuals for Oakland and Des Moines.









FIGURE 12. Running medians of monthly residuals for Minneapolis and




           Philadelphia.

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                                                   .  3t
 THE INFLUENCE OF AGE, YEAR OF BIRTH, AM) DATE ON MORTALITY FROf!




MALIGNANT MELANOMA IN THE POPULATIONS OF ENGLAND & WALES, CANADA




          AND THE WHITE POPULATION OF THE UNITED STATES
                       John A.  H.  Lee (1)




                     Gerald R.  Petersen (2)




                     Richard G. Stevens (3)




                        Kajo Vesanen (1)

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                                                              Page 2
                  FOOTNOTES FOP. TITLE PAGE
     Received for publication  :

     (1) From the Department of Epidemiology SC-36, University of
Washington Seattle, WA 98195.  Address reprint requests to Dr. Lee.
K. Vesanen is witli the Department of Biostatistics at the University
of Washington

     (2) San Jose State University, San Jose, CA 95192

     (3) The Fox Chase Cancer Center, Institute for Cancer Research,
7701 Burholme Ave., Philadelphia, PA 19111

     The study was supported by Environmental Protection Agency
Research Grant R805363010 from the Office of Health and Ecological
Effects.

     A preliminary summary of these findings was presented at the
1977 meeting of the Environmental Protection Agency, Program
Planning and Review Workshop for the Biological and Climatic Effects
Research Program.

     We are indebted to Mr. Larry C. Clark for data tapes.

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                                                              Page 3
     ABSTRACT
     The age-adjusted death rates from malignant melanoma of the




skin have increased from 1951 to 1975 by about 3% per year in the




populations of England & Wales and Canada, and in the white




population of the U.S.  This is due to large increases in risk of




successively later born cohorts.  Any effects of earlier diagnosis




or improved treatment within the period 1951-75 have been




sufficiently steady to to fail to alter these trends.  The slope of




the log rates with log age is about 3.5.   Projections of rates for




at least the next decade can be made with some confidence, and




provide a basis for evaluating control measures.







     MALIGNANT MELANOMA :  COHORTS : AGE DISTRIBUTION :  CANCER




CONTROL :  INTERNATIONAL COMPARISONS

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                                                              Page
      INTRODUCTION
      The  incidence of malignant melanoma has been rising rapidly in




 many  white  populations  in recent years  (e.g. 1-6).  The death rate




 has also  been  rising sharply in the populations of England ฃ Wales




 (7),  and  Canada  (8).  Mortality data for malignant melanoma for the




 U.S.  have been incompletely published for the years 1950-67 (9) and




 for later years  without specification by age.  There are indications




 from  these  data  that mortality is also  rising in the U.S.




 population.







      A major component  of the causation of this rising mortality is




 a systematic increase of risk of successively later born cohorts




 (7,8,10).   This  produces a large difference between the age




 distribution of  the mortality in a population,  and the age




 distribution within each cohort (11).  The prognosis of malignant




 melanoma  has improved (12), probably due to earlier diagnosis.  Such




 a change  may also affect the observed age distribution of a disease




 (13).







     Mortality from malignarit- melanoma in white populations is




 inversely related to latitude of residence (14,15),  an exception




 being the inhabitants of Sweden who have more melanomas than would




 be expected from their latitude of residence (16).   Long duration of




 residence in Israel of persons of  European origin leads to an




 increased incidence of melanoma (17,18).  The incidence of malignant




melanoma per unit skin area is greater on exposed than on unexposed




 sites (1,19).  The intensity of ultra-violet light  at  ground level




will rise due to the expected depletion  of the  stratospheric ozone

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                                                              Page 5


 layer  (20).  Although  the  action  spectrum of neoplastic change in

 the  human  melanocyte is unknown,  there are reasonable, grounds for

 assuming that  the  ultra-violet component is important.  At a time

 when administrative measures, such as the control of refrigerating

 agents, are under  consideration to protect the ozone layer, it is

 important  to have  projections of  the future trends of mortality from

 malignant  melanoma.  These can provide a base line for the

 evaluation of  control  measures.


     In this paper we  report the  U.S. mortality data for whites from

 1951 through 1975, and compare the rates and their trends and age

 distributions  with those from Canada and England & Wales.


     DATA AND  METHODS


     The numbers of deaths from malignant melanoma by age and sex

 for  England &  Wales and for Canada, and the population data for all

 three countries were obtained from the standard publications of the

 respective offices of vital statistics.  The U.S. melanoma mortality

 data for 1950-67 were obtained from special tabulations from the
                           V
 Department of  Epidemiology, University of North Carolina, using data

 originating with the National Center for Health Statistics.  Data

 for  1968-76 were obtained from special tabulations from the National

 Center for Health Statistics, who take no responsibility for the

 analyses that we have made.


     Rates  and rates of change were calculated  as shown in the Table

descriptions.   As a check that there were no  serious  interactions in

the data matrices,  multivariate methods were  also used to estimate

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                                                              Page 6



 the  parameters.  The  results are shown in the Tables.



     RESULTS



     The  age adjusted death rates from malignant melanoma of skin

 (ICD172)  for England  & Wales, Canada, and the United States white


 population by 5-year  periods 1951-55 through 1971-75 are shown in


 table  1.  The rates are rising most rapidly in Canada (Table 2), but


 the  changes are marked in all three countries.  The least

 proportionate increase is in U.S. females.



     The  prognosis of malignant melanoma has been improving (12), so


 that each cohort will suffer a smaller increment of mortality per


 unit time in the later time periods than in the earlier.  However,


 the mean  slopes of rate against age (Table 3) show no consistent

 changes with time.  A constant rate of improvement with time will


 not change the rates of change observed, and a steady improvement is


 probably what is occurring in the diagnosis and treatment of

 melanoma.



     The rates of change with increasing age appear to be similar at
                           i
 all ages  (Table 4).  They are lower in females than in males,


 probably reflecting the better female prognosis, and are higher in

 Canada than in the U.S.   or England & Wales.   Estimates using


multivariate techniques (Table 4) give similar results to the simple

means,  and indicate that  interactions between parameters are not


large.

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                                                              Page 7


      The  increase of mortality with increasing age is greater than

 increase  of  incidence with age reported from Connecticut (11).

 Case-fatality  increases with age  (12), which could account for the

 difference.  The differences between the cohorts in their mortality

 rates are large and consistent (Table 5 and the Figure as an

 example),  particularly when it is considered that neighboring

 cohorts are  only separated by 5 years in date of birth.  The decline

 in  the  1931  female cohort compared to the 1936 is consistent and

 interesting.


      Projections of mortality rates are an important guide to the

 effectiveness of administrative or therapeutic measures.  For

 malignant  melanoma rates they are likely to be reliable as the rates

 of  change  with age do not show any sharp changes with time,  and the

 current behavior of the cohorts that will  provide the bulk of the

 deaths in  the next decade is well established.   Projected rates

 derived from the present matrices of data,  using the rates for

 1971-75, and the rates of change for 1971,(13),  are shown in

 Table  1.
                           *,

     DISCUSSION


     The driving force behind the current  increase in the mortality

 from malignant  melanoma is a systematic increase in risk with

 successively later years of birth.  It  is  difficult to imagine a

 reasonable mechanism involving  better  diagnosis  or reporting that

 could produce these regular cohort differences,  particular  in the

light of the parallel  increase  in incidence  rates .  Why people born

five years later should go through life with a  substantially

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                                                              Page 8






increased risk of dying from malignant melanoma compared with  their




elder peers in the same population is unknown.   Whether  there  are




systematic differences in life style that are distributed in this




way, or whether there is some deeper biologic mechanism  at work,




will require,  in the first instance, a series of studies in clinical




epidemiology.

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                                                              Page 9
Table  I - Death Rates* and Projected** Death Rates from Malignant
Melanoma of Skin by Sex and Time Period: England & Wales, Canada,
and United States whites.

Time Period   1951-  1956-  1961-  1966-  1971-  1976-  1981-
               55     60     65     70     75     80     85

MALE
England&Wales  6.8    7.4    8.6   10.6   11.8 :  13.4   15.2
Canada         7.1   10.1   11.7   13.5   15.8 :  18.9   '22.1
U.S.white     14.5   16.6   19.9   22.9   26.3 :  28.7   33.5

FEMALE
England&Wales  7.2    8.5    9.8   10.8   13.3 :  16.3   19.8
Canada         6.0    8.2   10.1   11.5   12.3 :  13.0   14.1
U.S.white     11.2   12.6   13.8   15.4   16.5 :  17.7   18.8

*In this, and the subsequent tables,  rates are given per
million per year, age adjusted using  the UICC standard European
population (21).

** Rates to the right of the colons are derived from each national
set of age and sex specific rates 1971-75, and the sets of cohort
slopes for 1971.   These are the age-specific increments of the log
rates divided by  the increments of the log ages.  Rates for new cohorts
entering the projection at age 15-19  were given the 1971-75 values.
The method is discussed in (13).

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                                                             Page 10
Table 2 - Mean Annual Percentage Increase in Malignant Melanoma
Death Rate*, England & Wales, Canada, and U.S.  whites by Sex.

               Male                 %

        England & Wales            2.9
        Canada                     3.5
        U.S. whites                3.0

               Female

        England & Wales            2.9
        Canada                     3.3
        U.S. whites                1.9

* The slope of the line fitted to the national  period rates in
Table 1 as a % of the national means.

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                                                             Page 11
Table 3 - Mean* Cohort Slopes by Mid-year of Observation,
Malignant Melanoma Mortality Rates, England & Wales; Canada;
and United States whites.

                      MALE                    FEMALE
Mid-Year    England                   England
            & Wales  Canada   U.S.w   & Wales    Canada     U.S.w
1956
1961
1966
1971
3.59
3.41
4.43
3.48
6.34
2.98
3.92
3.94
3.54
3.87
3.76
3.66
3.82
3.43
2.64
3.91
5.38
5.01
3.56
2.69
3.21
2.92
3.25
2.96
^Arithmetic means of the age-specific increments of the log rates
divided by the increments of log ages (13).  The data for 1956
are the means of the age-specific slopes between the years
1951-55 and 1956-60.

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                                                             Page 12
Table A - Mean age-specific Cohort Slopes* for Death Rates from
Malignant Melanoma by Sex: England & Wales, Canada, and United
States whites, 1951-55 through 1971-75.
England
&
AGE
20
25
30
35
40
45
50
55
60
65
70
75
80
85
Mean
20-85
Estimates
Wales

7.01
3.79
2.05
3.44
1.88
3.09
4.90
2.24
3.94
4.30
4.48
2.31
7.24
3.32

3.86

fron least
squares
**

3.32
Estimates
from maximum
likeli-
hood***  3.58
                  MALE
                   Canada
                    6.37
                    2.36
                    3.71
                    1.81
                    4.14
                    4.56
                    3.83
                    3.49
                    3.27
                    7.51
                    4.43
                    4.28
                    2.21
                   16.33
                    4.88
                    5.06
3.67
            U.S.
           Whites
            4,
            3.
            3.
 ,83
 ,44
  31
2.65
2.27
3.33
3.51
              ,80
              ,86
              .00
              ,85
              .93
              59
            6.08
            3.89
            3.27
3.4!

England
& Wales
4.66
3.37
2.49
3.74
2.89
2.91
2.91
2.13
2.45
4.60
4.89
3.61
3.76
4.54
FEMALE
Canada

4.21
4.52
2.71
3.69
4.23
0.42
3.05
4.74
3.06
6.28
4.70
3.33
9.85
7.49
            3.50
            3.31
3.19
            4.45
            4.68
3.34
                                    U.S.
                                   Whites
                        4.27
                        3.82
                        2.57
                        2.
                        2.
                        2.
                        2.
                        2.
                        2.
                        3.
              ,63
              ,12
              ,50
              ,10
              ,69
              ,95
              ,48
            3.59
            4.79
            3.90
            6.72
            3.44
            3.15
2.93
*Cohort slopes as described in Table 2, arithmetic means of these
for successive time periods 1951-55 to 1956-60; 1956-60 to 1961-65
etc.

**Slopes of lines fitted to the age parameters for each data set (22).

***Slopes of lines fitted to the age parameters for each data set (23).

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                                                             Page 13
Table 5 - Mean Percentage Increase of Death Rates from Malignant
Melanoma between successive Birth Cohorts over the same Age Range
Cohorts
           Age
          Range
Mean % change
between cohorts
England
& Wales
1936/1931 20-39
1931/1926 25-44
1926/1921 30-49
1921/1916 35-54
1916/1911 40-59
1911/1906 45-64
1906/1901 50-69
1901/1896 55-74
1896/1891 60-79
1891/1886 65-84
1886/1881 70+
6
18
27
6
30
31
15
26
12
13
- 12
 MALE
Canada
                             26
                             23
                              9
                             28
                             27
                             22
                             29
                              4
                             25
                             23
                             16
 U.S.
whites

    1
   10
   19
   16
   24
   17
   21
   19
   12
   21
   11
England
& Wales

    - 6
     18
      8
     38
     21
     40
     22
      4
      9
     18
      6
 FEMALE
Canada   U.S.
        wh i t e s
1
13
20
15
27
13
16
7
52
1
17
- 5
10
18
11
12
10
13
13
8
5
1
                    15.6%    21.1%   15.6%     16.2%   16.4%   8.7%

  Mean of age-specific inter-cohort  ratios expressed as a percentage.

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                                                                Page  14
                  150
                  125

                  100
                   50-
                I
                a
                   25-
10-
                   5-
                   2-
                             '46
                     15
        \    25   i  35  I 45 i 55 16
       20      30   40  50  60
Figure:  Cohort  Diagram of Mortality Rates  from Malignant Melanoma

         in U.S.  white Males.  1951-75

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                                                             Page 15
                         REFERENCES
  1.      Elwood JH, Lee JAM: Recent Data on the Epidemiology of
         Malignant Melanoma. Sera in Oncology 2:149-154,1975

  2.      Lee JAH: The current rapid increase in incidence and mortality
         from Malignant Melanoma in developed Societies. Pigment Cell
         v.2 ed. Riley V pps.414-420 Basel Karger 1976

  3.      Magnus K: Incidence of Malignant Melanoma of the Skin in the
         Five Nordic Countries: Significance of Solar Radiation. Int
         J Cancer 20:477-485,1977

  4.      Teppo L,Pakkanen M, Hakulineri T: Sunlight as a Risk Fetor of
         Malignant Melanoma of the Skin. Cancer 41:2018-2027,1978

  5.      Malec E, Eklund C: The Changing Incidence of Malignant Melanoma
         of the Skin in Sweden, 1959-68. Scand J Plast Reconstr Surg
         12:19-27,1978

  6-      Soodalter-Toman DL, West DW,  Derrick LR:  Epidemiology of
         Malignant Melanoma in Utah 1966-76. In press

  7.      Lee JAM, Carter AP:Secular trends of Mortality from Malignant
         Melanoma. J Natl Cancer Inst  45:91-97,1970

  8.      Elwood JM, Lee JAH:Trends in  Mortality from Primary Tumours
         of Skin in Canada: J Canad Med Assoc 110:913-915,1974

  9.      Burbank F: Patterns in Cancer Mortality in the United States:
         1950-67. Natl. Cancer Inst Mono 33 Washington USDHEW

10.      Gordon T, Crittenden M, Haenszel K: End Results and Mortality
         Trends in Cancer II Cancer Mortality Trends in the United
         States. Natl Cancer Inst Mono 6 pps 133-350 Washington USDHEW

11.      Lee JAH:  Letter. Am J Epidera  107:259-260,1978

12.      End Results  Section:  Cancer Patient Survival No.5 pps 223-227
         Washington USDHEW

13.      Stevens RG,  Lee JAH:  Tuberculosis Generation Effects and
         Chemotherapy.  Am J  Epidera 107:120-126,1978

14.      Lancaster HO:  Some Geographical Aspects of the Mortality
         from Malanorna  in Europeans. Med J Aust 1:1082-1087,1956

15.      Elwood JM,  Lee JA1I, Walter SD, Mo T,  Green AES: Relationship
         of Melanoma  and Other Skin Cancer Mortality to Latitude
         and Ultraviolet Radiation  in  the United States and Canada.
         Int.  J.  Epidera 3:325-332,1974

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                                                             Page 16
16.      Lee JAK,  Issenborg HJ:  A Comparison between England and Males
         and Sweden in the Incidence and Mortality of Malignant Skin
         Tumours.  Brit J Cancer  26:59-66,1972

17.      Movshovitz M, .Modan B:  Role of Sun Exposure in the Etiology
         of Malignant Melanoma:  Epidemiological Inference. J Hatl
         Cancer Inst 51:777-779

18.      Anaise D, Steinitz R, Ben Hur N: Solar Radiation: A Possible
         Etiological Factor in Malignant Melanoma in Israel.
         A Retrospective Study (1960-72). Cancer 42:299-304

19.      Committee on the Impacts of Stratospheric Change: llalocarbons:
         Environmental Effects of Chloroflouromethane Release,  pps
         101-106 Washington National Academy of Sciences

20.      Committee on the. Impacts of Stratospheric Change: Response to
         the Ozone Protection Sections of the Clean Air Act Amemdments
         of 1977:  An Interim Report. Washington National Academy of
         Sciences

21.      Doll R, Cook P:  Summarizing Indices for Comp-.rison of  Cancer
         Incidence Data.  Int J Cancer 2:269-279,1967

22.      Barrett JC:  Age, time and cohort factors in mortality  from
         cancer of the cervix. J Hyg Camb 71:253-259,1973

23.      Breslou ME,  Day  NE: Indirect Standardization and
         Multiplicative Models for Rates with Reference to the  Age
         Adjustment of Cancer Incidence and Relative Frequency  Data.
         J Chron Dis  28:289-303,1975

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                NCI  -  Human Health Effects  Research  Project
                           for the BACER  Program
    Project Title:   Non  Melanoma  sk,in  cancer  surveys  in  the  United States
                         An  Environmental  Epidemiologic  Project
                Interim Report  -  As  of  December -IS,  1977



         TYPE  OF CONTRACT:   EPA/NCI  Cooperative  Interagency Agreement




      Amount of Funding Provided  by  EPA (for the BACER Program):  $840,000


                 Survey Period  -  One year, beginning June 1, 1977
              Funding Period - One year's funding applied through
                                 September 30, 1978
          Key Personnel:

            NCI:  Joseph Scotto, Senior Health Services Officer and
                  Thomas Fears, Ph.D., FieTd Studies and Statistics Program,
V                Division of Cancer Cause and Prevention

            EPA:  Herbert Wiser, Ph.D. and Alphonse Forziati, Ph.D.
                  Research and Development

-------
Summary
                                  c •
     In response to the BACER program's urgent need for more epidemiologi • -1
data on the possible human health effects of ozone depletion, the Natioru;
Cancer Institute is presently conducting simultaneous surveys on the annual
                                  *
incidence of r.onmelanoma skin cancers in various locations in the United
States.  All new cases of basal  cell carcinoma and squamous cell carcinoma,
diagnosed as of June 1, 1977 (through May 31, 1978) will  be reported.  In
addition, a sample interview of patients and the general  population will be
sought in each'location.  It will be necessary to evaluate the new morbidity
data in light of known and suspected etiological factors  (both genetic and
environmental).            •
     At this juncture the incidence phase is well under way and promises
fruitful  results.  A pilot test of the interviewing phase has already proved
beneficial in paving the way for a successful telephone interview survey.
Preliminary results from the pilot study indicate that there are important
questions to be taken into consideration, e.g.,  outdoor exposure patterns,
skin reaction to sunlight (burn, tan)5 skin conditions (freckles), ethnic
group (genetics), skin complexion, industrial exposure, etc.

-------
                         TABLE OF CONTENTS
                               v '
                                                           Page
Introduction			  1
Progress Update - Incidence Pha,se	  3
Interviewing Phase - Report on Pilot Study	  7
General Procedures		  7
Results	 11
Direct Responses vs. Proxy Responses	 12
Plan A vs. Plan B	 12
Telephone Interview vs. Personal Interview...	.'	 18
Patient Responses vs. General Population Responses	 18

References	.;.	....	 22
Tables and Figure
   Table 1..	................."...		....  4
   Table 2	  9
   Table 3..	 10
   Table 4................................................. 13
   Table 5	...16
                            %
   Table 6	.......................'.......	 19
   Figure 1			  8

-------
  Introduction

                                    o •

      The National Cancer Institute (NCI) in collaboration with the Environmental



  Protection Agency (EPA) is conducting basic epidemiologic research to study the



  potential human health effects of stratospheric ozone depletion.  It was

                                   4


  essential that this project be implemented as soon as possible in order to



  provide the Biological and Climatic Effects Research (BACER) program information
                                          ป


  which is urgently needed to decide on guidelines and restrictions for our



  country's use of certain chemicals known and suspected of depleting the earth's



  protective ozone "shield".



      The NCI had previously conducted a short-term study of the incidence of



  nonmelanoma skin cancer in four locations which were part of the Third National



  Cancer Survey (TNCS 1969-72):  Dallas-Ft. Worth, San Francisco-Oakland, Iowa



  and Minneapolis-St.  Paul.   '  While the results of this study were being



  analyzeds the Department of Transportation (DOT) had already embarked on its



  famous Climatic Impact Assessment Program (CIAP).  This program was initially



  concerned with the effects of stratospheric pollution (e.g., ozone depletion)



  from supersonic aircraft (SST) exhausts, such as nitrogen oxides.  Responding



-  to DOT's need, the NCI formed the Ultraviolet Radiation and Skin Cancer Working



  Group.  Members included representatives from Temple University; Queensland,



 Australia,  the National Oceanic Atmospheric Administration (NOAA), prominent



 scientists  in the field of medicine and phys'ics as well as the leaders of the



 DOT's CIAP  program.   Under the auspices of this working group, measurements



 of ultraviolet radiation (UV-B) were made in various locations of the United



 States.   By design,  the four locations of the TNCS's skin cancer survey were •


                                         (?)
 also included,   NCI  produced a. monograph^c/ of the measurements from each of



 ten locations  for one entire year (48S half-hour "readings per day),  1974, and



 compared UV-B  measurements  with available morbidity data:  four locations for

-------
 nonmelanoma  skin  cancer,  and  nine  locations  for melanoma of the skin.  As
                                  ป •
 expected,  NCI  showed  that the incidence  of skin cancer was associated with
 increased  UV exposure, and that  UV  count was  correlated with latitude,
 altitude sky cover, season of the year,  and  time of day.   '     '  '
                                  4
      Concurrently., other  scientists 'had  reported that chlorofluoromethane
 (CFMS)  gases such as  those used  as  propellants in aerosol cans and as
 refrigerants (freons) could deplete stratospheric ozone in potentially
 devastating  proportions.   One  estimate was that the earth may realize a 7 to
 9  percent  total ozone level depletion by the year 2000, if continued use of
 CFM's persisted at current levels.
      It was  further recommended  by another interfederal agency task force3
 the Inadvertent Modification of  the Stratosphere (IMOS) Committee., that
 much  basic research was needed to study and estimate potential harmful
 biologic effects of ozone  depletion both human and nonhuman in the very
 near future.   '  It was specifically noted that more epidemiologic data on
 skin cancer  were urgently  needed to provide better estimates of the amount
 of increased risk to human skin  cancer which may result from various amounts
 of increased UV-BS resulting from suspected ozone depletion.   Utilizing the
 existing data  from the NCIS the current or tentative estimates of the
                               V
 biological  amplification factor is put at 2,  for increases  of UV-B of less
 than 10 percent.  This means that a one percent increase in UV-B is expected
 to result in a two percent increase in skin cancer.   Combined with a physical
 amplification factor,  also put at 2 (the physical  amplification factor of 2
means that a one percent decrease of ozone  results  in a two percent increase
 in UV-B),  this  means  that a fourfold increase in skin cancer may result from
 low levels  of ozone depletion.  With the present state of the  art, it is  also
 known that  the  effects will be amplified to much greater degrees for ozone

-------
                                                                           3
 depletion  levels  greater  than  10  percent.  However, there still remains a
 great deal  of  variability in many"of these estimates — thus the need for
 quick-yielding basic  research  projects.
      Again,  the NCI responded  to  the need by recommending the utilization
 of its  continuing surveillance, epidemiology and end results (SEER) program.
 Seven SEER  participants are now engaged in the latest epidemiologic effort.
 In addition  to basic  morbidity information, the NCI will attempt to obtain
 relevant information  from the  general populations as well as a sample of
 patients in  each  location.  The new information sought deals with factors
 which may be related  to genetic susceptibility (e.g. ethnic group, eye
 color,  hair  color,, skin color, etc.) and environmental susceptibility (e.g.
 outdoor exposure  habits,  occupational exposure, sunburning, tanning,
 protective measures,  etc.).  The method of obtaining information employs
 sample  telephone  interviewing techniques.  The specific contractors engaged
 in  this project are listed in Table 1.   A pilot test of the interviewing
 phase is being conducted  at the University of Minnesota.  This  location was
 chosen  because the same contractor had provided NCI with incidence information
 from  the TNCS  and had independently followed-up the survival of skin cancer
 patients diagnosed in the earlier study, thus providing a test  data base for
 patient interviewing.   Computer support services  are provided by the Geomet
 Company, and sampling and interviewing  support are provided by  the Westat
 Company.
 Progress_Update -  Incidence Phase
     At a recent general  meeting  of the BACER program,  held at  the  University
of Maryland, a  presentation of all activities through  mid-September was  made (see
 attachment 1, written  transcript,  September  19, 1977).   As  of December  9,  1977

-------
                   TABLE 1
            List of Contractors
Location
Seattle
San Francisco
Detroit
Atlanta
New Mexico
New Orleans
Utah
Minneapolis-
St. Paul
Support Services
Bethesda, Md.
Bethesda, Md.
ซ
Contractor
ป
Fred Hutchinson
Cancer Research
Center
California Tumor
• Registry
Michigan Cancer
Foundation
Emory University
New Mexico
Tumor Registry
Tulane University
Utah Cancer
Registry
University of
Minnesota
Contracts
Geomet
Westat '
No. of Documents
submitted through
December 9, 1977
606
456
770
13257
965
460
1,100
688
Purpose
Data Processing
Sampling,
Funding
Appropriated*
$74,842
$85,720
$87,190
$123,469
$55S785
$91,201
$34,060
$71S505
$123,870
$92 , 358
                                 Interviewing
"Funding  appropriations  for the seven SEER field
 offices  cover  only  phase  1,  incidence  reporting

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 participating  areas have submitted  5664 incidence-related documents to NCI
 for  processing.  This  represents about 20 percent of the total amount we
 expect  for  one year's  survey.  Slow starts in some areas like
                                  i
 San  Francisco-Oakland, New Orleans  and Atlanta were due mainly to
 administrative problems.
      Each location has reported that cooperation from the medical community
 has  been received, and that each expects to provide meaningful incidence
 data  during the course of the survey.  Examples of the kinds of activities
 going on in several field offices are provided in attachment, 2.  There are
 a few individual physicians' in some locations who may not participate in this
 study;  however3 the amount of cases involved appears to be minimal and
 accountable by various means.
     As site visits were made to each field office, it became apparent that
 several new codes could be added to our abstracting'procedures to facilitate
 reports by cell type.   In particular, it v/as observed that some locations
were accessing a large number of in-situ carcinomas and "Bowen's disease" of
 the skin.  The item (#25) for cell type on our abstract forms was expanded
 to include several additonal codes.
                              V
     An interactive central  computer system has been developed and installed.
As documents are received from the field offices, they are edited manually as
well  as by computer.   Change documents are submitted by field office personnel
to correct errors and update files.   A summary of the computer system design
is given in attachment 3.

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      In  related work, the New Mexico field office has estimated that the
amount of malignant melanomas of the skin observed for "Anglos", i.e.,
Caucasians other than those Spanish ancestry, in the Albuquerque area,
was consistent with what would have been projected from the UV and melanoma
data  provided by the NCI.
      Further, the city of Albuquerque, Second Council., has endorsed the
New Mexico Melanoma project of the New Mexico Cancer Control program which
is engaged in a prevention program* making people aware of the sun related
association of increased risk to melanomas of the skin (see attachment 4).
     Obviously, it is too soon to provide new estimates of nonmelanoma
skin cancer morbidity from the new SEER locations.   Completion of
documentation for a full year's survey is expected by the end of the
summer of 1978.   Meanwhile, the NCI will evaluate melanoma data which is
being routinely collected by the same SEER participants.   Preliminary
indications are that the incidence of melanoma of the skin are continuing
to increase during the 1970's.  It is not clear,, however, to what degree
increased amounts of UV-B or changing lifestyles may have affected the
apparent rising trend.

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  Intervi ewi ng Phase - Reporb on Pj^ot Study
      There are various epidemiologic factors known or suspected of influencing
  the development of skin malignancies in humans.  The objectives of the pilot
  study conducted at the University of Minnesota were to develop a telephone
  interviewing technique which would yield-high response rates., and to produce
  a meaningful set of questions which would provide answers to pertinent factors
  relating to the individual's genetic and environmental susceptibility.
      The pilot study design is given in figure 1.  Definition of terms are
  given in Table 2.  Numbers of households and individuals interviewed are
  given in Table 3.
      The decision to attempt a short, 10 minute telephone interview was made
  because the alternate approach (person-to-persor. interview)  was determined to
• be too costlya both in time and money.   In the pilot study a systematic sample
 of telephone numbers from the Minneapolis-St. Paul area was  used to provide
 sample responses from the general  population.  In the final  study designs  we
 expect to employ a random digit dialing technique developed  by the Westat  Co.
  (see attachment 5).   This would allow the inclusion of samples of households
 with unlisted telephone numbers*   In the Minneapolis-St.  Paul  areas it was
 estimated that only a small  proportion  of numbers were unlisted and that over
 93% of the households have telephones.                              •

 General  Procedures
      Simultaneously,  samples  of households from the general  population were
 selected  and interviews  were  obtained via  Plan A or Plan  B.   After a  decision
 was  made  as  to  which  plan was  to be  used,  in this case.  Plan B was selected, a

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                                                             8
                   FIGURE 1
              PILOT STUDY DESIGN
 PLAN A
Telephone
150 Direct
 39 Proxy
 PLAN B
Telephone
198 Direct
 61 Proxy
 PLAN A
Personal
   63
 PLAN B
Personal
   75
                     PLAN B
                    Patients
                       84

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                          TABLE 2


            Pilot Study Plan Definition of Terms
1.  Instrument:  Questionnaire containing 24 questions or less,
     including skin complexion chart.

2.  Interview:  Telephone communication to obtain responses to
     questionnaire.

3.  Direct Interview:  Interview conducted directly with sampled
     household member.

4.  Proxy Interview:  Interview conducted with a household member
     who provides responses for another member of the household.

5.  Personal Interview:  Person to person interviews obtained after
     telephone interview is obtained (a repeat of the interview is
     given to confirm telephone responses).

6.  General population:  Households obtained from sampling of area
     telephone directory.

7ซ  Patients:  Sample of patients who were diagnosed with basal cell
     or squamous cell carcinoma of the skin in Minneapolis-St. Paul
   —(SMSA) during the TNCS (1971-72).   Physicians permission must
     have been obtained prior to initial contact.

8.  Plan A:

     1.  Telephone sampled household from General/Operation.
     2.  Obtain free and informed consent to interview.
     3.  Conduct interview of selected respondent,
                       V
9.  Plan B:

     1.  Telephone sampled household.
     2.  Obtain mailing address from proposed respondent.
     3.  Mail out questionnaire to household.
     4.  Obtain free and informed consent to interview.
     5,  Conduct.

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                                                                       10
                            TABLE 3


      Numbers of Households and Individuals in Pilot Study
                      	General Population   	Patients
                         Plan A          Plan B          Plan  B
No. Household
  contacted        \        144             170             157

No. Household
  Interviewed              117             145              84

No. Household                                     .          .
  Refused               '27              25              73

Response Rate (%)           81              85              54*

No. Individuals
  included                 189            _259_             84

  by direct interview      150             198          • •  ;19

  by proxy interview        39              61               65
     *
      This  response  rate was  low  mainly  because old patients
      (over 75 years)  refuse  to response for various reasons
      (senilitys  infirmity, etc.)ซ   The  patients, who were old
      to  begin with, were five years older  than they were at
      time  of diagnosis  (1971-72).   Although the response rate
      is  expected to improve  for  current patients, we may in
      subsequent  studies,  limit our  comparison group to those
      under 75 years,  if responses from  older individuals
      continue to be unreliable.

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                                                                           11
 sample of the patients were  interviewed according to the preferred plan.  To
 verify that responses received  by  telephone were valid and reliable, a sample
 of respondents were  also  given  person to person interviews.  Sample sizes
 are provided in Table 3.  Responses from person to person interviews were
 then compared with telephone responses.  Between 20 and 25% of the individual
 responses from the general population were obtained by proxy, interview.
 Therefore,  it was necessary to  verify that oroxy data were also reliable.
 Responses for each question  (see attachment 6) were evaluated in total and
 for each  plan type to determine if the questions were appropriate,
 understandable, and  productive.          .      .
 Results
     After  reviewing the  responses from each question and conferring with
 consultants  to the BACER  program, we found several problem areas which
 required  major changes.
     General  occupational history as well as industrial exposure questions
were found  difficult to administer and were therefore made more specific.
Questions on  childhood exposure patterns, use of sunscreens and sunlamps, use
of  hats or  long sleeves  were not as productive as had been anticipated.
                              V
Vacation  information questions produced variable response and required
improvement.  Questions  on outdoor exposure patterns on the job also needed
to  be spelled out more specifically for various ages.   The question of utmost
consideration was that on skin complexion.   Although the skin complexion
chart appeared to work,  there was room for improvement.  We have found we
could obtain better skin color tones by employing a printing process
recommended by representatives of the National  Geographic magazine.   As a
result of the pilot,  a new instrument has been  designed (see attachment 7),

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                                                                            12
 which  deletes  olds  nonproductive questions,, restructures the more meaningful
 questions, and provides an  improved skin complexion chart.

 Direct Responses  vs. Proxy  Responses
     The average  proxy response was found to be comparable to the average
 direct response for most questions (see Table 4).  Proxy responses, however,
 must be interpreted with caution, since most proxy interviews were obtained
 from female members of the  households.  Of particular concern are questions
 relating to sunburn effects and tanning ability.
     Any misgivings about proxy information that the investigators may have
 had were found to be outweighed by the increased sample sizes they provide,
 at minimal costs  (Table 3). ' We anticipate that proxy information will be
 obtained in subsequent study.                                  .
 Plan A  vs. Plan B                                                            .
     Questions asked in Plan A were identical  to those asked in Plan B except
 for those relating  to skin complexion.  Mo basic differences in responses
were found between Plan A and Plan B except of course for that skin complexion
 information (see Table 5).  In Plan As the individual  was asked whether he
considered his skin color to be "fair", "medium"9 or "dark".  In Plan B9 a
skin complexion chart was provided on the bottom of the questionnaire (see
attachment 4) which the individual received'in the mail, and he was asked to
compare his skin complexion against the chart.   The latter has  the apparent
advantage of providing a standard of comparison and thereby eliminating any
personal or regional biases which may exist with respect to skin color.   The
investigators were, however, concerned that two telephone contacts (i.e..
Plan B) would result in a lower response rate  compared to Plan  A.   However,
the response rate was 81% for Plan A and 85% for Plan  B — essentially no
difference (Table 3).

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                                   TABLE 4

                     Direct Responses  vs.  Proxy Responses


                     Telephone  Interviewing Method
 No.  responses
      PLAN A
      Proxy

        39
           PLAN A
           Direct

            150
             PLAN B
             Proxy

               61
                         PLAN  B
                         Direct

                          198
Hours - Outdoors^
  Weekdays     x"
  Weekends
               m
x
m
Hours - Outdoor^
  Vacation     x
               m
               0x

Sunbathe
  Frequently
  Occasionally
  Rarely
  Never

Reaction to Sun
  No Reaction--
  Some Redness
  Burn
  Painful Burn
  Not in Sun
  Other Reaction

Age of Respondent
    •   x"
       m
Type of Tan
  None
  Light Tan
  Average Tan
  Deep Tan
  Other
  Not in Sun

Freckles
  Yes
  No
19.2
13.0
 2.5

12.1
11.9
•1.0
       40.0
       33
        3.6
       17.9
       10.3
       23.1
       48.7
                     19.0
       35.9
       30.8
       20.
       10.
,5
.3
        2.6
       41.7
       38.0
        2.3
        2.6
       30.8
       23.1
       43.6
                     14,
                      1,
            11.5
             9.0
              .5
            43.3
            41.0
             1.8
            20.0
            27.3
            28,0
            24.7
            40:9
39
10
10
.6
,7
,7
,7
,7
       38.5
       61.5
           39.4
           35.0
             1.2
            6.7
            11.4
            47.0
            33.6
            1.3
           37.3
           62.7
              .21.6
              15.0
               2.1

              12.0
              11.3
               1.0
              42.9
              41
               2.8
               9.8
              26.2
              16.4
              47.5
                            36.1
                            49.2
                             8.2
                             1.6

                             4-9
              40.5
              35.5
               2.0
               4.9
              14.8
              35.1
              44.3
                          16.2
                          12.0
                            .9

                          10.6
                           9.0
                            .4
                          40.2
                          40.0
                           1.5
                          19.3
                          25.4
                          29.4
                          25.9
34.2
40.3
16.8
 5.6
 3.1
             "18.0
             82.0
                          5.6
                         19.2
                         41.4
                         31.3
                          1.0
                          1.5
                         45.7
                         54.3

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Table 4 (cont'd.)
                                                                          14
Eye Color
  Blue                43.6
  Brown               38.5
  Green               15.4
  Other                2.6

Hair Color
  Black                5.3
  Brown               60  •
  Red                  t'-
  Other               2f:.;

Years Worked on
Primary Occupation
     Y         .       16.1
     m                14.5
     ฐx                2.0

Skin Conditions
  Acne                35.9
  Psoriasis            5.1

Chemical Exposure
  Yes                 35.9
  No                  64.1

Ethnic Background
  Irish               17.9
  English  -—        10.3
  German              33.3
  Scandinavian        30.8

Skin Complexion
Chart
     3
     4
   •  5
     6
 •  '  7
     8
     9
    10

Skin Cancer
Complexion Chart
Matches
  Exactly
  Closely
  Not Closely
42.7
40.7
10.7'
 6.0
11.3
69.3
 2.0
17.3'
12.8
 9.5
 1.0
56.0
 3.3
26.0
74.0
25.3
20.7
SOoO
34.7
49.2
29.5
11.5
 9.8
 6.6
73.8
 1.6
18.0
13.9
10.0
 1.5
39.3
 1.6
31.1
68.9
14.8
26.2
47.5
24.6
               5.8
              21.2
               9.6
               5.8
              21.2
               3.8
              32.7
40.4
29.8
15.7
14.1
 3.5
62.6
 2.5
31.3
13.3
10.0
  .8
59.1
 2.5
18.7
81.3
25.8
21.7
49.5
39.9
               1.6
               3.2
               7.0
               7.0
               3.2
              11.9
              30.8
              35.1
               3.9
              86.3
               9.8
               5.5
              81.3
              13.2

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                                                                          15
Table 4  (cont'd.)
Chart Comparison
with SKIN
  Lighter
  Darker

Skin Color
  Fair
  Medium
  Dark
                            34.9
                            65.1
                            33.7
                            66.3
46.2
35.9
17.9
44.2
49.0
 6.8

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                      TABLE 5
                   Plan A vs. Plan B
                                                                    16
No. responses
Telephop.e Interviews  vs.
  PLAN A       PLAN B

    189          259
                                               Personal  Interviews
                        PLAN A

                          63
                        PLAN B

                          75
Hours - Outdoors
  Weekdays     x"
  Weekends
               m
               a—
                x
               m
               cr
19,0
14.5
 1.1

11.6
 9.6
 0.5
17.5
13.0
 0.8

10.9
10.0
 0.4
                             18.9
                             13.0
                              1.8

                             10.7
                              9.5
                               .7
17.7
10.0
 1.7

11.0
 9.0
 0.7
Hours - Outdoors
  Vacation     x"
               m
               a
42.6
40
 1.6
40.8
40
 1.3
                             44.1
                             41.0
                              2.5
41.0
36.0
 2.6
Sunbathe                 %
  Frequently           19.6
  Occasionally         23.8
  Rarely               27.0
  Never                29.6

Reaction to Sun          %
  No Reaction          39.9
  Some Redness         37.8
  Burn                 12.8
  Painful Burn          8.0
  Not in Sun            1.1
  Other Reaction        0.5

Age of Respondent        $
     x                 39.9
     m                 35.5
     ฐTT                 1.0
             17.1
             25.6
             26.4
             31.0
             34.6
             42.4
             14.8
              4.7
              CD Bป
              3.5
             40.9
             37.0
              0.9
            19.0
            22.2
            28.6
            30.2
            30.2
            47.6
            12.7
             9.5
            36.6
            34.5
             1.5
                                          13.3
                                          21.3
                                          30.7
                                          34.7
                                          32.0
                                          45.3
                                          17.3
                                           4.0
                                           1.3
                                          39.8
                                          34.0
                                           1.8
Type of Tan
  None
  Light Tan
  Average Tan
  Deep Tan
  Other
  Not in Sun

Freckles
  Yes
  No
 5.9
15.4
42.0
35.6
.1.1
37.6
62.4
 5.4
18.1
40.2
34.4
 0.8
 1.2
39.1
60,9
                              6.3
                             19.0
                             38.1
                             35.5
                             46.0
                             54.0
 5.5
17.8
41.1
32.9

 2.7
41.3
58.7

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Table 5 (cont'd.)                                                         . 17


        Eye Color               %            %           %            %
          Blue                  42.9         42.5        33.3         44.0
          Brown                 40.2 v .     29.7        46.0         25.3
          Green                 11.6         14/7        14.3         16.0
          Other                  5.3       •  13.1         6.3         14.7

        Hair Color             '  %            %           %            %
          Black                 10.1          4.2         7.9          8.0
          Brown                 67.6 '  .      65.3        69.8         64.0
          Red                   2.7          2.3         6.3          1.3
          Other                 19.7         28.2        15.9         26.7

        Years Worked on
        Primary  Occupation       %    '        %           %            %
              x               13.6         13.4        11.7         13.5
              m       '         9.0         10.0         9.5         10.0
              ฐ~.      '         0.9          0.7         1.1          1.0
                X

        Skin Conditions          %            %           %            %
          Acne                  51.9         54.4        58.7         58.7
          Psoriasis          ..   3.7          2.3         --           5.3

        Chemical  Exposure        %            %           %            %
          Yes                  28.0         21.6        39.7         25.3
          No                  72.0         78.4        60.3         74.7

        Ethnic Background        %            %           %            %
          Irish                23.8         23.2        27.0         29.3
          English             18.5         22.8        28.6         18.7
        •German               46.6    -     49.0        57.1         60.0
          Scandinavian         33.9         36.3        34.9         44.0

        Skin Complexion                       %           %            %
        Chart             1                            .              1.2
                          3                 1.3     .1.6
                          4                 3.8         1.6          1.3
                          5                10.1        19.0         21.3
    ...                  6    ' ,            7.6         6.3          9.3
                          7                 3.8
                          8                13.9         3.2          5.3
                          9                24.9    .    31.7         26.7
                         10                34.6        36.5         4.6

        Skin Color Complexion
        Chart Matches                         %           %            %
          Exactly                            5.2         3.2          2.8
          Closely                           82.4        84.1         83.3
          Not Closely                       12.4        11.1         13.9

        Chart Comparison with                 ซ           ป,            „/
        SKIN                                 %           k
          Lighter                           34.0        19.0         18.8
          Darker                            66.0   "     81.0         81.2

        Skin  Color               %            %           %            %
          Fair                 44.6         39.7        48,3         41.2
          Medium               46.2         52.6        45.0         52.9
          Dark                  9.1          7.7         6.7          5.9

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                                                                           18
 Telephone Interview  vs.  Personal  Interview
                                   o
      Responses  obtained  from telephone  interviews were found to be in agreement
 with  responses  obtained  in  person, within reasonable  limits for a great
 majority  of  questions.   The level of agreement ranged between 60% and 95%
 for telephonic  responses  compared to personal responses.  The proportion of
 responses in exact agreement varied by  question  (e.g., sunbathing, 63%,
 reaction  to  sun, 64%, type  of tan, 70%s eye color, 86%, state of major
 residence, 88%).  Just as importantly, however, the average response for
 telephone interviews was  comparable to  the average response for personal
 interviews,  in  Plan A and Plan B (see Table 5).

 Patient Responses vs. General Population Responses
      In addition to providing a test of the questions and methods of
 Interviewing, the pilot study also offers9 on a small scale, a comparison
 of responses from patients  and the general population.  It must be
 emphasized that these results are from" a small pilot study, arid not to be
 interpreted as definitive.
     All  patient interviews were administered via Plan B.   Because the
 patient group was relatively old (only five patients were under 50), averaging
 about 65 years, the comparison group used was that for Plan Bs among the
 general population over 50 years of age.  Comparisons were made separately
 for each sex group.   Table  6 shows the results for selected questions of
 particular interest.   It was surprising to find that the difference between
 comparison groups for hours of outdoor exposure were small.   A greater
surprise was  the finding that the proportion of individuals  of Scandinavian
ancestry in the patient population was  found not to be dramatically large
compared to that found for the general  population group.   Individuals of

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                                       TABLE 6      .                         Iy

 • Patients  compared to General  Population Surveys  Individuals  50 years  and  over
                                  Plan B Only

                       Telephone  Interviewing Method
 No. responses
       PATIENTS
         MALE  .

          46
             PATIENTS
              FEMALE

                35
            PLAN B over 49
                 MALES

                  33
               PLAN B over 49
                  FEMALES

                    42
Hours - Outdoors
  Weekdays     x"
               m
               a
  Weekends
                x
x
m
a
                x
21.1
16
 2.3


13.2
10
 1.6
13.8
10
 2.0


 9.9
 7.9
 1.1
26.6
20
 3.2


12.2
10
 1.1
                                                            14.2
                                                            10
                                                             1.3
8.9
7.9
 .76
Hours - Outdoors^
  Vacation     x"
               m
               cr
         41.1
         41.0
          3.0
               35.6
               29
                4.3
                 36.6
                 36
                  3.5
                   35.8
                   32.5
                    3.8
Sunbathe
  Frequently
  Occasionally
  Rarely
  Never      , __

Reaction to Sun
  No Reaction
  Some Redness
  Burn
  Painful Burn
  Not in Sun
  Other Reaction

Age of Respondent
      x"
      m
         19.6
          8.7
         21.7
         50.0
         28*3
         37.0
         17.4
         10.9

          6.5
         65.4
         66.8
               17.1
               14.3
               17.1
               5-1.4
               14.3
               37.1
               20.0
               17.1
                8.6
                2.9
               64.7
               54.5
                2.1
                  6.1
                 12.1
                 24.2
                 57.6
                 21.2
                 54.5
                 12.1
                  6.1
                  6.1
                 60.4
                 55
                  1.7
                   21.4
                    9.5
                   28.6
                   40.5
                   40.5
                   19.0
                   23.8
                   11.9

                    4.8
                   60.1
                   59.8
Type of Tan
  None
 . Light Tan
  Average Tan
  Deep Tan
  Other
  Not in Sun

Freckles
  Yes
  No
         10.9
         19.6
         43.5
         21.7
          4.3
         54,3
         45.7
               23.5
               23.5
               41.2
                2.9
                8.8
               71.
               28,
                 9.1
                 12.1
                 42.4
                 36.4
                18.2
                81.8
                   9.5
                   23.8
                   35.7
                   21.4
                   2.4
                   7.1
                  42.9
                  57.1

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                                                                             20
  Table 6 (cont'ci.)
 Eye  Color
   Blue                  57.8           54.3             51.5               40.5
   Brown                 17.8           17.1             30.3               26.2
   Green                  8.9           17.1             12.1                9.5
   Other                 15.6           11.4              6.1               23.8

 Hair Color
   Black                  8.7            2.9        .      9.1                2.4
   Brown                 58.7           54.3             72.7               57.1
   Red                   10.9            5.7  .
   Other                 21.7           37.1             18.2               40.5

 Years Worked on
 Primary Occupation
       x                28,2           27.6             27.9               21.6
       m                27.5           26               28                 22
       ฐ-                4.6            7.3              1.5                2.0
        A

 Skin Conditions
   Acne                  54.3           54.3             42.4               46.3
   Psoriasis              4.3                                                7.1

 Chemical Exposure
   Yes                   37.0            8.6             42.4               19.0
   No                    63.0           91.4             57.6       .        81.0

 Ethnic Background                      .
   Irish                 21.7           25.7             18.2               21.4
   English               34.8           28.6             18.2               14.3
   German                32.6           31.4             54.5               38.1
   Scandinavian          26.1           25.7             18.2               47.6

 Skin Complexion
 Chart
     .1                  2.2
      3                  2.2                                      .
      4                                                 .3.4                2.6
      5                  4.4            2.9  •            6.9                5.3
      6                  2.2            5.7              3.4                7.9
      7                  4.4            2.9              3.4                2.6
      8                  8.9            5.7             13.8               10.5
      9                 28.9           28.6             34.5               26.3
     10                 46.7           54.3             34.5               44.7

 Skin Color Complexion
 Chart Matches
   Exactly               11.4           11.4              3.6                7.9
  Closely               72.7           60.0       -      85.7               81.6
  Not Closely           15.9           28.6             10.7               10.5

 Chart Comparison
with SKIN
  Lighter               27.0           35.5             19.2           .    28.1
  Darker                73.0           64.5    •         80.8               71.9

-------
                                                                           21
                                   o •
Irish and English ancestry, however, did appear in greater proportions in

the patient group.  A physical characteristic which appears to predominate

among both patient, groups is freckles.
                                   •
     Since the pilot study was not large it offered little likelihood of

detecting small differences between patients and the general  population.

Thus the findings of the pilot's patient-general population study are not

unexpected.   In contrast to the pilot, the full survey in all  areas will

be greatly increased in sizes and therefore has much greater  likelihood of

detecting meaningful differences.

-------
                                                                           22
                                 REFERENCES

1.  Scotto, J., Kopf, A.M., and Urbach, F.:  Non-melanoma skin cancer among
    Caucasians in four areas of the United States.   Cancer 34:  1333-1338,
                                 •
    1974.
2.  Scotto, J.s Fears, T.R.S and Gori, G.B.:  Measurements of ultraviolet
    radiation in the United States  and comparisons  with skin cancer  data.
    U.S. DHEW, PHS, NIH, NCI, DHEW  Pub!.  No. (NIH)  76-1029, 1976,  120 pp.
3.  Fears, T.R.S Scotto, J., and Schneiderman, M.A.:   Mathematical models
    of age and ultraviolet effects  on the incidence of skin cancer among
    whites in the United States. Am. J.  Epidemic!.  105:   420-427, 1977.
4.  Scotto, J., Fears, T.R., and Gori, G.B.:  Ultraviolet exposure patterns.
    Environ.  Res.  12:  228-237s 1976.
5.  Scotto, J. and Fsars, T.R..:  Intensity patterns  of solar ultraviolet
    radiation.  Environ. Res.  14:   113-127,  1977.
6.  Fluorocarbons  and the Environment.  Report of Federal  Task Force  on
    Inadvertent Modification of the Stratosphere  (IMOS).   Council on
    Environmental  Quality, Federal  Council  for Science and Technology.
    U.S.  Govt. Print. Off., Washington, D.C.,  1975,  109 pp.

-------
                       FINAL REPORT
INSTRUMENTATION FOR MEASURING IRRADIANCE IN THE UV-B REGION
                       J. D. Rowan
                       K. H. Norris
             Instrumentation Research Laboratory
          Agricultural Marketing Research Institute
           Beltsville Agricultural Research Center
                 Beltsville, Maryland 20705
                     EPA-IAG-D6-0168
                     Project Officer:

                     R. J. McCracken
Agricultural Research, Science and Education Administration
                 U.S. Department of Agriculture
                   Washington, D.C. 20250
                       Prepared for
              Environmental Protection Agency
                        EAGER Program
                    Washington, D.C.  20460

-------
                             Annual  Report  1977

      EPA  Interagency  Program on  Biological and Climatic  Effects  (BACER)
        Instrumentation  for  Measuring  Irradiance  in  the  UV-B  Region


                                 SUMMARY

      The  responsibility  of the Instrumentation Research  Laboratory  (IRL)  in
 this  program was developing  portable instruments  for use by biologists to measure
 UV-B  irradiance in growth chambers, greenhouses,  and field plots.   A  simple
 UV-B  radiometer and two  UV-B spectroradiometers have been designed, constructed,
 tested, and put into  use in  the  UV-B research  program.   Each  of  these instru-
 ments is  now being manufactured  by  commercial  firms.

      The  two spectroradiometers  differ only in the monochromators:  one has a
 single holographic grating;  the  other, two holographic gratings  for greater
 stray light rejection.  The  spectroradiometers automatically  scan the 250-400 nm
 region in less than 5 minutes, printing a  tape of the corrected  irradiance  as
 a function of wavelength.  The input is cosine corrected by a specially designed
 teflon-bubble diffuser coupled to the input slits.   The  output of the monochro-
 mator is measured with a multiplier-type phototube and a logarithmic response
 amplifier.  The amplifier output is digitized with a digital voltmeter, and the
 digital output is interfaced with a desk-top programmable calculator.

      The design provided for the spectroradiometer system response to be  stored
 in the calculator so that, as the spectrum is  scanned, the calculator corrects
 the measured signal for instrument calibration and outputs the true spectral
 irradiance of the source being measured.

      The programmable calculator controls the operation of the spectroradi-
 ometer so that, on command, scanning is initiated and readings are recorded for
 each  nanometer interval.   The calculator prints the wavelength and irradiance
 for each wavelength interval and, at the end of the scan, reverses the wave-
 length drive and returns the monochromator to the starting wavelength.  The
 calculator is programmed to print an integral for a programmed action spectrum
 at the end of each scan.   At the completion of the scan,  the data can be stored
 on a magnetic tape, if desired,  for future analyses.

     Provision is included for a precise check of wavelength:   the spectrum
 of a miniature low-pressure mercury-arc lamp is scanned,  and the calculator
 computes the position of the 253.7 nm and the 296.7 nm lines to a precision of
i 0.01 nm.

     The performance specifications approach the requirements  for UV-B measure-
ments stated in NBS publication  #20, "Optical Radiation News," dated April 1977.
                                      iii

-------
                                     Annual  Report 1977

            EPA  Interagency Program on  Biological and Climatic Effects (BACER)
               Instrumentation  for  Measuring  Irradiance  in  the UV-B Region


            Our  UV-B measuring  instruments  developed  for this  project  meet the
        requested requirements.  The  broadband radiometer is  small,  hand-held,
        battery operated, and has fast response for rapid measurements.  The spectro-
        radiometers have the following features:

            (1)  Fast, accurate, and reproducible.

•>           (2)  Convenient, automatic  single-key-stroke operation.
i
t
V,
I            (3)  Programmable calculator-controlled scanning, Fig. 1.
r
I            (4)  Logarithmic amplifier for wide dynamic range.
                                       •
p            (5)  Calculates and prints true spectral irradiance for each nm
if                wavelength, Fig. 2.

I
i            (6)  Calculates and prints weighting function  (Aฃ9).

j:            (7)  Programmed-wavelength-calibration check.

I            (8)  Double monochromator unit has very low stray light.
I
|            (9)  Bandwidth of 2 nm.

)'           (10)  Temperature stable.

x          (11)  Receptor has excellent cosine response.
r
[;       -    (12)  Spectra are stored on magnetic tape for efficient data processing.

           (13)  Portable—operates  on small lab cart.

           (14)  Minor disturbance of area by the "measuring head."

            Our spectroradiometer performance specifications approach the requirements
       for UV-B measurements stated  in NBS publication  #20, "Optical Radiation News,"
       dated April 1977.   These requirements are cited  in attached copy of "Making a
       UV Measurement?,"  Electro-Optical  Systems Design, 9(6):17,  1977.  (page 7)

       Cosine Response for UV-B

            The spectroradiometers,  as well as other UV-B radiation meters developed
       in our program, incorporate a new  design for cosine-corrected input optics.
       We tested all available  cosine-correction schemes,  including integrating
       spheres, diffusing reflectors, and sintered-quartz diffusers.   None of these
       gave adequate performance for the  250 to 370 nm  region,  so  we developed the

-------
 teflon bubble diffuser.  The  spectral transmission of this teflon  diffuser
 is shown in Fig.  3.  Fig. 4 is a drawing of the  input optics  for the  Spectro-
 radiometers.  We  supplied a similar receptor to  our cooperators that  have  a
 Gamma Scientific  monochromator, Fig. 5.  The response of this type of diffuser
 to radiation from different angles is compared with that of a commercial
 instrument in Fig. 6.  The teflon bubble receptor provides excellent  cosine
 correction, and has a stable  surface which can be readily cleaned.  This
 type of diffuser  is now used  in commercial instruments.

 Wavelength Accuracy

     To check spectroradiometer wavelength accuracy, a low-pressure mercury-
 arc lamp is scanned and the mercury line location computed.  The wavelengths
 of the 253.7 and  the 296.7 nm lines are measured with a readout precision
 of +_ 0.01 nm, so  that wavelength shifts as low as 0.02 nm are readily detected,
 Fig. 7.  This field check can be made quickly and routinely between measurements
 if desired.

 Spectroradiometers

     Our UV-B Spectroradiometers are now used routinely by technicians to
measure UV-B irradiance in growth chambers, greenhouses,  and field plots.
These instruments are identified by the acronyms:  IRLSpec-S for the single
monochromator version;  IRLSpec-D for the double monochromator; and IRLSpec-SO
 for the commercial model single monochromator.   Numbers shown on the graphs—
 for example,  1770815.09—identify instrument (first digit), year (next two
digits),  month,  day,  and scan number for that day.   The first digit may be
1, 2, or 3 for IRLSpec-S, IRLSpec-D,  or IRLSpec-SO,  respectively.

     Measured performance specifications for the IRLSpec-S spectroradiometer
are as follows:

                  Wavelength range  -  250 to 370 nm
                  Readout interval  -  1  nm
                  Scanning speed -  0.5  nm/sec.
                  Spectral bandpass -  2  nm
                  Wavelength reproducibility -  +_ 0.02  nm
                  Wavelength accuracy  -  +_ 0.05  nm at  296.7 nm
                  Radiometric  reproducibility  - +_ 2%
                  Radiometric  accuracy  - +_ 5%
                  Radiometric  range - 0.001 to  2000 mW-m~2-nm~^
                  Stray light  - less than 2 x  10~4  at  285  nm as  tested
                   with a xenon arc lamp filtered with 0.5-mm-thick
                   cellulose  acetate
                  Cosine correction - within +_ 10%
                  Output - printed  tape  direct  reading  in  wavelength
                   and corrected irradiance at each nanometer interval
                   from 250 to 370 nm
                  Size  of measuring head -  less than 28 x  20  x 10 cm
                  Temperature  stability  less than 0.04 nm  wavelength  shift
                   for 25ฐC temperature change.

-------
      The IRLSpec-D spectroradiometer,  with double-grating ir.onochromator, •
 provides better stray light rejection.  This unit incorporates all the
 features of the single monochromator unit although the measuring head is,
 Of necessity,  slightly larger.   Observed wavelength instability of the
 double monochromator was  caused by grating-sync-drive cable temperature
 sensitivity.   Our technician,  George Button, solved this temperature insta-
 bility with his cable-stringing technique.   The grating-drive cable spring
 was eliminated, and the cables  were crossed to provide automatic temperature
 compensation and wavelength stability.

      Measured  performance specifications for the IRLSpec-D spectroradiometer
 are as follows:

                   Wavelength range - 250 to 370 nm
                   Readout interval - 1 nm
                   Scanning speed - 0.7 nm/sec.
                   Spectral bandpass -  2 nm
                   Wavelength reproducibility - +_ 0.02 nm
                   Wavelength accuracy  - +_ 0.05 nm at  296.7  nm
                   Ra
-------
      Measured performance specifications for this commercial IRLSpec-SO
 spectroradiometer are as follows:

                   Wavelength range - 250 to 370 run                        .  ,
                   Readout interval - 1  run                                 •  .
                   Scanning speed - 1.0  nm/sec.
                   Spectral bandpass - 2 nm
                   Wavelength reproducibility -  +_ 0.02  nm
                   Wavelength accuracy - +_ 0.05  nm at 296.7  nm
                   Radiometric reproducibility - +_ 2%
                   Radiometric accuracy  - +_ 5%                ,
                   Radiometric range - 0.001 to  200 mW-m~2-nm
                   Stray  light -  less than 1 x 10~4 at  285 nm as  tested
                    with a xenon arc lamp filtered with  0.5-mm-thick
                    cellulose acetate
                   Cosine correction - within +_  10%
                   Output - printed tape direct  reading in wavelength and
                    corrected irradiance  at each  nanometer  interval from
                    250  to 370 nm
                   Size of measuring head  -  less  than 25  x 21  x 13 cm
                   Temperature stability -  less  than 0.04 nm shift for
                    25ฐC temperature  change.

Spectroradiometer  Sensitivity and  Dark  Current

     The photomultiplier-tube  dark  current,  system correction, and standard-
lamp spectral irradiance  are  plotted  in Fig.  8 for the IRLSpec-S with
Hamamatsu R166 phototube  (solar blind response)  and Corion  SF-1.25 solar
blind filter.  The IRLSpec-D photomultiplier  dark  current (Hamamatsu R212, S-5
response and Corion SF-1.25 solar blind filter), system correction, and
standard lamp spectral irradiance are plotted in Fig.   9.   Plots of the IRLSpec-
SO photomultiplier-tube dark  current  (Hamamatsu R166,   solar blind response,
and Corning 7-54 filter),  system correction, and standard lamp spectral irradi-
ance are shown in Fig. 10.

     The instruments developed in our program are now manufactured.   A broad-
band UV-B radiometer is available from Optronic Labs,   Inc.   Spectroradiometers
similar to ours are advertised in Optical Spectra, 12(1):13,51, and 60,  dated
January 1978, and are available from three firms:

                                 EG&G, Inc.
                                 35 Congress Street
                                 Salem,  Massachusetts  01970

                                 Gamma Scientific, Inc.
                                 3777 Ruffin Road
                                 San Diego, California  92123

                                 Optronic Labs,  Inc.
                                 7676 Fenton Street
                                 Silver  Spring,  Maryland  20910

Irradiance Spectra

     Typical spectra from our IRLSpec-D are plotted in Fig.  11, 12,  13,  and 14;
from our IRLSpec-S, Fig.  15 and 16; and from our IRLSpec-SO, Fig. 17.

-------
      Irradiances of FS40 and FBZS40 lamps are compared in Fig.  11.   A typical
 curve for an FS40 lamp with and without a cellulose acetate (CA)  filter is
 shown in Fig.  12.   CA filters with FS40 lamps are compared in Fig.  13.   Measured
 irradiances of four FS40 lamps used to enhance the sun's UV-B at  Snowmass,
 Colorado's 2980 m (9777 ft.)  site, are plotted in Fig.  14.   The fixtures were
 two lamps mounted in pairs at a Z of 110 cm and filtered with 0.127-mm (0.005-in.)
 CA.   The change in irradiance of a FBZS20 lamp after aging 16.5 hr.  is  shown
 in Fig.  15.   The irradiance measurements of two Sylvania F15T8  CW lamps at
 Z = 8 cm are compared for the IRLSpec-S, Fig.  16;  the commercial  IRLSpec-SO,
 Fig.  17;  and the IRLSpec-D, Fig.  18.   Irradiance for a  15-watt  daylight lamp
 is shown in Fig.  19.

      Spectral  data of the sun at Beltsville,  Maryland,  during the year  are
 plotted  in Fig.  20 and 21.   The sun curve on  a very clear day illustrates the
 stray light at wavelengths below 289 nm for the IRLSpec-D,  Fig. 22.   The action
 spectra  of measured Beltsville sunshine with  weighting  function 9 are shown
 in the region  of 294  to 318 nm at the bottom  of Fig.  20.

      The  weighting function plotted on the  graphs  is  the  AE9  equation developed
 in cooperative research at BARC:
                =   I 0.25(A/228.178)9'ฐJ       exp   [ 4-(A/228.178)9-ฐJ
The equation was used to compute UV-B sun equivalent  (UVSEB), indicated on the
graphs.  Details of its development are presented in the Agricultural Equipment
Laboratory report.

     Spectral irradiance data are now plotted automatically with the HP9815A
calculator interfaced directly and controlling a HP9872A plotter.  After the
data are recorded on the cassette, selected scans are loaded into the calculator;
and the plotter draws and labels the graph to programmed dimensions, and then
automatically plots the data  (Fig. 11, 12, 13, and 14).  A curve is plotted from
250 nm to 370 nm in 25 seconds.

Narrow-band Radiometers

     Two narrow-band, probe-type radiometers were commercially manufactured to
our specifications.  These radiometers have solar-blind photo-multiplier-type
detectors (Mfg.  No. R166), a peak response at 294 nm and 299 nm, respectively,
and  a half-band width of 5 nm.  Irradiance readout is a 3 1/2 digit display
located in the remote electronic unit with switched ranges from 10~^ to 10~H
        O    1
watt'cm~^'nm~J-.   The small sensor head (5x5x16 cm),  with teflon dome receptor,
is attached by 2.75-m cable for easy placement within growth chambers.

Broad-band UV-B Radiometers for Routine Monitoring

     A small battery-powered radiometer,  the IRLMeter, was designed and con-
structed for measurement of total UV-B radiation from artifical sources.   The
instrument has a solar-blind vacuum photo-diode (Hamamatsu R403), an integrated
circuit amplifier,  and a microammeter packaged in a  meter case.   The circuit,
as shown in Fig.  23, provides for four decades of range switching.   The relative
wavelength response, as shown in Fig.  24,  peaks at 300 nm and is relatively flat
from 280 to 320 nm and the radiometer is  not sensitive to radiation longer than
400 nm.  Typical comparative data of the  IRLMeter with the IRLSpec-S spectro-
radiometer are shown in Fig. 25.

-------
      This broad-band UV-B radiometer sensitivity is adjusted to a full scale
 reading of 2.0 UV-B sun equivalents, Beltsville (UVSEB)  when measuring the
 output of FS40 fluorescent lamps  filtered with 0.127-mm (0.005 in.)  CA.
 Correction factors  were developed for use with FS40 lamps  with 0.254-mm
 (0.010 in.)  CA filter,  and for use with B2S fluorescent lamps.

      The IRLSun-meter radiometer  is a modification  of the  IRLMeter.   The new
 calibration reads directly in sun equivalents,  Beltsville  (UVSEB. Aฃ9).   Cali-
 brations for two suns (UVSEB  Aฃ9)  full scale were developed  for four sources:

      (1)   Sunshine  for  a clear day (6/8/77),  1:00 p.m.  EOT

      (2)   FS40 lamps  filtered with 0.127-mm (0.005  in.)  CA

      (3)   FS40 lamps  filtered with 0.254-mm (0.010  in.)  CA

      (4)   FBZS40 CLG  lamps  (unfiltered)

      A commercial version of  the  IRLMeter  has been  manufactured,  the Optronic
 Model 725.   These Model  725 radiometers were  calibrated  by IRL  for our  coopera-
 ting  laboratories from  IRLSpec-D  irradiance values  for five  sun equivalents
 full  scale, with FS40 source  filtered  by 0.127-mm (0.005 in.) CA  aged 6 hr.
 A calibration  scale factor was developed for  0.191-mm  (0.0075 in.) CA,  0.254-mm
 (0.010.in.) CA, 1.52-mm  (0.036 in.) petri  dish, and FBZS40 and FBZS20 lamps.

      An Optronic Model 725 broad-band  radiometer was calibrated from IRLSpec-D
 irradiance values for 10 UVSEB full  scale with a Rayonet F8T5 RPR3000A  (8 watt)
 lamp  source  filtered with 0.127-mm (0.005  in.) CA supplied by the Peoria coop-
 erators and then aged 6 hr.   This  Peoria CA filter absorbed  less  at shorter
 wavelength than Beltsville CA, Fig.  26, 27, and 28,  significantly changing the
 Aฃ9 action integral.

      Rayonet F8T5 RPR3000A  (8 watt) lamps have strong energy in the 254 nm
 region, the energy approaching the peak value at 313 nm  (ratio 1.4).   The peak
 energy of the FS40 at 313 nm  is approximately 700 times the energy at 254 nm.
 This  254 nm energy can be removed with CA filter, but it probably accelerates
 the aging of the CA filter.  The irradiance of the Rayonet RPR3000A with the
 CA filter at 20 cm approximates the energy of the FS40 with CA filter at 50 cm
 and 75.1 cm, Fig.  29, 30, 31,  and  32.

Eye Protectors

     Personal eye protectors were evaluated for their UV-B attenuation,  and
two FS40 lamps at 50 cm from the IRLSpec-D spectroradiometer receptor were used.
An aperture through black cloth was necessary to prevent significant leakage
between lens and receptor, even though they were placed as close as possible.
Plots of plastic goggles No. 6, 7, and 8 (dashed line)  are at the 0.001 irradiance
 level in Fig. 33.   No. 5 was a pair of plastic goggles, and No. 2, a plastic
 face shield.  UV-B blocking of prescription eyeglasses (glass and plastic)  and
sunglasses are shown in  Fig. 34.

-------
 MILT.* S. KIVKB
Tin- Engineering Magazine of Electro-Optical and Laser Technology
June, 1977. Vol. 9, No. 6
        SKILNYK
            & Vice President
 Kditnr

 WILLIAM S. HUDSPETH
 Atxotiate Editor

 WILLIAM D. ASHMAN

 Lois ESMAIL
 Circulation Manager

 CKNE BRIKSKE
 Marketing Services
 EDITORIAL ADVISORY BOARD
 L. lieiser, Consulting Physicist
 I). Belforte, Aveo Everett Research Lab I
 R. Buzzard, Lexel
 D. Casasent, Carnegie-Mellon Univ.
 W. Hunter, Naval Research Lab
 G. Klauminzer, Molectron
 H. Lavin, General Electric
 D. Lockie, Watkins-Johnson
 M. Pasture!, Coherent Radiation
 W. Ruderman. Interactive Radiation
 R. Watson, ฃGiซn.. |ni..  Published mimlhly  by Miltnn S.
 Kivpr Publications, Inc. Printed in U.S.A. Control- t
 lซi circulation paid at Pontiac. Illinois and Chicago,'
 llhnoi. 60607. Subscrintion price: $3 per copy, $20
 pf  year. POSTMASTER: Send  Form 3679 to,
 tl.ECTRO-OPTlCAL SYSTEMS DESIGN, 222 West
 Adims. Chicago. III. G060S. (312) 263-1866.
           OFFICIAL PUBLICATION
•JUNE. 1977
      4
      4
      6
      6
      6
      Q
     12
     12
     12
     13
     14
     17
     17
Laser "Writes" in Liquid Crystal
And Now: The Free Electron Laser
The Latest in Wrist Wear
Start Clicking for Nikon's Photo Contest
Heat-Generated Ultrasonic Waves
c..f..-n O,I~Kป~~<. ซ~. i /irvซ.      „ 	
Quality Control Session to be Featured
Coherent Laser Radiation Shrinks to 38 nm
Unconventional Form for CRT Terminal
Need a Laser Diode?
Instant Movie Camera Debuts
BRH OKs First Laser Safety Standard Variance
Making a UV Measurement?
 taking a UV Measurement?
 Washington, D. C. — The increas-
 ing interest in ultraviolet  optical
 measurements has spurred  the
 National Bureau of Standards' Op-
 tical  Radiation and Radiometric
 Physics groups to  look into  the
 matter.  (A special chapter of the
 NBS  Self-study Manual on Optical
 Radiation Measurements will be
 devoted to making state-of-the art
 accuracy UV measurements and is
 planned for later this year.) Over
 the past few months, NBS has in-
 vestigated and characterized sev-
 eral  currently  available spec-
 troradiometers suited  for  use in
 the UV, in a program sponsored by
 the EPA.
  To summarize their findings (see
 Optical  Radiation News, April
 1977), the current state-of-the-art
 for spectral  irradiance measure-
 ments in the 250-350 nm range var-
 ies widely. The uncertainty for a
 highly controlled laboratory mea-
 surement of radiation of a simple
 character is estimated at about 3%.
 At the  other extreme, the mea-
 surement of very complex  radia-
tion under very unfavorable field
conditions could hit 26%. Typical
                            uncertainties for lab and field are
                            estimated to be about 6% and 15%,
                            respectively.
                              What can be done to improve
                            these "best" UV measurements?
                            NBS believes that what is needed
                            most is a field-portable  spec-
                            troradiometer with  some  impres-
                            sive characteristics. This instru-
                            ment would not only have negligi-
                            ble out-of-band leakage (=* 10~8) and
                            high wavelength accuracy (— 0.03
                            nm) but would also be much less
                            sensitive  to  temperature  and
                            mechanical shock, be hermetically
                            sealed and have an easily replace-
                            able window on its entrance port.
                            Difficult field measurements could
                            be made to within 10% uncertainty
                            with such an instrument. NBS also
                            suggests  microprocessor  control
                            for this device.
                              (Ed. note: The technology for such
                            a device exists. But such a spec-
                            troradiometer would necessarily be
                            a "big ticket" item even if we disre-
                            gard the mechanics of just how the
                            development expenses are amor-
                            tized. There is some question of
                            whether the potential market is big
                            enough to be able to  absorb the de-
                            velopment costs.)O
                                L
                                                                                                     -47

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                                LIST OF FIGURES

 Fig.  1  -  Spectroradiometer components

 Fig.  2  -  Typical  UV spectra

 Fig.  3  -  Teflon spectral  transmission

 Fig.  4  -  IRLSpec-S  receptor optics

 Fig.  5  -  IRL receptor  optics for gamma

 Fig.  6  -  Receptor cosine  response

 Fig.  7  -  Hg  line  wavelength check -  IRLSpec-D

 Fig.  8  -  IRLSpec-S  sensitivity  and dark  current

 Fig.  9  -  IRLSpec-D  sensitivity  and dark  current

 Fig.  10  -  IRLSpec-SO sensitivity and  dark current

 Fig.  11  -  Lamp spectra  (unfiltered) FS40,  FBZS40CLG1076 and weighting
             function 9

 Fig.  12  -  Lamp spectra  FS40  and  cellulose acetate 6.5 hr.  Z = 30 cm,
             Z = 50 cm

 Fig.  13  -  Lamp spectra  FS40  and  0.127 mm,  0.191 nan, 0.254 mm cellulose
             acetate

 Fig.  14  -  Lamp spectra, Snowmass,  Colorado

 Fig.  15  -  Lamp aging -  BZS20CLG0377

 Fig.  16  -  Sylvania F15T8 CW  lamp spectra  - IRLSpec-S

 Fig.  17  -  Sylvania F15T8 CW  lamp spectra  - IRLSpec-SO

 Fig.  18  -  Sylvania F15T8 CW  lamp spectra  - IRLSpec-D

 Fig.  19  -  GE F15T8D  lamp spectra - IRLSpec-D

Fig.  20  -  Sun spectra - Beltsville, MD

Fig.  21  -  Sun spectra - Beltsville, MD -  IRLSpec-S

Fig.  22  -  Sun - Beltsville, MD - IRLSpec-D

Fig.  23  -  UV-B IRLMeter  circuit

Fig.  24  -  IRLMeter  spectral response

Fig. 25 -  Regression IRLSpec-S vs. IRLMeter

-------
Fig, 26 -  Rayonet F8T5 RPR 3000Aฐ spectra new cellulose acetate, Z = 16.3  cm

Fig. 27 -  Rayonet F8T5 RPR 3000Aฐ spectra cellulose acetate aged 6 hr., Z  =
             16.3 cm

Fig- 28 -  Rayonet F8T5 RPR 3000Aฐ spectra cellulose acetate aged 6 hr., Z  =
             20 cm

Fig. 29 -  Rayonet F8T5 RPR 3000Aฐ  and sun  (Beltsville) spectra

Fig. 30 -  Spectra FS40 and Rayonet F8T5 RPR 3000Aฐ

Fig. 31 -  Spectra FS40 and cellulose acetate aged 0.5 hr., Z =.50 cm

Fig. 32 -  Spectra FS40 and cellulose acetate aged 6 hr., Z = 50 cm

Fig. 33 -  Spectra - eye protective device

Fig. 34 -  Spectra prescription eyeglasses and sunglasses

-------
 r
           TEFLON RECEPTOR
                 SINGLE

                 MONO-
               CHROMATOR
 I
1
 j	  ^LOTฃMEIฃR 11  _         	 	I
                  A/D
               INTERFACE
                  +
                 POWER
                 SUPPLY
         I
             PROGRAMMABLE
              CALCULATOR
        UV-B  IRLSpec-S
                            3-31-77
Fig.  1. Spectroradiometer components
                             r
Jy1E_ASURINฃ

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        DOUBLE

        MONO-
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                                                                                ^
                                                                                 MOTOR
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                                           INTERFACE
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                                             POWER
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                                         PROGRAMMABLE
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                                     UV-B   IRLSpec-D
                                                                                  3-31-77

-------
FILE -17 J) 3
TYPE 2
USED 1208
MflX 1203
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2780203.09
250000, 0039
251000.0048
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253000. 1084
254000.2177
255000. 1071
256000.0087
257000. 0030
2 5 8 0 0 0 . 0 0 2 7
259000.0027
260000. 0022
2 6 1 0 0 0 . 0 024
262000. 0031
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265000. 0339
2660-00.0499
267000. 0438
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270000. 1144
271000. 1838
272000.247 3
273000.3516
274000. 5030
275000. 7620
276001.0323
277001.3153
278001.6617
279002. 1661
2 ft 0 fi fi ft . fi S ft 6
281004.0457
282004. 7257
233005.6125
28400'6.8764
285008.8472
286011.0359
287012. 6380
283014.3190
289016.9874 .
290020.5 9 7 1
291023.4209
292025.7890
293028.3414
294031.4599 \
295035.6576
296041.5418
297044.7439
. 298047.0842
2 9 9 fi 4 9 . ft 5 5 6
300052.6190
301057.2066
302060. 8413
303063.8780
304065. 5053
305067.2258
ft H f. fi 6 ft . 959 9

307071 . 0456
308073. 4446
309074.8453
310075.3275
311078.3118
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313095. 7436
314095. 1361
315078.5403
316074. 1841
317073. 4798
318074. 7080
319073. 8314
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3 21067. 2 6 7 5
322066. 0442
323066. 6018
324065. 1532
3 2 5 0 6 0 . 8 3 9 3
3 26057. 3 4 9 9
327055.6234
3 2 30!=; 5. ft ft fi ft

329054. 1393
330050. 1 182
331046. 6747
332044.9649
333045.0813
334044.2513 .
335040. 3120
33 6 M 36. 6656
337034. 9533
338034.4372
339032. 7606
340030. 0634
341027.7691
342026.5048
343025.9462
344024.6910 '

*

345022.5070
346020.5725
347019.5569
348019.0976
349018.1677
350016.6673
351015. 1344
352014.3364
353014.0250
354013.3521
355012.0807
3 56011.0 3 2 7
357G10. 4609
358010. 2614
359009.6726
360008.3263
ft f, 1 fi fi ft . fi 6 ft !=!
362007. 6653
3 6 3007.5 3 5 2
364014.7553
365033. 0470
3 6 6 0 2 3 .8414
3 67012. 3 3 6 3
368007.0350
369005. 1119
I NT. 9 FROM
250
80.2453
SUNS = 26.2240
SUM FROM
250
TO
369
MILLIWflTTS/MSQ
3428.0341
SUM FROM
•-' 7 fi
L. 1 U
TO
290
MILLIWflTTS/MSQ
117. 1260
SUM FROM
290
TO
320
MILLIWflTTS/MSQ
1880. 9133
SUM FROM
320
TO
340
MILLIWflTTS/MSQ
1059.7177
Fig. 2,  Typical UV spectra

-------
  1.4-
                   0.020" TEFLOW DISK
    \
^ 1.0--*
t; .8
   '200
300
400
   Fig. 3.  Teflon spectral transmission
500
600
                      MONOCHROMATOR
                      —r
                             AL. HOUSING

                              0.020" TEFLON
           FRONT
          SURFACE
          MIRROR
     rig. 4.  !RLSpec-S  RECEPTOR  OPTICS
                                              3-31-77
                         12

-------
      FRONT
     SURFACE
     MIRROR
                                 0,020" TEFLON
   GAMMA MONOCHROMATOR
rig. 5.  IRL  RECEPTOR  OPTICS FOR GAMMA
                                            3-31-77
                      13

-------
                 TRUE COSINE
                    CURVE
                 DATA POINTS (•) ARE
                 IRL COSINE RESPONSE
            TEFLON (0.020ฐ) BUBBLE RECEPTOR*
          80  SO 40 20  0  20 40  60 80

                  ANGLE - DEG.


Fig.  6.  Receptor cosine response
11-3-76
                     14

-------
 H  RUN  SPECTRH
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   2 7 8 S 2 03.81
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MERCURY  LINE  FT
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MERCURY LINE  R
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MERCURY  LINE  ft'


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 2978047.488
 2988829.754


MERCURY  LINE  RT
W. L. CRL.

  2730283.87

 2948082.192
 2950021,214
 2960043,419
 2978047.278
' 2930629= 117
                           MERCURY  LINE  RT
 Fig. 7.  Hg line wavelength check - IRLSpec-D

                 15

-------
IE-SB-
                         HAVB1OJ na IRL AK?I FR USJA



       Fig. 8.  IRLSpec-S sensitivity and dark current



                               16

-------
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                            WAVELENGTH ™ IRL AKRI FR USDA




       Fig.  9.   IRLSpec.D sensitivity and dark current
                                  11 7

-------
 1E+02
 1E+01
1E-01
1E-02
1E-03-
lE-g
1E-05-
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                                            .AMP
                                    \
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     uf   
-------
                 fllaJ ft  1 FBZS40CLG1076 lUnfilqered
                                      - 75,1 cm
                                      = 75.1 cm
                 filฎf 3 i_ES4Q_unfiU:eied
           filo* 21 1
Meiakting function
                              WAVELENGTH  n*>       IRL AMRI  ARS USDA
Fig.  11   Lamp spectra  (unfiltered) FS40,  FBZS40CLG1076 and weighting function 9

                                 *..  19

-------
                                       IRL AM FR (HJA
Fig. 12   Lanp spectra FS40 and cellulose  acetate 6.5 hr.  Z ป 30cm, Z = 5Cbm
                                  . 20 •<

-------
I
 E

3*
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LU
O
           2770923, ฉ3 filofl 3 I_J^4J)jinfiltered

           277@92aง7 filotf 4 i^JSAHJ- O^IO" CA-8h

           277S92155 filo^ 5

                            6 1—

                filol? 21 1Wln8 function (9)
WAVELENGTH  nm
                                                         IRL AMRI ARS  USDA
    Fig.  13  Lamp spectra  FS40 and 0.127 mm,  0.191 mm, 0.254 mm cellulose acetate

                                            21

-------
                           Snowmass,  Colorado
                                                                              „ ,   .
                                                                              Z(cm)
0.001
                                            C53     SJ    (S3


                        CM     CM    CO     CO     CO    CO


                         WAVELENGTH nm  IRL  AMRI  FR USDA




                        Fig.  14.   Lamp spectra,  Snowmass, Colorado



                                            22   ,
CO

-------
I  i
           Fig. 15.  Laiap aging  - BZS 20 CLG0377

-------
2-3
LJ - .'„
a a -:•
N < >
                                          J  >?rn
                    Fig. 16    Sylvania F15T8 CW lamp spectra - IRbSpec-S

                                                   24

-------
.00/1
               Fig.  17.  Sylvania  F15T8 CW lamp spectra IRLSpec-SO
                                            25

-------

.901.
                Fig. 18.  Sylvania F15T8 CW lamp  spectra  IRLSpec-D
                                           26

-------
    I.
                 go   90  300  /
                X YI
,ooo(
                                            770S-I9-7D

                                         XRLSpec-& : U v- B .
                                          B.:W.  ir  z 71 yn;.
                                         z. GE  FISTS  p::. .;:

                                          (g>  S  cm/^lz/- 2.37
                                          A  (SAL. 2 96.7.5;   :

                                                 - 77
                                           J  P R. . • :  ..: .  ;..::: . • ::
                                            :       .    .  . ;	
                                          IRL—  AMRl
                                          A R5  -  US
                                                                  I too.
35-0
           Fig. 19.  GE F15T8D lamp spectra IRLSpec-D

                                  27

-------
        2770630. BSfflf i ladL 76*J3 (Int. 9)_18
                          5*_0(Int_91_L
                            flCTnfc fl)
0. 001 ,
                           .   WAVELENGTH nm
               Fig. 20.   Sun spectra  - Beltsville, MD
                                      28
IRL AMRI.ARS USDA

-------
                                                                          'e&e
Fig. 31.  Sun spectra - Baltsville, MD - IRLSpec-S

-------
    R I i  ;—:—;_i
    'M444-l-H-i
                                                      V.  L L Ei. M b
                                                     IRLSpec-D UV-B
                                                      Z Y\ m  B w
                                                    IRL-AMRI-ARS-USDA
                                                    77P630-

.OO9|
                                            2.0   30   40   356  go
                  Fig.  B2.  Sun - Beltsville,  MD -  IRI-Spec-D
                                          30

-------
                                          + 4.05V
  !00-
   10-
>-
fr-
Cft

-g.
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                                               2-7-77
               RESPONSE  OF  UV~B IRL METER
               PT-R403 "  0-0625 TEFLON  DISK
               t   1
                       I   I   I
                                  t   I  I _ 1   i  I
         220   240  260   280
300  320   340   360

Xnrn
380  400
               Fig.  24-  IRLMeter spectral response
                               31

-------
                                                         ND.  3-J1-2O  DIETZGEN  GRAPH  PAPER

                                                                    2O X 2O PER  INCH
EUGENE DIETZGEN CO.

     MADE IN U. 5.  A.
U)
to

-------
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                                                                 ^
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110.5
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16.3
16.3
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-------
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    10.000
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                             WAVELENGTH nm IRL AMRI FR USDA

         Fig.  27.  Rayonet F8T5 RPR 3000Aฐ spectra cellulose acetate  aged 6 hr.,
                      Z = 16.3 cm
                                              34ซ

-------
                                                                    AE9  Zfcnd
         2Z11103J5.  FILE 0-3z
         2221103.20. EILEJht3L
0.601
                                                                       oa
                                                                 in    ID
                                                                 CD    CO     CO
                        WAVELENGTH nm IRL AMRI FR USDA
            Fig.  28. Rayonet F8T5 RPR 3000Aฐ  spectra cellulose acetate  aged 6 hr.
                     Z = 20 nm
                                        35

-------
•S3.
 IS
                                                                        5.67
                                                                   =  95.1  Zฐ20
2778330,09 fila? 76 S
       11 filo# 14 1_
77B9S2o IS f iiofl 15
     filฎ# 21
                  1:15 EDT
01d_Rayonet  //I  Unfiltered
           tfl +
                                                             CO    CO

                                  ' WAVELENGTH nm        IRL AMRI ARS  USCA
             Fig.  29.   Rayonet FOT5 RPR 3000Aฐ and  sun  (Beltsville) spectra
                                            36

-------
 ฎ 1
 lฃj j[

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      1    .
                      4-
277S923.B8
77S^2.11 filel 14 U
77B9S2.10-file# 15 l_oid

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                             ^ 4  rsmj umiltered
                                 Old Rayonet #1 unfiltered
        4*8.1  !Z~^   7^5.1 c
        95.1  Z  = 20 cm ,
                                             h + O.OOSA'n, CA-lOh  AE^= 7.43
                                                                             20 c
                                            1:15 EOT
                                                                    = 5.67

        ID    CD
        CM    (M
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             Fig.  30.   Spectra  FS40 and Rayonet B8T5 RPR 3000Aฐ
                                            37

-------
                          FS_40+Peo
                          FS40+Bel
                          Weiahtin
               WAVELENGTH nm IRL AMRI FR USDA
Fig.  31.  Spectra FS40 and cellulose acetate aged 0.5 hr. Z = 50 cm
                                  38

-------
                                                                      AM   7. (am)
                        FI1F
             2H1102JZ. FJLE
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            Fig.  32- Spectra FS40 and cellulose  acetate aged 6 hr  Z = 50 cm
                                              39

-------
       IOC
2 ; .
u - •
o -• :
N < :
j: ^- .

0 C
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d l'l
                                                                                   IRLSpec-D
                                                                              B w = 2 n
                                                                           ; -7 7ซ603-20pHi2->|-l 17. u
                                                                          : 77dfi03rl7DI
                                                                         -: Window glajsa: ' :  j •
                                                                                       AE  = 0.800J
                                                            770&03-14D
                                                                       4 1- Mylar  (0.005")
                                                         I  ....:..:.
                                                        7 r UV :Goggle |(ajnt>er>) N
                                                                          iTQYOOSl
                                                                       8 :- UV Goggle j(yellcwlsh) .
60    7o   8Q     To
                                               /a    2o
                                            A    "h  rn
~44   3^0   60    70    80   W
                                                                                      .*••
                    Fig.  33.  Spectra  -  eye protective device
                                                    40

-------
                                  IRLSpec-D UV-B
                                      W = Z
i ••• :  :- Source f- FS40
         Plabtic lens (Nancy) :
         Glabs lens (Don)
       : :770B03-11D
     3-- Glass lens (George)
  •  : --f v770ioP3-;-lDp
   : ~5f r-;Refaectlve
      i .:(Randy)
     60    70 I  80
           Fig. 34.  Spectra prescription eyeglasses and sunglasses

                                         41

-------
                                APPENDIX A


Figure

Ala  - Ale   -  Operate Program,  IRLSpec-D

A2 - Calibrate Program,  IRLSpec-D

A3a  - A3b - Operate Program, IRLSpec-S

A4a  - A4d - Operate Program, IRLSpec-SO


     Spectral Response Curves

AS - Standard Lamp Plotted Each nm

A6 - FS40, FBZS40CLG1076, Sun,  IRLSpec-S

A7 - Filtered FS40 and FBZS40CLG1076, IRLSpec-S

A8 - FS40, FBZS40CLG1076, Z = 73 cm, IRLSpec-D

A9 - FBZS40 WLG, FBZS20 WLG, Vitalite, IRLSpec-D

A10 - F15T8 CW Westinghouse, IRLSpec-D

All - F15TO CW Westinghouse, IRLSpec-SO

A12 - F15T8 CW Sylvania,  IRLSpec-D

A13 - F15T8 GRO-LUX Sylvania F15T8 WW GE, IRLSpec-D

A14 - F40R GE, F40IR Westinghouse, F1.5T8R GE, Z - 50 cm, IRLSpec-D

A15 - F40IR Westinghouse, Z = 20 cm, IRLSpec-D

A16 - F15T8BL Sylvania,  F15T8BLB GE, IRLSpec-D

A17 - F40 BL + FBZS40 CLG1076,  IRLSpec-D

A18 - F20T12/2021 Sample  No.  3176-2, IRLSpec-D, IRLSpec-SO

A19 - F20BL Phillips phosphor,  F4T5BL conventional phospher,
        IRLSpec-S
                                   42

-------

fill TO STflRT
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        Fig. Ala.   Operate  Program  IKLSpec-D   (Cassette D4)
                         43

-------
0125 LINE
0126 H
0127 -
0128 S
0 1 2 9 U
0130 H
0131 S
0179 ttREGS
0180 G U T 0 G 6 9 5
0182 CLERR
0183 6
0184 + ฃ-
0185 ENTER!
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0 1 3 3 H ! 0 1 8 9 S T 0 P
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0135 C 	 C
0136 0 ' 0192 LBL
0137 H . 	 C
0 1 3 8 T ; 0 1 9 4 F R N T <:•:
0139 . i 0196 N
0140 0197.
0141 R 0198 L
0 1 4 2 E . i 0 1 9 9 .
0 1 4 3 H i 0 2 0 U
0144 B f fi201 f:
0145 LINE 0202 H
0 1 4 6 E I 020 3 L
0147 N 0 2 H 4 .
0143 T
0149 E
0150 R
0151
0152 Y
0153 R
0154 .
0155 M
0 1 5 6 0
i 0157 .
0153 D
0159 fl
0160 V
; 0161 #
i 0162 END*
0163 STOP
0164 FIX 2
0 1 6 6 2
0167 EEX
0:68 6
0169 +
0170 S T 0 R 0 ij 0
0172 PRINT
0173 5
0174 MfiSK 4
0176 1
0177 5
0173 1
0 205 E N H *:
0 2 U 6 5
0 2 070
0203 STO F
0209 SFG 2
0210 LBL
---- D
0212 LBL
• 	 D
0214 FIX 2
0 216 .
02170
0213 1
0219 S T 0 + R 0 O 0
0 2 2 1 R C L R 0 U 0
0223 PRINT
0224 FIX 4-
0226 1
'0227 S MJ fl
0228 IF SFG 2
0 2 2 9 G 0 T 0 G' 2 3 5
0231 1
0 2 3 2 2
0 2 3 3 0
0234 STO F
02353
0 2 3 6 9
0 2 3 7 S T 0 G
0233 1
0 2 3 9 E N T E R t
O 2 40 0
0241 + $-
0242 LORD
0243 5
0244 NBYTE
0 246 R E R D
0248 EHTERt
0249 4
0 250 + r -
0251 IF X=Y
0 2 5 2 G 0 T 0
0 2 5 4 G 0 T 0
U 2 5 6 3
0257 WBYTE
O 2 5 9 F 0 R
0 2 6 0 1
0 2 6 1 S T 0
• 0262 RERD
0264 IF 0
O 2 6 5 L 0 T 0
O 2 6 7 G 0 T 0
0269 X ฃ Y
0270 IF S F G
0271 G 0 T 0
O 2 7 3 S T U •
0274 6
0275 WBYTE
0277 FOR
0278 RERD
0 2 3 0 X ^ Y
0 2 3 1 S T 0 +
0232 NEXT
0233 3
0284 WBYTE
0236 RCL
0287 RCL I
0 2 3 9 -
0290 1 0 1 X
. 0291 STO I
0293 CFG
0294 RCL
0 2 9 5 2
0 2 9 6 4
0297 9
0 2 9 8 +
0299 ENTERt
0 3 U 0 1
0301 EEX
0302 3












(-
r


l-

r.
^

r
f

j
r
(

J
t
t

r
E.

4
i_
h


H
4
R








Fig. Alb.  Operate Program IRLSpec-D    (Cassette D4)   (cont'd)
                         44

-------
 .i_-< *^ ...,,'ปa*aฃซ.,-i
0303 *
0 3 04 +
0305 PRINT -
0306 IF CFG 3
0 3 0 7 G 0 T 0 0 3 1 6
0 3 O 9 .
0 3 100
0311 1
0 3 1 2 S T 0 - R O 0 U
0314 G 0 T 0 0317
0316 NEXT R
0317 1
0313 WBYTE 4
0320 CFG 3
0321 RCL fi
0 3 2 2 8
0 3 2 3 U
0324 -
0325 IF +
0326 GOTO 1
0323 IF SFG 2
0329 GOTO K
0331 RETURN
0332 LBL
	 	 F
0334 3
0335 WBYTE 4
0337 RETURN
0338 LBL
	 p
0340 CLEfiR
0341 WBYTE 4
0343- RETURN
0344 LBL
	 B
0346 1
0347 WBYTE 4
0349 1
0350 RETURN
0351 LBL
	 I
0353 PRNT*
0 3 5 5 I
0356 N
0357 T
0 3 53.
0 3 5 9 9
0 3 6 0
0361 F
O 3 6 2 F:
0 3 6 3 0
.0364 N
0365 END*
0 3 6 6 2
0 3 6 7 8
0 3 6 3 0
0 3 6 9 S T 0 P
0370 F I X 0
0372 PRINT
0373 ENTERt
0374 2
0375 4
O 3 7 6 9
0 377 -
0 3 7 8 S T 0 R
6 3 7 9 8
0 3 8 O 0
0 3 8 1 S T 0 F
0332 CLERR
O 3 8 3 S TO. I
O :I 3 4 F 0 R fi * F
0C-85 RCL fi
O :I 8 6 2
0 :1 8 7 4
0 '~i 3 3 9
0 3 3 9 +
0 3 9 U 2
0391 2
0 3 9 2 8
0393 .
0 3 9 4 1
0 3 957
O ''- 9 6 8
0 :] 9 7 -
0 :: 9 8 9
U :: 9 9 Y t X
0 4 0 0 S T 0 H
0401 4
O 402 -i-
0 4 0 3 4
0404 Y t X
0 4 0 5 S T 0 . J
0406 RCL H
04074
0 4 0 8 -
0 409 + ฃ -
0410 e t X
0411 R C L J
0412 *
0413 RCL I 'R
0415 *
0416 R C C +
0417 NEXT R

0413 RCL I
0419 FIX 4
0421 PRINT
0422- ENTERt
042 3 3
0424 .
0 4 2 5 0
0 4 2 6 6
0427 r
0423 PRNT*
O 4 3 0 S
0 4 3 1 U
0432 N
0433 3
0434
0435 =
0436 PRINT
0437 END*
0 4 3 8 G 0 T 0 H
0440 LBL
	 fi
0442 .
044 3 0
0444 1
0445 3 T 0 + R 0 0 O
0447 FIX 2
0449 RCL ' R000
0451 P R I N T
0452 FIX • 3
0454 1
0455 STO H
0456 1
0457 5
045 3 0
0 4 5 9 S T 0 F
0460 4
O 4 6 1 0
0462 3 T 0 G
0 4 6 3 1
0464 S T 0 B
0 4 6 5 5
0466 WBYTE 4
0463 REflB 4
0470 ENTERt
0471 4
0472 + i -
0473 IF X=Y
0474 G 0 T 0 047 8
0 4 7 6 G 0 T 0 0
0473 3
0479 WBYTE 4
O 431 F 0 R H -* F
Fig. Ale.   Operate  Program  IRLSpec-D   (Cassette  D4)   (cont'd)
                                        t




                            45

-------

0432 RERD 4
(3484 IF 0
6 4 3 5 G 0 T 0 0 4 8 9
0 4 3 7 G 0 T 0 0
0 4 8 9 X ฃ V
0490 IF SFG 4
0491 G 0 T 0 0 5 3 0
0 4 9 3 C L. ERR
0494 8 T 0 C
0 4 9 5 6
0496 MBYTE 4
0493 FOR B + G
0 4 9 9 R E R D 4
0501 X ? Y
0502 S T 0 + C
0503 NEXT B
0 5 0 4 3
0505 WBYTE 4
0 5 0 7 1
0 5 0 8 S T 0 B
0 5 0 9 R C L C
0510 STO+ 1 R
0512 + ?-
0513 RCL fl
0514 2
0515 4
' 051 6 9
0517 +
0513 EHTERt
0519 EEX
05204
0 521 #
0522 +
0523 PRINT
0524 IF SFG 8
0 5 2 5 G 0 T d 0 5 4 1
0527 NEXT fl
O 5 2 8 G 0 TO 0541
0530 PRNTo:
0532 0
0533 ','
0534 E
0535 R
0536 L
0537 0
0538 fl
0539 D
0540 EH Do:
0541 1
0542 NBYTE 4
0544 CFG 3
0545 CFG 4
0546 RETURN
0547 LBL
	 L
0549 LBL
	 L
0551 CLEfiR
0552 1
0553 L H & G 0
0554 LBL
	 	 i/
r\
0556 RCL R047
0553 RCL R043
0 5 6 0 H-
0561 LOG
0562 EHTERt
0 5 6 3 4
0564 *
0565 +?-
O 5 6 6 2
0 5 6 7 9
O 5 6 8 6
0 5 69.
0 5 7 0 5
0571 +
0572 FIX 2
0574 PRHTo:
0576 LINE
0577 L
0578 I
0579 N
0530 E
0581
0 5 3 2 R
0583 T
0534 PRINT
05S5 EN Do:
0536 F I X 4
0583 CFG 2
0539 3TOP
0590 LBL
	 H
0592 PRNTo:
0594 3
0595 U
0596 H
0597
0598 F
O 5 9 9 R
0 6 0 0 0
0 6 O 1 M
0 6 0 2
0603 EH Do:
0 6 0 4 3 T 0 F'
0605 F I X
0607 PRINT
0 6 0 3 2
0 6 0 9 4
0 6 1 O 9
0611 -
0612 S T 0
0613 PRNTo:
0615 T
0 6 1 6 0
0617
0613 EN Do:
0619 STOP
0620 PRINT
0621 2
0 6 2 2 4
0 6 2 3 9
0624 -
0 6 2 5 S T 0
0626 CLERR
0627 S T G
O 6 2 3 F 0 R
0629 RCL
O 6 3 0 2
0631 4 .
0 6 3 2 9
0 6 3 3 -
0634 RCL I
0 6 3 6 S T 0 +
0637 NEXT
0638 FIX
O 6 4 0 R C L
0641 P R H T o:
0643 M
0644 I
0645 L
0646 L
0647 I
0643 W
0649 R
0 6 5 0 T
0651 T
0652 3
0653 /
0654 M
U 6 5 5 S
0 6 5 6 Q
0657 PRINT
0658 ENDo:

Fig. Aid. Operate Program IRLSpec-D (Cassette D4) (cont'd)
46 ' '



\'





Fi











F

i
h
R




H
I
H
1
1
i




















r ~

-------
6bby
8 6 6 1
	
8 6 6 3
8 6 6 4
8 6 6 5
8 6 6 6
6 6 6 8
6 6 6 9
8 6 7 8
8671
8672
6673
8674
8 6 7 6
6677
6678
6679
8 6 3 6
6681
6 6 8 2
8 6 8 3
8 6 8 4
8 6 8 6
	
8 6 8 8

8 6 9 6
8 6 9 1
8692
8693
8 6 9 5
8 6 9 6
8 6 9 8
8 6 9 9
. 8781


ij U T U
LBL '
N
CLEfiR
STO
4
MBYTE
4
6
STO
1
STO
FOR
REfiD
•'• ^— V
S T 0 +
NEXT
RCL
PfiUSE
PfiUSE
C L E ft R
STO
G U T 0
LBL
U
LBL
u
B
8
i
STO-
CLEfiR
MBYTE
1
MBYTE
PRNTv:


H



c

4


G

B
B*G
4

C
B
c



c
6671







R 0 8 8

4

, 4



o 7 y o
8784
8 7 6 5
6 7 6 6
8787
8788
6 7 8 9
671 1
	
6713
	
6715
6717
6719
6 728
6721
8722
6723
6724
6725
872 6
672 3
8729
6731
8 7 3 2
6733
6734
8735
6736
8737
6 7 3 3
8 7 3 9
6746
8741
8742
674 3
6744
8745
R
E
O
E
T
EN Do:
.GOTO 6182
LBL
G
LBL
L,
R C L R 8 6 8
F I X 2
PRINT
1
STO fi
1
ฃ
M
STO F
FIX 4
FOR fi + F
RCL I fi
RCL H
i
4
4
+
ENTERt
1
EEX
.Ji
*
+
PRINT
NEXT fi
STOP
END
Fig. Ale.  Operate Program IRLSpec--D   (Cassette D4)  (cont'd)





                          47

-------
FILE I
YYPฃ i>
USED 218
f'l fl x s e 0


0880 PRHT'M
0 0 0 2 C
0 0 U 3 fl
0 004 L
0005 I
0 0 0 6 E:
0 0 0 7 R
0 0 0 S fl
0 0 0 9 T
0010 I
0 0 1 i u
0912 N
0013 LINE
0014 C
0 0 5 1
3 0 5 2
0 0 5 3 -
0 0 5 4 0
0 0 5 5
0 0 5 6 fl
0057 H .
0 o 5 s n
0059
0 0 6 0 -
0 061 1
0062 LI HE
0063 LINE
0064 LINE
0 0 6 5
0 0 6 6
U U 6 7 N
U U 6 8 0
0069 .
0 0 7 0
i _ _ .
0015 0 ! 0071 R
t
0 0 1 ۥ R [ 0 0 7 2 U
0 0 1 7 R ! 0 0 7 3 N
0013 E
0 0 1 9 C
0020 T
0021 I
0022 0
0023 N
0 0 2 4
0025 W
0 0 2 6 I
0027 L
0 0 2 3 L
0029 LINE
0 0 3 0 B
0031 E
0 0 3 2
0 0 3 3 R
0034 E '
O 0 3 5 C
0 O 3 6 0
0037 R
0 0 3 S B
0039 E
0 0 4 0 L
0041
0 O 4 2 0
0043 H
0044 LINE
0045
0 O 4 6
0047 F
0 0 4 3 I
0 0 4 9 L
0 0 5 0 E
0074 S
0075 =
0 0 7 6 '?
0077 EN Do:
0 0 7 8 3 T 0 p
O 0 7 9 S T 0 E
0080 1
0 0 S 1 S T 0 fl
0 0 8 2 1
0 0 S 3 5
0084 0 I
0 1 0 5 2
.0106 1
0107 4
0 1 0 3 S T 0 L'
0 1 0 9 .
0 1 1 0 2
0111 1
0112 4
0 1 1 3 6
0114 S T 0 H
0115 FOR fl*F
0116 RCL fl
0117 2
• 01184
0119 9
0 120 +
0121 RCL I
0122 *
0123 RCL .i
0124 +
0125 10tX
0126+?-
0127 RCL B
0123 +
0129 16t X
n 1 :-: w :-; T n f
0131 RCL fl
0 1 3 2 2
0133 4
O 1 3 4 9
0135 +
0136 RCL D
0137 -
0161 ~
0162 FTi
- •- " '. L
0163 LC-:
0164 -
0165 3Tf
fi 1 6 7 P p i ,
- *• - 1 I T ;
0 1 6 3 HF'
' i L.
0 169 1
0170 c,
0171 0
0172 Etif.
0 1 7 3 1
0174 El.'!''
01750
0176 + ? -
0177 RCIi;-
0 178 1
0 179 5
0 1 8 0 0
0181 EHTE'
0 1 8 2 1
0133 EHTE;
0 1 S 4 1
0135 + ? -
0186 RCBiV
fi 1 8 "' P P N T
- A - i 1 1 •. 1 1 !
0 1 8 9 C
0 1 9 0 H
0 1 9 1 L '
0192. .
0 1 9 3
O 1 9 4 C:
0 1 9 S n
0133 . 0196 N
0035 STO Fl 0139 5 019? s
0 0 36. 16146* '101 9 R T
0 0 8 7 0
0 0 8 8 0
0 0 3 9 3
0 0 9 O 5
0 091 + v ~
0092 STO I
0093 1
0 094 .
0 0 9 5 3
0096 .1
0 097 7
0 0 9 3 3 T 0 J
0 0 9 9 2
0 100 .
0101 3
0102 7
U 1 0 3 6
* 1 * 4 3 T U B

0141 1/X
0142 STO- f:
0143 RCL fl
0144 3
0145 1
0146 -
0147 RCL H
0148 *
0149 EHTERt
0150*
0151 + ? -
0152 8 1 X
0 1 5 3 .
0154 1
0155 + ฃ -
0156 *
0157 S T 0 + C
0153 RCL I fl
0160 RCL E

0 1 9 9 .
0 2 0 0
0 201 I
0202 N
0203 LINE
U 2 0 4 1
O 2 0 5
0 2 0 6 T
0 2 O 7 0
0 2 0 8
0 2 0 9 1
O 210 5
0211 0
0212 E H D •:••
0213 CLEH?
0214 S T 0
0215 CLEfiF
0216 L D i. b i'
0217 END

Fig. A2.  Calibrate Program IRLSpec-D
                      48
(Cassette  D2)

-------
DRTE           '•
E   UV SPECTRR
1780210.01
  OPERATE PROGRAM IRLSpec-S
0000
0001
0002
0003
OOOU
0005
0006
0007
0003
0009
0010
0011
0012
0013
001U
0015
0016
0017
0018
0019
0020
0021
0022
0023
00 2 U
0025
0026
0027
0028
0029
0030
0031
00^2
0033
OO 3 U
0035
0036
0037
0038
0039
OOUO
OOUl
OOU2
OOU3
00 UU
OOU5
00 U 6
OOU7
OOU8

yyus
0050
CHR
D
R
T
E
CHR
E
+
1
EXP
p,
SM
10
E
,— .r-i
bP
8 a
3
"7
0
SM
06
FLG
10
0
SM
00
1
U
0
SM
01
2
5
O
SM
02
f9
106
CHR
2
CHR
102
m
0
1
EM
10
r~iM
RM
10
0051
0052
0053
005U
0055
0056
0057
0058
0059
0060
0061
0062
0063
006U
0065
0066
0067
0068
0069
0070
0071
0072
0073
007U
0075
0076
0077
0078
0079
0080
0081
0082
0083
008U
0085
0086
0087
0088
0089
0090
0091
0092
0093
OO 9 U
0095
0096
0097
0093
0099

0100

CHR
IJ
V

.-
P
E
c
T
R
fl
CHR
FIX5
02
LUL
10
CHR

W
L


I
R
R
R
D



W
L



I
R
R
ft
D

CHfi

01
106
CHR


LHH

0101
0102
0103
01 OU
0105
0106
0107
0108
0109
0110
0111
0112
0113
011U
0115
tvcl "1 C
*— • JL -LC1
0117
0118
0119
0120
0121
0122
0123
012U
0125
0126
0127
0128
0129
013y
0131
0132
0133
013U
0135
0136
0137
0138
0139
01 UO
01U1
01U2
01U3
01UU
01 U5
01U6
01U7
01U8
01U9

-A 	

101
FLG
fifi
IFER
01
106
101
~
RM
11
L
IND
RM
00
™~
iex
L
IND
SM
01
SM
fi-:
102
RM
02
FIX5
fif-l
COL
0-:
RM
03
FIX5
03
COL
08
•?
01
1
EM
02
RM
02
FIX5
M0
COL
03
1 06
101



0151
0152
0153
015U
0155
0156
0157
0158
0159
0160
0161
0162
0163
016U
0165
yit't
0167
0168
0169
0170
0171
0172
0173
017U
0175
0176
0177
0178
0179
0180
0181
0182
0133
013U
0185
0186
0187
0188
0189
0190
0191
0192
0193
019U
0195
0196
0197
0198
0199

-' 	

RM
11
—
R
IND
RM
00
=
10"
R
IND
SM
01
102
FIX5
y — '
COL '
08
•>•
01
1
EM
OM
EM
01
EM
02
RM
02
™
RM
06
—
IF-
00
FLG
01
106
CHR
0
CHR
102
LF
RM
02
—
RM
07
ซ
T P -4-
1 r ~

0201
0202
0203
02 OU
0205
0206
0207
0208
0209
0210
0211
0212
0213
021U
0215
0216
0217
0218
0219
0220
0221
0222
0223
022U
0225
0226
0227
0228
0229
0230
0231
0232
0233
023U

0236
PP-17
0238
0239
02UO
02U1
02U2
02U3
02UU
02U5
02U6
02U7
02U8
02U9

0250

OU
SP
8b
71
R
RM
Ul

L
RM
U2
LOG
x
1
=
SC:

7O
RM
1&
71
. i j.
'f 1X5
02
COL
07
L
RM
63

R
RM \
63

LOG
•;..-
^
_
SC
70
RM
1U
_
FIX5
02
COL
07

LF

Fig.  A3a

   49

-------
OPERATE PROGRAM IRLSpec-S    (cont'd)
0251 E
0252 FLG
0253 00
0250 SP
0255 8C
0256 CM
0257 02
0258 CHfl
fi^cjq .-•

fi^fil
| T
1 1
13
0262 R
0263 T
flap's
0266
0267

FPF '3
0270
0271
0272
0273
0270
0275
0276
0277
0278
0279
0280
0281
• 0282
0283
0280
0285
0286
0287
0288
0289
0290
fisqi
0292
0293
0290
0295
0296
0297
0298
0299
0300

T
1
N
T
1
q

H~
1
<~-
CHfl
E
IFE
12
-X.l_
a
8
0
0
FLG
12

1
f
0
2_
^,
2

SM
01

1
0
0

SM
00

0301
0302
0303
0J00
0305
0306
0307
0308
0309
0310
0311
0312
0313
0310
0315
0J16
0317
0318
0319
0326
0321
0322
0323
0320
0325
0326
0327
0328
0329
0330
0331
0332
0333
0330
0335
0JJ6
0337
0338
0339
0300
0301
0302
0303
0300
0305
0306
0307
0308
0309
0350
0351
FLG
02
L
IND
RM
00
',•'.
L
IND
RM
01
=
EM
02
R
IND
RM
00
X
R
IND
RM
01
=
EM
02
1
EM
00
EM
01
RM
01
-
7
9
=
IF-
02
RM
02
FIX5
0J
COL
08
CHfl

s
l.l
N
9
' 0352 CHfl
0353 -
0350 RM
0355 15
0356 =
0357 FIX5
0358 05
0359
0360
0361
0362
0363
0360
0365
0366
0367
0368
0369
0370
0371
0372
0^73
0370
0375
0376
0377
0378
0379
0380
0381
0382
0383
0380
0385
0386
0387
0388
0389
0390
0391
0392
0393
0390
0395
0396
0397
0398
0399
0000


COL
16
LF
SP
Sd
FLG
07
CHfl
S
IJ
M

F
R
o
M
CHfi
E
SM
03
CHfl
T
o
CHfl
E
SM
00
CM
02
RM
03
-r
2
+
1
5
:=
SM
03
FLG
03
L


0001
0002
0003
0000
0005
0006
0007
0008
0009
0010
0011
0012
0013
0010
0015
0016
0017
0018
0019
0020
0021
0022
0023
0020
0025
0026
0027
0028
0029
0030
0031
0032
0033
0030
0035
0036
0037
00^8
0039
0000
0001
0002
0003
0000
0005
0006
0007
0008
0009
0050

IND
RM
03
EM
02
R
IND
RM
03
EM
02
1
SM
03
RM
03
—
1
5
zz
v
2
_
RM
00
:=
IF-
03
LF
RM
02
FIX5
03
?}
GT
07
SP
8e
3
0
0
SM
06
GT
10
CHfl
M
E
M


0051
0052
0053
0050
0055
0056
0057
.—.. | f- f.
U058
0059
0060
0061
0062
0063
0060
0065
0066
0067
0068
0069
0070
0071
0072
0073
0070
0075 '
0076
0077
0078
0079
0080
0081
0082
0083
0080
0085
0086
0087
0088
0089
0090
0091
0092
0093
0090
0095
0096
0097
0098
0099
0500

F
R
0
M
CHH
LF
E
SM
08
CHH
T
Q
CHH
E
LF
SM
09
FLG
11
L
IND
RM
08
FIX5.
03
COL
12
R
IND
RM
08
FIX5
03
COL
10
LF
1
EM
08
RM
08
™
RM
09
=
IF-
11
EP
00

           Fig.  A3b
            50

-------
flUTO STflRT
H SPECTRfl
B REVERSE
C S L CflL.
E STOP
F FQRWfiRD
G PRINT DflTfl
I INTEGRflL
J RUN WLGTH
K WLGTH EQUfiT.
L STB LHMF EQUfi
M SUN
0 OPERflTE
' ENTER YR.MQ.DRYtt

3780120. 00
REflBY
.HIT fl C OR J
FILE 0
.- TYPE U
'USED 720
HflX 300

0000 PRNTo:
0002 fl
0 0 O 3
0 O 0 4 S
0 0 0 5 P
0 0 0 6 F
r_' V V '— ' U_
0 U 0 7 C
0003 T
0 0 0 9 R
0010 fl
0011 LINE
0012 B
0013
0014 R
0015 E
0016 V
0017 E
0013 R
0019 S
0 O 2 0 E
0021
0022 LINE




0 0 2 3 C
0 0 2 4
0 0 2 5 S
0026
U027 L
0028
0029 C
: 0 0 3 0 fl
0 O 3 1 L
O 0 3 2 .
0 0 3 3 LINE
0 0 3 4 E
0035
0 0 3 6 S
0 0 3 7 T
0 O 3 8 0
0039 P
0040 LINE
0041 F :
0 0 4 2
0 O 4 3 F
0044 0
0045 R
0 0 4 6 W
0047 fl
0 0 4 8 R
0 0 4 9 B
0050 LINE
0 0 5 1 G
0052
0 0 5 3 P
0054 R
0055 I
0 0 5 6 N
0057 T
0 0 5 8
O 0 5 9 B
0060 fl
0061 T
0062 fl
0063 LINE
0064 I
0 0 6 5
0 0 6 6 I
0067 N
0068 T
0 0 69 E
0 0 7 M G
0071 R
0072 fl
0073 L
0074 LINE
0 0 7 5 J

0 0 7 6
0 077 R
0073 U
0079 N
0 0 3 0
0031 W
0 0 8 2 L
0 0 8 3 G
O O 8 4 T
O 0 8 5 H
0 O 3 6 LINE
O 0 3 7 K
U 0 g 8
fi o K '3 u
W %•' — ' ป' H
0 0 9 U L
0 0 9 1 G
0 092 T
0093 H
L~1 L"1 Q 4.
U U y T"
0095 E
O O 9 6 Q
0097 U
O 0 9 8 fl
O 0 9 9 T
0 100 .
0101 LINE
0102 L
0 1 0 3
M 1 pi 4 '-:
v 4 v i •— '
0105 T
0106 B
0107
0103 L
0109 fl
0110 H
0111 P
0112
0 1 1 3 E
0114 Q
0115 U
0 116 fl
0117 LINE
0 1 1 8 M
0119
0120 S
0121 H
0122 M


0123 LINE
0124 0
,-> 4 .-, cr
0 1 ฃ. •-'
0126 0
0127 P
0123 E
0129 R
0130 fl
0131 T
0132 E
0133
0134 LINE
0135 E
0136 N
0137 T
0138 E
0139 R
0140
0141 Y
• 0142 R
0 143 .
13144 M
0145 0
0146 .
0147 B
0143 fl
0149 Y
0150 tt
0151 LINE
0152 END*
015:': STOP
0154 3
0155 EEX
0156 6
0157 +
0153 FIX
0160 PRINT
0161 STO
0163 2
0164 5
0165 4
0 1 6 6 0
0167 S T U
0168 2
0169 5
0170 1
0171 O
0172 STO
. 0173 FIX


Fig. A4a.  Operate Program IRLSpec-SO





                              51  '
(Cassette B)
                                                                           R 0 0 0

-------
6175 1
i~i 4 ~~* •"* "~
0 1 r' b D
0177 1
0173 #REGS
0179 LBL
	 0
0131 LBL
	 0
0 1 3 3 C F G 3
9134 CFG 3
0185 F'RHTo:
0137 R
0133 E
0139 R
0 1 9 0 D
0191 Y
0192 LINE
.- '. 4 ,•"! .— | 1 1
u i y J H
0194 I
0195 T
0 1 9 6
0 197 ft
0 1 9 3
0 1 9 9 C
0 2 0 0
0 2 0 1 0
0202 R
0203
0204 J
0205 LINE
0206 EN Do:
0 2 0 7 S T 0 p
0203 LBL
0210 LBL
	 ft
,-j .-, J •-, 4
tlili 1
0213 ENTERt
0214 HNDSK 4
U 2 1 1" 2
0217 WBYTE 4
0219 1
0220 ENTERt
0'"' O 1 ivf
_ ii- tZ. 1 U
0222 +r-
0 2 2 3 L 0 H D
0224 FIX 2
0226 .
0227 0
0223 1
U 2 2 9 S T 0 +
0231 RCL
0233 PRINT
0234 FIX
0236 3
0237 WBYTE
0 2 3 9 1
0 2 4 0 S T U
0241 IF SFG
0242 G 0 T 0
0244 1
0 2 4 5 2
0246 0
0247 3 TO
024 3 F 0 R
0249 STRRT
0251 REflD
0253 1
0254 EEX
0255 3
0256 +
0257 STO- I
0259 RCL I
0261 + *-
0262 10tX
0263 STO I
0265 RCL
0 266 -
0267 IF -
•' 0263 CLEflR
0 2 6 9 R C L
0 2 7 0 2
0271 4
0 2 729
0273 +
0274 ENTERt
0275 1
0276 EEX
0277 3
0 2 7 3 *
0279 +
0230 PRINT •
0231 IF SFG

0232 GOTO
0234 NEXT
0285 IF SFG
0 2 3 6 G 0 T 0
0288 G OS UB


R00t
R 0 0 t
4

4
H
4
024



F
fi + F
4
4




ft
ft

H
E


fl









.-.

B
fl
4
K
B '
I
I
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j 	 i
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	 j
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0296 I
0297 N
0293 T
0 2 9 9 .
0 3 0 0
0 3 U 1 F
0 3 0 2 R
0 3 0 3 0
0 3 0 4 M
U 3 O 5
0 3 0 6 ?
0307 EN Do:
O 3 0 8 2
0 3 0 9 3
0 3 100
0 3 11 S T U P
0312 FIX fi
0314 PRINT
0315 FIX 4
0317 ENTERt
0 3 1 3 2
0 3 1 9 4
0 3 2 0 9
fi "•'. ''• 1 -
V '-' 1— 1
0322 STO ft
fl 3 '"' '•! ft
'-' •_' L. •_' '_'
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T— •—' tl_ j '_>
0325 STO- F
0326 CLEflR
0 3 2 7 S T 0 I
0328 FOR ft*
0329 RCL ft
0 3 3 0 2
0331 4
• 0 3 3 2 9
0 3 3 3 +
fl ':; • -; 4 •?
1J •-' •_' *T i^
0 3 3 5 2

O 3 3 6 8
0 3 3 7
0 3 3 ft 1
'-' •-' •-' '.' i
0 3 3 9 7
0 3 4 0 3
0 341 -H

0342 •--.
0343 V,
0344 XT-
0345 4
0346 r
0347 4
0343 rr
0349 STi<
0350 pfL"
0351 4
O 3 5 2 -
0353 + = -
0354 ei:1
0355 RCL
0 3 56 *
0357 RCL
0 3 5 9 *
0360 ft CO
0361 NEXI
0362 RCL
0363 PRUT
0365 LINE
0 3 6 6 I
0367 N
0363 T
O 3 6 9 E
0370 G •
0371 R
0372 ft
0373 L
0374
0375 9
0376
0 3 77 =
0378 PR HIT
F 0379 LINE
0330 LIME
0381 END'-
0332 EHTEP
0383 3-
0384 .
O 3 8 5 0
0 3 8 6 6

0 3 8 7 +
0338 PRNT*
0390 S
0 3 9 1 U
0392 N
0 3 9 3 S

Fig. A4b.  Operate Program IRLSpec-SO    (Cassette B)   (cont'd)
                            52

-------
1
I
8394
8 3 9 5
8 3 9 6
8 3 9 7
0393
0399
0 4 0 0
6401
0 4 0 3
	
0405
	
8487
8 4 8 9
041 1
0412
0 4 1 3
0414
8415
8416
6417
0418
8419
0420
0421
0422
0423
0424
0425
0427
8428
0 4 3 0
0432
0433
0434
0435
8 4 3 6
8437
0433
8439
8 448
6441
8442
644 3
8444
6445
8446
6447
6449

=

PRINT
LINE
LINE
EHD*
0 4 5 0
U 4 5 2
0454
	
'0 4 5 6
	
'0 4 5 8
GOTO H 6459
LBL
K
LBL
K
R C L R 6 6 4
RCL R685
-^
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+ i-
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.
4
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t
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PRINT
RCL R647
RCL R648
r
LOG
+ ฃ-
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p
IT
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2
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0 4 6 0
0461
0 4 6 2
0464
	
0 4 6 6
0467
0 469
0476
0472
0473
	
8475
8476
6473
8 4 7 9
8431
6 4 8 2
	
8 4 8 4
	
8 4 8 6
6 4 8 7
6 4 8 3
8 4 8 9
8491
8 4 9 3
6 4 9 5
8 4 9 6
6497
0 4 9 8
0 4 9 9
0 5 8 8
6561
6 ; 0502
0503
5 '0504
+ 0 5 0 5
PRINT
FIX 4
CFG 4
8 5 8 7
0 5 0 3
0510
GOSUB
G 0 T 0
LBL
..1
LBL
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5
0
S T 0
SFG
G 0 T 0
LBL
F
2
WBYTE
.-J
WBYTE
B
0






F
4
R



4

8511
8512
. 0513
0514
0 5 1 6
0517
0513
0 5 1 9
0 5 2 0
0522
052 3
0524
0525
0 5 2 6
0527
052 3
4 i 8 5 2 9
RET U R H i 0 5 3 0
LBL
E
C.
WBYTE
C L E R R
WBYTE
0531
i ' 0532
', 0 5 3 3
4 ; 0534

O 5 '3 6
4 8 5 3 7
RETURN 0538
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.
8
1
STO +
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F I X
PRINT
1
STO
1
5 •
6
S T 0
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ENTER
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: 	
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6546
R 6 0 6 0547
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2


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F
•~i

T
4

4

054 9
0 5 5 0
0552
055 3
0554
0 5 5 6
0 5 5 7
0 5 5 8
6 5 6 6
0 5 6 1
0 5 6 2
0 5 6 4
0 5 6 6
0563
0 5 6 9
0 5 7 0
PflUSE
PflUSE
F 0 R
RERD
1
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3
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4
9
+
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1
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*
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NEXT
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B
LBL •
B
1
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HHDSK
ฃ
WBYTE
PflUSE
0
W E: Y T E
PR USE
1
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5
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fl
4




R

R










4

8
B
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4

4


4


4


4
4
4
B


                     Fig. A4c.  Operate Program IRLSpec-SO    (Cassette B)   (cont'd)

                                                  53

-------
0 5 7 1 G 0 T 0 6 5 6 6
'"1 5 7 ::! 1
'. '..' T '
'.' '. '.
y i ,•' <:• K E R Ii 4
0573 RCL D
0579 +
0580 IF -
0531 GOTO 0576
0583 0
0534 MBYTE 4
0536 IF SFG 3
0587 STOP
0588 IF SFG S
0 5 3 9 G 0 T 0 0712
0591 RETURN
0592 LBL
	 M
0594 LBL
	 M
0596 PRNTo:
0598 R
0599 E
0 6 0 0 C
0601 0
0602 R
0603 D
0604 ? :
0 6 0 5
0 6 0 6 0
0607 R
0603 LINE
0 6 0 9 S
0610 U
06 if M
0612
0613 F
0614 R
0 6 1 5 0
0616 M
.: 0617 EN Bo:
0613 CLEflR
0619 STOP
0620 IF 0
' 0 6 2 1 G 0 T 0 0
• 0623 FIX O
0625 PRINT
0626 2 f 0K76 LBL
062? 4 ; - 	 -,
f • f~ ' •"' '• '
-•-*—-
G 6 2 9 -
0 6 3 0 S T H R
0631 PRNTtf
' 0633 T
0 6 3 4 0
0635 EN Bo:
0636 STOP
0637 PRINT
0638 2
0639 4
0 6 4 y 9
0641 -
0.642 STO F
0643 CLEflR
0644 STO I
0645 FOR fl+F
0646 RCL I fl
0 6 4 8 S TO-*- I
0649 NEXT R
0650 FIX 4
0652 RCL I
0653 PRNTo:
0655 M
0656 I
0657 L
0658 L
0659 I
0660 N
0661 fl
O 6 6 2 T
0 6 6 3 T
O 6 6 4 S
O 6 6 5 /
0666 M
O 6 6 7 S
f} j-r ".- ;.-.; i r-

— cT
0680 1
0681 STO
0682 1
0-': 3 5
0V.'-! O
0 6 0 ':< S T 0
0636 FIX
0 6 3 3 F 0 R
0689 RCL I
0 6 9 1 R C L
0692 2
O 6 9 3 4
0694 9
0 695 +
0696 ENTER!
• 0697 1
O 6 98 E E X
0 6 '•"• "
0 7 0 y *
0701 + .
0732 PRINT
0703 NEXT
0704 S T 0 P •
0705 LBL
	 L
0707 LBL
	 L
0709 CLERR
0710 1
0711 L D I G 0
0712 .
0 7 1 3 O
0714 1
0715 STO-
0 6 6 3 Q 0717 G 0 T 0
0669 071Q END
0670 PRINT
0671 LINE
0672 LINE
0673 ENDo:
0674 GOTO M











^



p
4
fi-

ll











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PC
o







Fig. A4d.  Operate Program IRLSpec-SO    (Cassette  B)   (cont'd)
                                54

-------
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                                             55
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-------
Fig. A6 - FS40, FBZS40CLG1076, Sum, IRLSpec-S
                   56

-------
                FS4M-40O Fibergilas
Fig. A7 - Filtered FS40 and FBZS40CLG1076,  IRLSpec-S



                      57

-------
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                      58

-------
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-------
7^   80    $0   3oo  /c   Za
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          Fig. A1.0 - F15T8 CW Westinghouse,  IRLSpec-D

-------
o-o
80    9&
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                                                        -30    4-0    3.5-8    60   7^?    00
                          Fig. All - F15T8 CK Westinghouse, IRLSpec-SO

-------
2 -'
t >
                                              71
                               Fig.  A12 - F15T8 CW Sylvania, IRLSpac-D

-------
                                                     •       •
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Fig, M.3 - F15T8 GRO-LUX Bylvania F15T8 WW GE,  IRLSpec-D

                        ,.63

-------

                         IRLSpec-D UV-B (2nm BW)
                            IRL-AKRI-ARS-USDA

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                       Fig. &X4 - F40R C3S, F40IR  Kestinghouse,  F15T8R GE,  2 - 50 cm,
                                  IRLSpec-0
                                             64

-------
fT^^'r;??^^^
             IRLSpec-D UV-B (2nm BW)
                IRL-A>mi-ARS-USDA
       Fig. R15  -  F401R SestJ.ngte>ซser 3 =ป 20 can, IRLSpec-D

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                                Fig.  A16 - F15TOBL Sylvania, P15T8BLB GE, IRLSpec-O
      .uprn^TJM-ctT-

-------
Pig. A17 - F40 BL + FBZsVo CIX31076,  IRLSpac-D

-------

6 L-4S%-.|^.,4- .	-^_-3jJ:.4'


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                                               68

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...-*, i  ••<*   • i  : 	   i_ .

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                                                      IRL-M1RI-ABS-USDA
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-------
                                 r tt
                      FINAL REPORT
       BIOLOGICAL EFFECTS OF ULTRAVIOLET RADIATION
               ON. PLANT GROWTH AND FUNCTION
                    M. N. Christiansen
                 Plant Stress Laboratory
                Plant Physiology Institute
         Beltsville Agricultural Research Center
                Beltsville, Maryland 20705
                                                            3t c
                                                             ป ^
                      EPA-IAG-D6-0168
                      Project Officer:

                      R.  J.  McCracken
Agricultural Research,  Science and Education Administration
                 U.S.  Department of Agriculture
                   Washington, D.C. 20250
                      Prepared for
             Environmental Protection Agency
                      BACER Program
                   Washington, D.C.  20460

-------
                     FINAL REPORT
    DIFFERENTIAL SENSITIVITY OF TWO CULTIVARS OF
      CUCUMBER (CUCUMIS SATIVUS L.) TO INCREASED
                UV-B IRRADIANCE:
            I.  DOSE-RESPONSE STUDIES
                    D. T. Krizek
                Plant Stress Laboratory
              Plant Physiology Institute
        Beltsville Agricultural Research Center
              Beltsville, Maryland 20705
                 EPA-IAG-D6-0168
                 Project Officer:

                 R.  J. McCracken

Agricultural Research, Science and Education Administration
           U.S.  Department of Agriculture
             Washington, D.C. 20250
                    Prepared for

             Environmental Protection Agency
                   BACER Program
                 Washington, D.C.  20460

-------
                             CONTENTS


Abstract	iii

Tables	    iv

Figures 	      v

Acknowledgments 	  vii

      Introduction 	 	     1

      Materials and Methods 	    3

          Plant Material	    3

          Cultural Conditions 	    3

          UV Source	    3

          UV Measurements 	    4

          Harvest and Data Analyses	    5

      Results and Discussion 	    7

          Comparative Phytotoxic Effects of UV-B
               Irradiation 	    7

          Comparative Influence of UV-B Irradiation
               on Vegetative Growth 	   8

      Conclusions	1.1

      Literature Cited 	   12

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                                ABSTRACT

     Dose-response  studies were conducted  at  Beltsville,  Maryland,  on

 two  cultivars  of  cucumber  (Cucumis  sativus L.)  exposed  to a UV-B irradiance

 gradient representing  an increase of  40%  to 770%  in  biologically

 effective UV  (BUV)  radiation over normal  sunlight.   Plants were irradiated

 in a fiberglass greenhouse in April employing FS-40  fluorescent sunlamps

 filtered with  0.127 mm Mylar (UV-A) or  0.127  mm cellulose acetate

 (UV A&B).  UV  treatment was given 6 hours  per day  (from 1000 to 1600)

 for 19 days from  the time of seeding.   Marked differences in UV-B

 sensitivity were  observed between 'Poinsett1  (extremely sensitive)  and

 'Ashley' (slightly  sensitive).  Increasing the  UV-B  level induced

 chlorosis of the  leaves, inhibited leaf and shoot growth,  and reduced

 biomass.  These effects were especially marked  in  'Poinsett'.  'Ashley'

 plants required approximately twice the level of BUV as 'Poinsett'  to

 exhibit a 20-25%  reduction in dry weight or leaf area.   Based on linear

 regression analysis of the 'Poinsett' data, it  x^as estimated that a

maximum proposed  decrease in stratospheric ozone content  of 20% (or a

 40% increase in BUV) would cause a 10%  reduction in  dry matter production

with a 15% decrease in leaf area.  A  100%  increase in BUV or greater
                           t
was needed to  cause pronounced chlorosis of the leaves  and a marked
                                        *
 reduction in dry  matter production.    Such  increases  would be far in

 excess of the  projected BUV levels expected as  a result of stratospheric

ozone reduction caused by chlorofluoromethane emissions.
                              iii

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                                TABLES    '                               Page




Table 1.  Weighted and unweighted UV  spectral  irradiance  under each of   16




          four UV set-ups in  the greenhouse.   Each  set-up contained




          eight FS-40 fluorescent sunlamps  filtered  with  0.127 mn Mylar




          (M-5) or cellulose  acetate  (CA-5).   Data  shown  are  mean




          values with their standard  errors and  ranges  for 48 pot




          locations per cultivar per  set-up.




Table 2.  Criteria for scoring chlorosis  in cucumber leaves.              17




Table 3.  Influence of increased UV-B irradiation on index of leaf       18




          injury, fresh weight and percent  dry weight of  tops in  'Poinsett*




          (P) and 'Ashley'  (A) cucumber plants irradiated for 19  days  in




          the greenhouse  (April 1-20, 1977).   Plants exposed  to a UV




          gradient provided by FS-40  fluorescent sunlamps filtered with




          0.127 mm Mylar  (M-5) or cellulose acetate  (CA-5)  at various




          distances above the canopy.  Data shown are riean values with




          their standard errors and ranges  for 48 plants  of each  cultivar




          per treatment.
                                IV

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                                FIGURES
                                	                                    Page

Figure 1.   'Poinsett'  (green label) and  'Ashley'  (yellow label)  cucumber    20


           plants grown for 19 days from  seeding  in  the  greenhouse under


           FS-40 fluorescent sunlamps filtered with  0.127 mm Mylar.


           Plants received UV-A irradiation but no supplemental  UV-B


           irradiation.  Note absence of  chlorosis.


Figure 2.   'Poinsett'  (green label) and  'Ashley'  (yellow label)  cucumber    22


           plants after 19 days of UV-B irradiation  (6.73 mean weighted

               _2
           mW-m   of BUV) provided by FS-40 fluorescent  sunlamps filtered


           with 0.127 mm cellulose acetate.


Figure 3.   'Poinsett'  (green label) and  'Ashley'  (yellow label)  cucumber    24


           plants after 19 days of UV-B irradiation  (11.02 mean  weighted

               -2
           mW-m   of BUV) provided by FS-40 fluorescent  sunlamps filtered


           with 0.127 mm cellulose acetate.


Figure 4.   'Poinsett'  (green label) and  'Ashley'  (yellow label)  cucumber    26


           plants after 19 days of UV-B irradiation  (15.30 mean  weighted

               _2
           mW-m   of BUV) provided by FS-40 fluorescent  sunlamps filtered
           with 0.127 mm cellulose acetate.


Figure 5.  Comparative sensitivity of  'Poinsett'  (top row) and  'Ashley'
                             v

           (bottom row) cucumber plants to increased UV-B irradiation.


           Plants irradiated for 19 days from time of seeding  (17  days


           from emergence) in the greenhouse under FS-40 fluorescent


           sunlamps filtered with 0.127 mm Mylar  (M-5) or 0.127 mm


           cellulose acetate (CA-5).  The latter  plants received a mean


           level of biologically effective UV (BUV) irradiance of  6.73,

                                        -2
           11.02, or 15.30 weighted mW-rn  , respectively.
28

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                                                                        Page
Figure  6.   Linear  regression  of  the  index of  injury for 'Poinsett'      29
            cucumber  leaves  vs. biologically  effective UV (BUV)  radiation .
                            -2
            in weighted mW'ra  .   Plants  were  irradiated in the greenhouse
            for  19  days from seeding  under eight  FS-40 lamps filtered
            with 0.127 mm  cellulose acetate under a UV gradient  ranging
                                 _?
            from 4.9  to 26.6 iaW-m  BUV  (1.6  to  8.7 UV-B sun equivalents).
                                                         _7
            (One UV-B sun  equivalent  = 3.06 weighted mW-m   of BUV).
Figure  7.   UV dose-response relationship  under  greenhouse conditions.    30
            Comparative leaf areas of  'Poinsett1  and 'Ashley'  cucumber  plants
            expressed as percentages  of  Mylar  controls.   Plants  irradiated
            for  19  days from seeding  in  the greenhouse under eight  FS-40
            lamps filtered with 0.127 mm cellulose acetate.   Lamps  mounted
            in separate set-ups at 1.43, 0.92, and 0.54 m above  the plants.
            Means and standard errors are  shown  for 48 plants  within  each
                                            -2
            set-up  for 6.7,  11.0  or 15.3 mW m    BUV (2.2,  3.6, or
            5.0  UV-B  sun equivalents) respectively.
Figure  8.   UV dose-response relationship  under  greenhouse conditions.    31
            Comparative dry  weights of tops of  'Poinsett'  and  'Ashley'
            cucumber  plants  expressed as percentages of  Mylar  controls.
            Plants  exposed ten the UV-B irradiation gradients described
            in Fig. 7.                                                        ;
Figure  9.   Linear  regression of  dry weight of tops  of 'Poinsett' cucumber 32
            plants  vs. exposure to biologically  effective  UV (BUV)  radiation
                   -2
            in mV?-m  .  See  Fig.  6 legend.
Figure  10.  Linear  regression of  total leaf area  of  'Poinsett' cucumber     33
                                                                        _2
            plants  vs. biologically effective UV  (BUV)  radiation in mWm
            See  Fig.  6 legend.
                             vx

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                       ACKNOWLEDGMENTS




     Appreciation is  extended to Richard Griffin, Nancy Maher, Scott




Ravitz, and Randy Rowland for their excellent technical assistance;




to E. James Koch and  Helen Herlich for their help in obtaining




statistical analysis  of the data; and to Jesse Bennett, Sterling




B. Hendricks, E. James Koch, and Olga v.H.  Owens for their critical




reviews of the manuscript.
                             Yii

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         DIFFERENTIAL  SENSITIVITY OF  TWO  CULTIVAKS  OF  CUCUMBER



            (CUCUMIS  SATIVUS  L.) TO  INCREASED  UV-B  IRRADIANCE:



                        I.  DOSE-RESPONSE  STUDIES


                                        I/  21  3/
                        Donald T. Krizek-  '  -  '  -



                              INTRODUCTION



     The influence of  stratospheric ozone reduction and  the  attendant



increase in solar ultraviolet-B irradiation (UV-B,  280-320 nm)  on the



biosphere have been  of  recent concern (Molina and Rowland, 1974).



     During the Climatic Impact Assessment  Program  (CIAP) in 1972-1975,



numerous studies were  conducted on  the  response of  higher plants  to



increased UV-B irradiation (Ambler  et al.,  1975; Basiouny et al., 1978;



Biggs, 1975; Biggs and  Basiouny, 1975;  Brandle  et al., 1977; Caldwell,



1977; Krizek, 1975b; Nachtwey, 1975;  Sisson and Caldwell, 1976,  1977;



Van and Garrard, 1975;  and Van et al.,  1976).   Despite the wealth of
— Plant Physiologist, USDA, Science and Education Administration,



  Agricultural Research, Plant Physiology Institute, Plant  Stress



  Laboratory, Beltsville, Md. 20705.



2/
— Research supported in part^ by the U.S. Environmental Protection Agency



  under interagency agreement EPA-IAG-D6-0168.



3/
—Abbreviations:  PAR: photosynthetically active radiation  (400-700  nm);



  UV-B:  280-320 nm region; BUV:  biologically effective UV irradiance  in


               -2                                                     -2
  weighted mW m  ; UV-B SE:  UV-B sun equivalent =  3.06 weighted mW'm



  BUV; FS-40 lamps:  Westinghouse FS-40 fluorescent sunlamps; M-5:



  0.127 mm (0.005 in. = 5 mil) Mylar; CA-5:  0.127 mm  (0.005  in. = 5 mil)



  cellulose acetate.

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data collected  during  this  period,  relatively  little  is  known about




UV-B dose-response  relationships  for  higher  plants.   Such information is




critical  in  order to assess  the biological  impact  of  increased UY-B




irradiation  caused  by  stratospheric ozone reduction  (Anon.,  1977;  Krizek,




1975a, 1976,  1977a).




     In the  course  of  screening a range  of  selected species  for




comparative  sensitivity  or  resistance  to broad-band UV-B irradiation, two




cultivars of  cucumber,  'Poinsett' and  'Ashley,  were  discovered that




differed markedly in their  response to increased levels  of UV-B




irradiation  (Krizek, unpublished results, 1976).   The present study was




conducted to  establish dose-response relationships for these cultivars




and to determine threshold  levels of biologically  effective  UV (BUV)




irradiation  required to  induce UV-B damage  in  these two  cultivars.




     Regression equations are presented  to  provide a  means for assessing




the potential biological impact of a projected  increase  in UV-B irradiance




on leaf growth and  dry matter production using  'Poinsett1 cucumber  as a




model of a highly sensitive plant.

-------
                          MATERIALS  AND  METHODS  .


 Plant Material.   'Poinsett1  and  'Ashley'  cucumbers were investigated:


 1)  because  of  their  differences  in  UV-B sensitivity observed in an


 earlier  screening  program;  2)  their rapid growth rate;  3)  their uniformity


 in  size;  and 4)  their  prostrate  habit for the  first 2-3 weeks.   Experiments


 were repeated  three  times (February, April and  June).   Data reported


 here (obtained during  April  1-19, 1977)  are representative of the dose-


 response  relationships obtained.


 Cultural  Conditions.   Plants were grown for 19  days from seed in 12.5 cm

                                                                      4/
 dia. white  plastic pots containing  a peat-vermiculite mix  (Jiffy Mix)— .


 Five seeds  were  planted in each  pot, using a special template.   After


 7 days,  the seedlings  were thinned  to one per pot.   UV-B irradiation was


 begun at  the time  of planting  the seed.   Minimum night  temperatures in


 the greenhouse did not go below  20  C; day temperatures  did not  exceed


 35 C.  Natural daylight and  photoperiod  were used.   The plants  were


 fertilized  daily with  a 1/4  strength ASMS Hoagland solution (Hammer


 et al., 1978).


 UV Source.  In order to provide  a gradient in UV-B irradiance,  four


 set-ups were constructed  in  a  fiberglass-covered  greenhouse (Table 1).


 Each set-up contained  four fixtures, each containing two FS-40  lamps.


 One set-up  contained eight FS-40 lamps  filtered  wฑth Mylar (M-5)  as a
4;
—  Mention of a trademark, proprietary product, or vendor  does  not


  constitute a guarantee or warranty of  the product by  the USDA and  does


  not imply its approval to the exclusion of other products or  vendors


  that may also be suitable.

-------
   UV-B control with the center four lamps maintained  at  a  distance of


   1 m and the outer four lamps at a distance of 0.75  m above  the plants.


   Each of the other three set-ups contained eight FS-40  lamps  filtered


   with cellulose acetate (CA-5) with the center four  lamps  kept  at a


   distance of 1.43, 0.92, or 0.54 m and the outer four lamps  kept at a


   distance of 1.22, 0.70, or 0.54 m respectively above the  plants.


        The pots were arranged in 8 rows x 12 columns  per set-up,  each

                             2
   covering an area of 1.73 m  (see Cams et al., 1977).  This  arrangement


   was divided, into four quadrants (replicates) and  the pots were positioned


   equidistantly from the center of each set-up.  Pots of  'Poinsett'  and


   'Ashley' were alternated at 0.15 m intervals along  the x  and y axes


   (corresponding to rows and columns) in order to obtain comparable  levels


   of UV-B irradiance for each cultivar (Table 2).


        The experiment was conducted according to a'  standard protocol


   described by Krizek (1977b).  The lamps were aged 100 hours  and the


   CA filters were aged for 6. hours prior to use.  The CA filters  were


   changed twice weekly (because of degradation by the short wave  UV).


   The heights of lamps above canopies were adjusted as the  plants grew
\

   to maintain the specified levels of UV-B irradiance.


      Measurements.  Broad-bandi UV-B irradiances were determined  at every


  ', \ot location at the beginning and end of. each experiment  by  means  of a

    \                             (
  ', mad-band radiometer developed by the Instrumentation Research
   I  boratory (IRL UV Meter) (Morris, 1977; Rowan and Morris, 1978).  Mean


   values for each of the four set-ups are given in Table 1.


        The IRL UV Meter was used to obtain the readings presented in


   Table 1.  The instrument consists of a solar-blind vacuum photo-diode


  ' (Hamamatsu R403),  an integrated circuit amplifier, and a microammeter

-------
packaged  in  a  meter  case  (Rowan  and  Korris,  1978;  Norris, 1977).   The



spectral  sensitivity of  the  IRL  Meter  in  the UV-B  .region is relatively



flat, with maximum sensitivity at  300  nm.   The detector is insensitive



at wavelengths  longer than 400 nm.



     Narrow-band UV  irradiances  were determined at selected pot locations



for every nm wavelength from 250 to  369 nm  with an automated spectro-



radiometer developed.by IRL  (Norris, 1977;  Rowan and Norris, 1978)  and



commercially available from  Optronic Laboratories, Inc., Silver Spring,



Md.                                                      .


                                                   -2
     BUV weighted irradiances are  reported  as inW-m  ,  the biologically



effective UV irradiances  derived from  the AE9 weighting function



described by Thimijan et  al., 1978 and Cams et al., 1977.  Since UV



irradiation  employed  in this study was obtained by filtering FS-40



lamps with CA-5, BUV  was  essentially confined to the UV-B region.  Mean



BUV values for  each  set-up are given in Table 1.



     Unweighted spectral  irradiances in the  UV-B region (Table 1) were



obtained by  summing measured values  at each  nanometer  from 280-320 nm



or using the regression equations  developed  to estimate the UV-B


                                                      _2
exposures (Krizek and Koch,  1978).   Dividing the mW-m    of biologically


                             v       -2           -2
effective UV irradiance by 3.06  raW-m  [the  raW-m   BUV of one Beltsville



control sunshine; i.e., 1 UV-B sun equivalent (SE)]  provides the



fraction of  BUV relative  to  that of  1 SE  (Thimijan et  al., 1978). .



Harvest and  Data Analyses.   Plants were harvested  after 19 days of



UV-B irradiation.  An index  of injury scale  was developed for scoring



the extent of leaf chlorosis (Table  2).  Leaf areas  were then measured



with a Lambda LI-COR  leaf area meter.  Fresh and dry weights of the



shoots were  taken; the latter were recorded  after  drying the samples in

-------
a forced draft oven at 80 C for 48 hr.  Data on plant height  and  node


number were also taken but are not reported since .they  showed  little or


no differences.


     Means, standard deviations, standard errors of the mean,  and


linear and quadratic regressions on weighted BUV were calculated  for


all parameters.  Since quadratic regression of the data yielded no


significant improvement of the correlation coefficient  (r values),  only


linear regression data are presented.  Data were analyzed separately by


cultivar and set-up.                          .


     Since the steepest UV gradient was obtained under set-up  4

                                                         _2
(Table l)-i.e., that having a mean BUV level of 15.3 mW-m    (or 5.0 UV


SE)-data obtained for this set-up were used in calculating the regression


equations presented.

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                                                                             . ป'.'..,T.K
                         RESULTS AND  DISCUSSION



Comparative Phytotoxic  Effects of UV-B  Irradiation.   Leaves of 'Poinsett'



cucumber plants irradiated  for 19 days  under  CA-5 filtered FS-40 lamps



developed marked interveinal  and marginal  chlorosis  with crinkling



distortion evident at the  tip and along the margins  of the leaves (Fig.



1-5).  UV damage was observed within 1-2 days after  seedling emergence



and increased in severity  as  the leaves expanded  and UV-B irradiance



increased (Fig. 5).  The index of injury under a  maximum of 6.7,


                  -2
11.0, or 15.3 mW-m   BUV (2.8, 5.4,  or  8.7 UV-B SE)  was 3, 5,  or  9,



respectively, (Table 3,  Fig.  6).  These values represented about  15, 25,



or 45% chlorosis, respectively (Table 1).  Leaves of 'Ashley'  cucumber



plants, on the other hand,  never reached an index of injury above 3


                                               _2
(15% chlorosis) even when  exposed to 15.3 mW'm   BUV (Table 3).   For



corresponding mean UV-B  doses, 'Ashley'  plants exhibited one-third



to one-half as much chlorosis as those  of  the cultivar 'Poinsett1



(Table 3).                                                       .



     Under a 40% increase  in BUV or  (1.4 UV-B SE)  (Table 1) which



corresponds to a 20% decrease in 0   content in the stratosphere,  leaves



of 'Poinsett' cucumber plants showed an injury scale of 1 (5%  or  less



chlorosis, while 'Ashley' plants showed an injury scale of 0 (no



chlorosis) (Table 3).  The  negative  regression of injury index for the



'Poinsett' cultivar, as measured by  leaf chlorosis and weighted BUV


                  _2
irradiance in mW- m  , is shown in Fig.  6.  The r  value (i.e.,  0.81)



indicates that the regression equation  described  may be used to estimate



leaf injury expected at a given BUV  irradiance level (Krizek and  Koch,



1978).  Since no significant regression could  be  established for  leaf



injury on BUV for the cultivar 'Ashley'  (r =  0.35),  the regression is



not shown.

-------
     The  threshold  level  for  chlorosis  in 'Poinsett'  cucumber leaves



.varied with  season,  increasing  in  the  spring and summer and decreasing



in the fall  and winter, suggesting a difference in photorepair capability



with season  and amount  of  PAR.



Influence of UV-B Irradiation on Vegetative  Growth.   At a mean BUV level



of 6.73 mWm~2  (a 120%  increase in BUV,  or  2.2  UV-B SE),  (Table 1)



and 'Ashley' cucumber plants  showed approximately equal (6-10%) reduction



in fresh weight of  tops  (Table  3)  and  total  leaf area (Fig. 7) as



compared to  the Mylar control plants.  Mean  dry weight loss, however,



at this BUV  level was greater in the case of 'Poinsett' (5.8%) than for



'Ashley1  (1.4%) when compared to their Mylar controls (Fig. 8).


                                                      -2
     UV-B irradiances in  excess of 6.73  weighted mW'rn .  caused greater



reductions in leaf area and dry weight of tops  of 'Poinsett1 plants than



for 'Ashley' plants  (Figs.  7, 8).  The  'Ashley1  cultivar  required about



twice as much BUV as 'Poinsett' to produce a 20-25% reduction in leaf



area or dry weight  (Figs.  7,  8).   Leaf size  was reduced by  increased UV



irradiation  to a greater  extent that was  dry weight of tops.  At 11.02


    -2
mWm   BUV or greater, vegetative  growth  as  measured  by fresh weight



of tops (Table 3), total leaf area (Fig.  7), and dry weight  of tops



(Fig.  8),  was markedly impaired in both  cultivars.



     When dry weight data  for the  48 'Poinsett'  plants under the UV


                                                       _2
set-up with the widest exposure range-4.9 to 26.6 mW- m   of BUV



(1.6 to 8.7 UV-B SE) and a mean of 15.3 mW-nf2  BUV (5.0 UV-B SE)-were



subjected to linear regression analysis,  a correlation coefficient of



0.77 was obtained (Fig. 9).   For each unit increase in BUV,  the loss in



dry weight would be predicted to be 30.60 mg. On the  basis  of one SE,


                               -2
a 37% increase in BUV (4.2 mU-m    BUV or  1.37 UV-B SE)  would be expected

-------
                                                                     _o
to reduce  dry  weight  of  'Poinsett1  cucumber plants by 10%; 10.5 mW-m
BUV  (or  3.43 SE) would reduce  it  by 25%;  and 21.0 mW'm~2 BUV (or 6.86
UV-B SE) to reduce  it by .50%.   Since actual reductions in dry weight
obtained at the higher BUV  levels were less than this prediction and
since relatively few  data points  were collected  for UV irradiances in
                    -2
excess of  15.3 mWm  BUV  (5.0 UV-B SE) ,  the equation is of greatest
value below this point.
     Assuming a 20% decrease in stratospheric 0   reduction caused by
chlorofluoromethanes  (CFM's) with a 40% corresponding increase in surface
level BUV  [actually likely  to  be  higher than this since the curve is non-
linear above 10% 0  reduction  (IMOS,  1975)],  one finds on the basis of
this regression curve an approximate  10%  decrease in dry weight for a
highly sensitive cucumber cultivar  such as  'Poinsett1.
     Linear regression analysis of  the leaf area data for 48 'Poinsett1
cucumber means on BUV resulted  in a correlation  coefficient of  0.81
(Fig. 10).  For each unit increase  in BUV,  the decrease in leaf area
                                2                                  -2
would be predicted  to be 8.84  cm  .   On the  basis of one SE 2.8  mW'm
(0.90 UV-B SE) or 90% of present  BUV  levels can  reduce leaf area in
'Poinsett' cucumber under the  conditions  of the  experiment by 10%;
6.7 inW-in   BUV (2.2 UV-B SE) ,*  a 120%  increase, would be needed  to reduce
leaf area  by 25%; 13.8 mWir~   BUV (4.5 UV-B SE),  a 350% increase, would
be required to reduce leaf  area by  50%.   Again,  assuming a 20%  maximum
decrease in stratospheric ozone reduction from CFM's,  one could predict
an approximate 15%  decrease in  total  leaf area for this plant.
     The inhibitory effects of  high UV-B  irradiance on leaf growth in
cucumber are consistent with the  findings of  Sisson and Caldwell (1976,
1977) and Dickson and Caldwell  (1978)  for Rumex  patientia L.,  previous

-------
work in our laboratory on  cotton  (Ambler et al.,  1975);  Alaska  pea




(Krizek et al., 1975b) and a  number of bedding  plants  (Krizek and




Semeniuk, 1975); and early work reviewed by Caldwell  (1968,  1971).




     Preliminary measurements of  stomatal resistance did  not indicate




a significant difference between  Mylar control  plants  and the green




portions of those exposed  to  increased UV-B, or between  'Ashley1 and




'Poinsett' leaves.  Moisture  content of the tops  was slightly,  but not




significantly, higher in 'Ashley1 than 'Poinsett', suggesting that




differences in turgor were not responsible for  the differences  in




leaf growth observed (Figs. 8, 10).




     Measurements of carbon dioxide exchange rates (CER)  made on selected




'Poinsett' cucumber plants at increasing BUV levels indicated a significant




reduction in CER which was related to the amount  of chlorosis observed.




•BUV irradiances in excess of  40%  enhancement levels expected to result




from CFM-catalyzed destruction of stratospheric ozone  content were




required to obtain statistically  significant differences  (Bennett, 1978).




     Additional studies are underway to determine the anatomical,




physiological, and biochemical bases for the differences in UV-B




sensitivity observed between the two cucumber cultivars.   Possible




explanations to account for these differences might include differences




in optical properties of the leaves with 'different degrees of screening




of the responding sites;  differences in photoreactivation; differences




in biochemical make-up  including peroxidase  activity;  and differences




in growth regulator  activity.
                              10

-------
                               CONCLUSIONS'


      Significant  differences  in  UV-B  sensitivity were found between two


cucumber  cultivars;  'Poinsett' was  extremely  sensitive and 'Ashley' was


slightly  sensitive.   Evidence  was obtained  for  UV-B induction of:  1) leaf


chlorosis;  2)  inhibition  of leaf growth;  and  3)  reduction in fresh and


dry weight  (biomass).   These  effects  were most  pronounced under conditions


of low PAR  and high  UV-B  irradiation.


      Based  on  regression  analysis of  plant  data  obtained  under a range

                                                 _2
of UV irradiances  from  4.6 to  26.6  weighted mW'm   of biologically


effective UV  (BUV),  it  was estimated  that a maximum decrease in


stratospheric 0  content  of 20% could cause a 10% reduction in dry matter


and a 15% decrease in leaf area in  the highly sensitive 'Poinsett'


cucumber  cultivar.   Whether reductions in growth of this  magnitude could


even  be detected in  nature is  questionable.

                                                                       _2
      Increasing the  BUV level  by at least one SE (i.e., from 3.06  mW-m

            -2
to 6.12 mW-m   or  greater) was required to  obtain pronounced chlorosis


of the leaves  (> 10% chlorosis) with  comparable  reductions  in biomass;


these levels would be far in excess,  however, of the  projected levels of


biologically effective  UV-B irradiances occurring from CFM-catalyzed


reduction of stratospheric ozbne.


     Further work  is needed to elucidate  the site and  mechanisms of UV-B


induced injury in  these cucumber cultivars.  It  is  clear  that the  choice


of plant material  is a  critical factor to the environmental decision-


maker in assessing the  biological implications of stratospheric ozone


reduction and  the  attendant increase  in UV-B irradiation.
                                 11

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                           LITERATURE CITED




Ambler, J. E., D. T. Krizek, and P. Semeniuk.  1975.  Influence of UV-B




  radiation on early seedling growth and translocation of   Zn from




  cotyledons in cotton.  Physiol. Plant 34(3):177-181.




Anonymous.  1977. United States investigations to evaluate the potential




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Basiouny, F. M., T. K. Van, and R. H. Biggs.  1978.  Some morphological




  and biochemical characteristics of C_ and C. plants irradiated rath




  UV-B.  Physiol. Plant 42:29-32.




Bennett, J. H.  1978.  Effects of UV-B radiation on photosynthesis and




  growth of selected agricultural crops.  Final EAGER Report submitted




  to the Environmental Protection Agency.   14 pp.




Biggs, R. H.  1975.  Effects on plants of  increased UV-B radiation.




   p. 62-65.  In Fourth Conference on CIAP.  U.S. Department of Transpor-




  tation (T. M. Hard and A. J. Broderick,  ed.) Washington, D.C.




Biggs, R. H.,  and F. M. Basiouny.  1975.  Plant growth responses to




  elevated UV irradiation under growth chamber, greenhouse, and solarium




  conditions.   In Impacts of^Climatic Changes on the Biosphere.  I.




  Ultraviolet radiation effects.   Climatic Impact Assessment Program




  Monograph 5:195-248.




Brandle, J. R., W. F. Campbell, W. B. Sisson, and M. M. Caldwell.  1977.




  Net photosynthesis, electron transport capacity, and ultrastructure of




  Pisum sativum L. exposed to ultraviolet-B   radiation.   Plant Physiol.




  60:165-169.
                                12

-------
Caldwell, M. M.  1968.  Solar ultraviolet radiation as an  ecological  factor




  for alpine plants.  Ecol. Monogr. 38:243-268.




Caldv.-ell, M. M.  1971.  Solar UV irradiation and  the growth and




  development of higher plants.  In (A. C. Giese, ed.) Phytophysiology,




  Acad. Press, New York.  6:131-177.




Caldwell, M. M.  1977.  The effects of solar UV-B radiation (280-315  ran)




  on higher plants:  Implications of stratospheric ozone reduction.   p.




  597-607.  _Tn Research in Photobiology (A. Castellani, ed.) Plenum Press,




  New York.  726 p.




Cams, K. R., R. Thimijan, and J. M. Clark.  1977.  Outline of irradiance




  distribution of fluorescent lamps and combinations.  Paper 5.6.  Presented




  at Symposium on Ultraviolet Radiation Measurements for Environmental




  Protection and Public Safety.  National Bureau of Standards, June 8-9,




  1977.  p. 74-76.  In Final Program and Abstracts.




Dickson, J. G. and M. M. Caldwell.  1978.   Leaf development of Rumex




  patientia L. (Polygonaceae) exposed to UV irradiation (280-320 nm),




  Amer. Jour. Bot. 65(8):857-863.




Hammer, P. A., T. W. Tibbitts, R. W. Langhans, and J. C. McFarlane.




  1978.  Base-line growth studies of 'Grand Rapids' lettuce in controlled




  environments.  J. Amer. Soc.^Hort. Sci.  103(5):649-655.




Interagency Task Force on Inadvertant Modification of the  Stratosphere




  (IMOS).  1975.  Fluorocarbons and the Environment.  White House Council




  on Environmental Quality and Federal Council for Science and Technology.




  Washington, D.C.,.NSF 75-403.  109  p.




Kriiiek, D. T. (Chairman), IMOS Subcommittee, on Biological  and Climatic




  Effects Research.  1975a.  A proposed federal program to determine  the




  biological and climatic effects of stratospheric ozone reduction.




  Federal Council for Science and Technology, Washington, D.C. Draft  Report.




  93  p.                                                    .




                               13

-------
Krizek, D. T.   1975b.   Influence  of  ultraviolet  radiation  on  germination




  and  early  seedling growth.  Physiol.  Plant  34(3):182-186.




Krizek, D. T.   1976.   Influence of increased  UV-B  radiation on  agricul-




  tural production,  p.  336-365.  In Stratospheric Ozone Research  and




  Effects.   Hearings before  the subcommittee  on  the Upper  Atmosphere of




  the  Committee on Aeronautical and  Space Sciences.  U.S.  Senate,  94th




  Congress,  2nd session.




Krizek, D. T.   1977a.   Biological and climatic effects  of  stratospheric




  ozone reduction:  A  progress report on the  EAGER program.   Presented




  at Amer. Society for  Photobiology  Meetings.  San Juan, Puerto  Rico,




  May  15, 1977.




Krizek, D. T.   1977b.   Current UV measurement methodology  and future needs




  in photobiological research.  Paper No. 5.2.   Presented  at  Symposium




  on Ultraviolet Radiation Measurements for Environmental  Protection and




  Public Safety.  National Bureau of Standards.  June 8-9, 1977. p. 49-52.




  In Final Program and  Abstracts.




Krizek, D. T. and E. J. Koch.  1978.  Use of regression analysis in




  obtaining  estimates  of UV  spectral irradiance  under FS-40 fluorescent




  sunlamps filtered with cellulose acetate.  Final BACER Report  submitted




  to the Environmental  Protection Agency. 24 p.




Krizek, D. T., R. L. Schaefer, and R. A.'Rowland.  1975.   Influence of




  UV-B radiation on vegetative growth of Pisum sativum L.  'Alaska'.




  HortScience 11(3).-22.  (Abstract).




Krizek, D. T. and P. Semeniuk.  1975.  Comparative sensitivity of




  bedding plants to UV-B radiation.   HortScience 10(3):323.  (Abstract).




Molina, M. J. and F. S. Rowland.  1974.   Stratospheric sink for




  chlorofluromethanes:   chlorine atom-catalyzed  destruction of ozone.




  Nature 249:810-812.
                                14

-------
Nachtwey, S. L.  (ed.).  1975.  Climatic Impact Assessment  Program

   (CIAP).  Monograph 5, Impacts of Climatic Change on  the  Biosphere.

  Part 1.  Ultraviolet Radiation Effects.  U.S. Department  of .Transpor-

  tation. Washington, D.C.

Norris, K. H.  1977.  Development of a portable, automated  UV-B spectro-  '

  radiometer.  Paper No. 5.5.  Presented at Symposium  on Ultraviolet

  Radiation Measurements for Environmental Protection  and  Public Safety.

  National Bureau of Standards.  June 8-9, 1977.  p. 72-73.  In Final

  Program and Abstracts..

Rowan, J. D. and K. H. Norris.  1978.  Instrumentation for  measuring

  irradiance in the UV-B region.  Final BACER Report submitted to  the

  Environmental Protection Agency.  69 p.

Sisson, W. B. and M. M. Caldwell.  1976.  Photosynthesis,  dark respiration

  and growth of Rumex patientia L. exposed to ultraviolet  irradiance

  (288 to 315 nanometers) simulating a reduced atmospheric  ozone column.

  Plant Physiol. 58:563-568.

Sisson, W. B. and M. M. Caldwell.  1977.  Atmospheric  ozone depletion:

  reduction of photosynthesis and growth of a sensitive higher plant

  exposed to enhanced UV-B radiation.  Jour. Expt. Bot. 28:691-705.
                            \
Thimijan, R. W., H. R. Cams, and L. E. Campbell.  1978.  Radiation sources

  and related environmental control for biological and climatic effects

  UV research (BACER).  Final BACER Report submitted to the Environmental

  Protection Agency.  78 p.

Van, T. K. and L. A. Garrard.  1975.  Effect of UV-B radiation on  net

  photosynthesis on some Cซ and C  crop plants.  Proc. Soil and Crop

  Sci. Soc.  Fla. 35:1-3.

Van, T. K.,  L. A. Garrard,  and S. 11. West.  1976.  Effects of UV-B

  radiation on net photosynthesis of some crop plants.  Crop Sci.  16:715-718,
                              15

-------
      1.   Weighted and  unweighted. UV spectral  irradiance  under each of  four  UV set-ups




          in the greenhouse.   Each set-up  contained  eight FS-40 fluorescent  sunlamps filtered with




          0.127 mm (0.005  in.  =  5  mil)  Mylar  (M-5) or  cellulose acetate (CA-5).   Data shown are




          mean values with their standard  errors  (SE)  and ranges of 48  pot  locations per cultivar




          per set-up.
Set-Up UV Treatment Cv

1

2

3

4

Filter Max Ht.
Above
Canopy
cm
M-5 97 'Poinsett'
'Ashley'
CA-5 143 'Poinsett'
'Ashley'
CA-5 92 'Poinsett1
'Ashley'
CA-5 54 'Poinsett'
'Ashley'
IRL UV Meter
Reading
scale
3.1
3.1
17.6
17.6
29.6
29.6
41.0
40.9
+ 0.1
+ 0.1
+ 0.5
+ 0.5
+ 1.3
+ 1.3
+ 2.6
+ 2.6
UV-B
Mean + SE


2.2
2.2
3.6
3.6
5.0
5.0

-
+ 0.1
+ 0.1
+ 0.2
+ C.2
+ 0.3
+ 0.3
SE
Range
_
-
1.4-2.8
1.4-2.8
1.7-5.4
1.7-5.4
1.6-8.7
1.5-8.7
BUV-/


Irradiance
Weighted
mW-m
Mean + SE
_
-
6.73 +
6.73 +
11.02 +
11.02 +
15.30 +
15.30 +


0.
0.
0.
0.
0.
0.


3
3
6
6
9
9
UV-B

Spectral
Irradiance
Unweighted
mW • m
_
-
548.
548.
920.
920.
1273.
1270.


37
37
32
32
68
58
-  BUY = 3.06 x UV-B SE

-------
Table 2.  Criteria for Scoring Chlorosis
               in Cucumber Leaves
Index of
Injury
0
0.2
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
Percent
Chlorosis
None
Trace
5%
10.0%
15.0%
20.0%
25.0% .
30.0%
35 . 0% .
40.0%
45.0%
50.0%
            17

-------
                 Table 3.  Influence of increased UV-B irradiation on index of leaf injury, fresh weight and
                           percent dry weight of tops in 'Poinsett'  (P) and 'Ashley' (A) cucumber plants
                           irradiated for 19 days in the greenhouse (April 1-20, 1977).  Plants exposed to a
                           UV gradient provided by FS-40 fluorescent sunlamps filtered with 0.127 mm  (0.005 in.
                           = 5 mil) Mylar (M-5) or cellulose acetate (CA-5) at various distances above the
                           canopy.  Data shown are mean values with their standard errors and ranges  of 48
                           plants of each cultivar per treatment.
oo
Set-Up UV Treatment



1

2

3

4

Filter Max Ht.
Above
Canopy
cm
M-5 97

CA-5 143
*
CA-5 92

CA-5 54

Cv

..
'Poinsett'
'Ashley'
'Poinsett'
'Ashley'
'Poinsett'
'Ashley'
'Poinsett'
'Ashley'
Index of Injury
Mean + SE


0
0
1.5 + 0.1
0.7 + 0.1
2.8 + 0.2
0.9 + 0.1
4.5 + 0.3
1.5 + 0.2
Range


0
0
1-3
0-3
1-5
0-3
1-9
0-3
Mean


12.0
12.7
11.1
11.7
9.2
11.3
8.6
10.2
Fresh weight %
of tops (g)
+ SE


+ 0.3
+ 0.3
+ 0.2
+ 0.2
+ 0.3
+ 0.3
+ 0.4
+ 0.3



8.
8.
8.
8.
4.
6.
4.
6.
Range


5-17.6
3-19.3
4-15.2
9-14.7
4-13.2
7-15.6
0-13.2
6-15.6
Mean


9.2
9.3
9.5
10.0
9.4
10.0
9.5
10.1
Dry weight
of tops
+ SE


+ 0.9
+ 0.1
+ 0.1
+ 0.1
+ 0.1
+ 0.1
+ 0.2
+ 0.1
Range


7.5-11.6
7.0-10.5
8.4-11.0
8.9-11.4
8.5-11.1
9.2-11.3
6.3-15.7
9.4-11.0

-------
Fig.  1   Appearance of 'Poinsett'  (green label) and 'Ashley' (yellow




         label)  cucumber plants grown for 19 days from seeding in the




         greenhouse under FS-40 fluorescent sunlamps filtered with 0.127




         mm (0.005 in.)  Mylar.   Plants received UV-A irradiation but




         no supplemental UV-B irradiation.   Note absence of chlorosis.
                                 19

-------
w   ^





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                                                   .
                                                                  •.-
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.

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j

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•
i
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f ""." -- *r*i ' -i *-"x
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.




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                                                                                                                  i


                                                                                                               ,


                                                                                                               t     -



-------
Fig.  2  Appearance of 'PoinseLt1  (green label) and 'Ashley' (yellow


        label) cucumber plants after 19 days of enhanced UV-B irradiation

                                _2
        (6.73 mean weighted mW-m   of BUV) provided by FS-40 fluorescent


        sunlamps filtered with 0.127 mm (0.005 in.) cellulose acetate.
                               21

-------
                                                   XUaJfc,
                          ......   -   : ,* .    11 f_^~-
                  -,        ..  -^K    -    ซ"4ป vT/,    -^

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                  3  *-J.,  '.  ^^^ +&&

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\, j > • .. v
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-

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-------
Fig. 3   Appearance of 'Poinsett1 (green label) and 'Ashley' (yellow


         label) cucumber plants after 19 days of enhanced UV-B irradiation

                                  _2
         (11.02 mean weighted mW'm   of BUV) provided by FS-40 fluorescent


         sunlamps filtered with 0.127 mm (0.005 in.) cellulose acetate.
                                 23

-------
r
                                                                 •*•*•" -ซ * "*
                                                                             ,

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                     ซ•"ป.
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                                                                                                                       |

-------
Fig.  4   Appearance of 'Poinsett' (green label) and 'Ashley* (yellow



          label) cucumber plants after 19 days of enhanced UV-B


                                               -2
          irradiation (15.30 mean weighted mW-m   of BUV) provided by



          FS-40 fluorescent sunlaraps filtered with 0.127 ram (0.005 in.)



          cellulose acetate.
                                  25

-------


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-------
Fig.  5   Comparative sensitivity of 'Poinsett1 (top row) and  'Ashley'



          (bottom row) cucumber plants to increased UV-B irradiation.



          Plants irradiated for 19 days from time of seeding in the



          greenhouse under FS-40 fluorescent sunlamps filtered with



          0.127 mm Mylar (M-5) or 0.127 mm cellulose acetate (CA-5).



          The latter plants received a mean level of biologically



          effective UV (BUV) irradiance of 6.73, 11.02, or  15.30 weighted


           „  -2         .   .
          row-in  , respectively.
                              2 7

-------
                          P
                                                                                                            '
                                                                               17 DAYS OlD L



                                                                               T R T 17 DAYS I
                                                                                  C f N T E R f
                                                                                          r •
                                                                                                                           t
                                                                                                                        *-*ซ*•;.


NJ
CO
                                                                                               '
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                                                                                                                                           .>

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                                                                                                                                    .— -i~. ...   . ^

-------
    10

3   8
z
o   6
X
S   4
Z
               FS40 & CAS
                         CUCUMBER

                         POINSETT
Y =0.9018 + 0.2324XXป.
r = 0.80 8 9       „.••*****




                        10      15      20     25

                         BUV (WEIGHTED mWm'2)
                           30
Figure 6.  Linear regression of the index of injury for 'Poinsett'
           cucumber leaves vs. biologically effective UV (BUV)  radiation
           in weighted mW-nT .  Plants were irradiated in the greenhouse
           for 19 days from seeding under eight FS-40 lamps filtered
           with 0.127 mm cellulose acetate under a UV gradient  ranging
           from A.9 to 26.6 mW-m   BUV (1.6 to 8.7 UV-B sun equivalents).
           (One UV-B sun equivalent = 3.06 weighted mW'nT2 of BUV).
                                  29

-------

cs
<
ki-
ll
O

100
ง90
ง 80
o 70
60
50
FS40 & CAS
ASHLEY CUCUMBER
X,
\. \
\x sfi
U POINSETT %(S V
•"• *ซ4>
"^^
n J^
1 1? * I_ _J___ ^co)
   6         9        12

BUV (WEIGHTED mWm'2)
                                                           15
Figure 7.   UV dose-response relationship  under  greenhouse  conditions.
           Comparative leaf areas  of  'Poinsett* and  'Ashley' cucumber
           plants expressed as  percentages  of Mylar  controls.  Plants
           irradiated for 19 days  from  seeding  in  the  greenhouse under
           eight FS-40 lamps filtered with  0.127 mm  cellulose acetate.
           Lamps mounted in separate set-ups at 1.43,  0.92, and 0.54
           m above the plants.  Means and standard errors  are shown   __
           for 48 plants within each set-up for 6.7, 11.0  or 15.3 mW-m
           BUV (2.2,  3.6, or 5.0 UV-B sun equivalents)  respectively.
                             30

-------


to
a. cr
O o
•-ฃ
u. z
n ฐ
W U
H- a:
O >^
UJ ^
5 o
o



100


90

80

70
60
50
FS40 & C&5
•wb xv^v n ASHLEY CUCUMBER
****** ;ป. ^ ^ ^ ^ V X r-*T-l
***/^ XxLJ \
\ XVVVXV -r
\ . . XVD
*%
*Xr
POINSETT CX
*Vป T*
** JL
****aป - ^^
— ^
-
0 B S I 9
0 36 9 12 15
                                                 -2,
                          BUY (WEIGHTED mWm'z)
Figure 8.  UV dose-response relationship under greenhouse  conditions.
           Comparative dry weights of tops of 'Poinsett' and  'Ashley1
           cucumber plants expressed as percentages  of  Mylar  controls,
           Plants exposed to the UV-B irradiation  gradients described
           in Fig.  7.
                               31

-------

o
ง
CA
W 4
D.
o
H-
UL.
0
I
o
UJ
ฃ
Qi
Q



1200

1000

800

600

400

200
FS40 & CAS CUCUMBER
POINSETT
'*X V ~ 1285.09-30.5980X
**X r - 0.7662
-
\
\

" ******
\
M

J 8 B I B |
0 5 10 15 20 25 30
                         BUV (WEIGHTED
Figure 9.   Linear regression of dry weight  of tops of 'Poinsett'  cucumber
           plants vs.  exposure to biologically effective UV (BUV)  radiation
           in mW-rn  .   See Fig. 6 legend.
                               32

-------



-------
                  FINAL REPORT
      USE OF REGRESSION ANAYLSIS IN OBTAINING ESTIMATES
      OF UV SPECTRAL IRRADIANCE UNDER FS-40 FLUORESCENT
            SUNLAMPS FILTERED WITH CELLULOSE ACETATE
                  D. T. Krizek
                  E. J. Koch
               Plant Stress Laboratory
             Plant Physiology Institute
       Beltsville Agricultural Research Center
              Beltsville, Maryland 20705

                      and

              Northeastern Region
                EPA-IAG-D6-0168
                Project Officer:
                R. J. McCracken
Agricultural Research, Science and Education Administration
           U.S. Department of Agriculture
             Washington, D.C. 20250
                Prepared for:

           Environmental Protection Agency
                 EAGER Program
             Washington, D.C. 20460.

-------
                           CONTENTS


Abstract	iii

Figures and Tables	iv

Acknowledgments	vi

     1.  Introduction	   1

     2.  Materials and Methods	   3

             UV Source	3
             UV Instrumentation	   3
             UV Measurements	4
             Regression Analysis	  5

     3.  Results and Discussion	6

             Weighted and Unweighted Measurements of
                UV-B Irradiance  •  •'	6
             Relationship Between IRL-UV Meter Readings
                and UV-B Spectral Irradiance	7
             Relationship Between UV-B Sun Equivalent and
                UV-B Spectral Irradiance	8

     4.  Literature Cited  	  10

-------
                          .   ABSTRACT





     Weighted and unweighted UV spectral measurements x^ere obtained under



two Westinghouse FS-40 fluorescent sunlamps filtered with 0.127 mm (0.005 inch



or 5 mil) cellulose acetate using newly developed broad-band UV radiometers



and an automated UV spectroradiometer in 10 cm increments from 20 cm to



110 cm.  Correlations were determined between sets of data obtained x^ith



the broad-band radiometer and the UV spectroradiometer.



     Linear regression analyses were performed on the weighted and unweighted



spectral data to obtain regression equations for predicting UV-B irradiance


                -2                                                 -2
(unweighted mW-rn  ), biologically effective UV (BUV) (weighted mW.m  ) in



the 280-320 nm (UV-B) region, UV-B sun equivalents, and incident UV flux


                               -2     21
in the UV-B region in photons-m   x 10   integrated over a 6-hour exposure.



     Examination of the correlation coefficients (r values) indicated



excellent agreement between measured and predicted values for all comparisons



(r values of 0.9972 to 0.9998).



     Use of the regression equations should permit accurate and rapid



estimates of both weighted and unweighted UV irradiances at any location in



an experimental set-up and provide a useful means of making interlaboratory



comparisons of spectral measurements.
                                iii

-------
                    List of Figures and Tables                              Page



Figure 1.  Regression  of UV spectral irradiance  in  the  280-320 nra            14



           (UV-B) region versus IRL UV-meter  (10  scale) reading.



Table 1.   Weighted and unweighted measurements  of  UV-B radiation            15



           obtained on Nov. 8, 1977, under two Westinghouse FS-40



           fluorescent sunlamps filtered with 0.127 mm  (0.005 in.)



           cellulose acetate  (aged 6 hours).  Measurements taken



           at 10 cm intervals from 20 cm to 110  cm.



Table 2.   UV sun equivalents obtained at various distances under            16



           Westinghouse FS-40 fluorescent sunlamps  filtered with



           0.127 mm (0.005 in.) cellulose acetate (aged 6 hours).



Table 3.   Biologically effective UV (BUY) radiation in the 250-279,         17


                                                        -2
           280-330, and 250-369 nm region (weighted mW-en  ) under two



           Westinghouse FS-40 fluorescent sunlamps  filtered with 0.127



           mm (0.005 in.) cellulose acetate (aged 6 hours).


                                               _2
Table 4.   Total UV irradiance (unweighted mW-m  )  under two Westinghouse    18



           FS-40 fluorescent sunlamps filtered with 0.127 mm (0.005 in.)



           cellulose acetate (aged 6 hours).   Measurements taken every



           nm from 250 to 369 nm with an Optronic UV spectroradiometer.



Table 5.   Actual and  predicted relationship between IRL UV-meter reading    19



           (10  scale) and unweighted UV spectral irradiance in the 280-320



           nm (UV-B) region.  Two FS-40 sunlamps plus 0.127 mm (0.005



           in.) cellulose acetate (aged 6 hours).



Table 6.   Actual and  predicted relationship between IRL UV-meter reading    20



           (10  scale) and weighted UV spectral irradiance in the 280-320



           nm (UV-B) region.  Two FS-40 sunlamps plus 0.127 mm (0.005 in.)



           cellulose acetate (aged 6 hours).
                               IV

-------
                                                                             Page




Table 7.   Actual and predicted relationship between  UV-B  sun                  21



           equivalent as measured with an Optronic Model 725 broad-band



           radiometer (0-10 scale and 0-5 scale)  and  unweighted  UV  spectral


                           _2
           irradiance (mW-m  ) in the 280-320 nm  region.   Two  FS-40



           sunlamps plus 0.127 mm (0.005  in.) cellulose acetate (aged 6



           hours).



Table 8.   Actual and predicted relationship between  UV-B  sun  equivalents      22



           as measured with an Optronic Model 725 broad-band radiometer



           (0-10 scale and 0-5 scale) and biologically effective UV



           (BUV) radiation in the 280-320 nm region.  Two  FS-40  sunlamps



           plus 0.127 mm (0.005  in.) cellulose acetate  (aged 6  hours).



Table 9.   Actual and predicted relationship between  UV-B  sun  equivalent       23



           as measured with an Optronic Model 725 broad-band radiometer



           (D-10 scale) and incident UV flux in the 280-320 nm  (UV-B) region


                                -2     21
           measured in photons'm   x 10   integrated  over  a 6  hour  day.



           Two FS-40 sunlamps plus 0.127 mm (0.005 in.) cellulose acetate.



Table 10.  Actual and predicted relationship between UV-B  sun  equivalent       24



           as measured with an Optronic Model 725 broad-band radiometer



           (0-5 scale) and incident flux in the 280-320 nm (UV-B) region
                             ^

                                -2     21
           measured in photons-m   x 10   integrated over  a 6  hour  day.



           Two FS-40 sunlamps plus 0.127 mm (0;.0.0.5 :in.) cellulose acetate.
                                 v

-------
                              ACKNOWLEDGMENTS




     We gratefully acknowledge Karl H. Norris, James D. Rowan, and




George F. Buttons, Instrumentation Research Laboratory, for their




outstanding cooperation in developing the instrumentation used in this




study and assisting in the calibration of these instruments; we thank




Scott J. Ravitz for his competent technical assistance in obtaining




these measurements.  We also appreciate the assistance of Lowell E. Campbell,




Stanley Holliday, and Richard W. Thimijan, Agricultural Equipment




Laboratory, in constructing the UV irradiation set-ups.
                                vi

-------
 USE OF REGRESSION ANSYLSIS  IN  OBTAINING ESTIMATES  OF  UV  SPECTRAL  IRRADIANCE



       UNDER FS-40 FLUORESCENT  SUNLAMPS  FILTERED  WITH  CELLULOSE ACETATE


                                 I/                  21
                 Donald  T. Krizek-   and  E.  James  Koch-



                             INTRODUCTION



      Broad-band  studies on  the influence of ultraviolet  radiation  in the



 280-320 nm (UV-B)  region on plant growth and  development  conducted since



 1972  during the  Climatic Impact  Assessment Program (CIAP) have demonstrated



 the urgent need  for  improved UV  sources  and instrumentation  (Ambler et al.,



 1975;  Biggs,  1975; Brandle  et  al.,  1977; Krizek, 1975a, b; 1977a, b;



 Caldwell,  1971,  1972, 1977;  Ormrod  and  Krizek, 1978;  Sisson and Caldwell,



 1975,  1976,  1977;  Skelly et  al., 1978; Anon;,  1977; Nachtwey, 1975; Van



 and Garrard,  1975, 1976.



      With  the recent development of broad-band radiometers and an automated



 UV  spectroradiometer (Norris,  1977; Rowan and Norris, 1978; Cams et al.,



 1977)  and  improvements  in spectroradiometers  used  in  CIAP (Kostkowski



 and Saunders, 1977)  the researcher  now has the means  of obtaining greatly



 improved UV measurements.



      The objective of the present study was to provide the investigator



 involved in the  EPA  Interagency  Biological and Climatic Effects Research (EAGER)



 program a  means  of obtaining Estimates of weighted and unweighted spectral



 irradiance in the UV-B  region.   The use  of regression analysis of spectral



 data  obtained with both broad-band  radiometers and an automated UV spectro-



 radiometer is described.
—  Plant Physiologist, Plant Stress Laboratory, Agricultural Research, Science



   and Education Administration, USDA, Beltsville, Maryland 20705.


2/
—  Biometrician, Agricultural Research, Science and Education Administration,



   USDA, Beltsville, Maryland 20705.




                                -1-

-------
     A series of regression equations is described for relating weighted




and unweighted spectral data in the UV-B region.   Use of these equations




should facilitate comparison of spectral measurements obtained in




different laboratories.

-------
                        MATERIALS AND METHODS




 UV Source



                                               3/
      UV radiation was provided by Westinghouse—  FS-40  fluorescent  sunlamps




 that  had been aged at least 100 hours according to  a standard  protocol




 (Krizek, 1977b).   The lamps were mounted  in  a  single 1.2  meter (A foot)




 fluorescent fixture without a  special reflector (Thimijan et al., 1978)




 and covered with  0.127 mm (0.005 in.  = 5  mil)  cellulose acetate  (CA).




 The CA  filters were aged  for 6 hours  on a specially  designed lamp rack




 before  being used in the  study (Thimijan  et  al.,  1978).




 UV Instrumentation




      Broad-band UV-B irradiance levels were  determined by means of  three




 instruments developed by  Norris and his associates in the USDA Instrumentation




 Research Laboratory (IRL)  at Beltsville,  Maryland, or based on his  specifications




 (Norris,  1977;  Rowan and  Norris,  1978):   (a) an IRL Meter UV-B radiometer




 (IRL  UV meter); (b)  an Optronic Laboratories,  Inc. Model  725 UV-B radiometer


                                                                    _2

 calibrated  to read from 0  to 5  UV sun equivalents [or 0 to 15.3 mW'm   of




 biologically effective UV  (BUV)  radiation (Cams et al.,  1977)]; and (c)




 the same instrument  calibrated  to read  from  0  to 10 UV sun equivalents (or



               -2
 0  to  30.6 mW-m   of  BUV radiation).




      The IRL meter was used  to  obtain unweighted UV-B measurements.   The



                                                                        3/
 instrument  consists  of a solar-blind vacuum photo-diode (Hamamatsu R403)— ,




 an integrated circuit  amplifier,  and a microammeter packaged in a meter case




 (Rowan  and  Norris,  1978; Norris,  1977).   The circuit provides  for four decades
3/
—  Mention of a trademark, proprietary product, or vendor does not constitute




   a guarantee or warranty of the product by the USDA and does not imply its




   approval to the exclusion of other products or vendors that may also be




   suitable.
                               —3—

-------
of range  switching,  referred  to as 10  , 10  , 10  , and 10  .  In the present

              7        8
study,  the 10  and 10 scales were used.  The spectral sensitivity of the


IRL meter in  the 280 to  320 nm region  is relatively flat with maximum


sensitivity at 300 nm.   The detector is insensitive at x^avelengths longer


than 400 nm (Rowan and Norris, 1978).


     UV spectral irradiances were determined every nm from 250 to 369 nm


with an automated spectroradiometer  (with a 2 nm bandwidth) developed by


IRL and commercially available from Optronic Laboratories, Inc. Specifications


for this instrument  are  described by Norris, 1977, and Rowan and Norris, 1978.


UV Measurements


     UV measurements were taken with each of these three broad-band radiometers


and the automated spectroradiometer described at 10 cm intervals from 10 cm


to 110 cm.  The sensor was placed under the center of the lamp fixture and


adjusted by means of a standard laboratory jack.  All measurements were


taken in a darkened  room with only UV lamps on.   Air temperature was maintained


at 25ฐC.

                                                    _2
     Weighted irradiance levels are reported as mW'm   BUV, the biologically


effective UV radiation derived from the weighting function (AZ9)  described


by Thimijan et al.,  1978, and Cams et al., 1977.


     Since UV irradiation used in this study was obtained by filtering FS-40


lamps with CA, BUV was essentially confined to the UV-B region.  Unweighted


irradiances in the UV region were obtained by summing measured or calculated


values at each nanometer from 250-279 nm (UV-C), 280-320 nm (UV-B) ,  a.nd


321-369 nm (UV-A).   Although spectral data were taken previously at  every


nm from 321-400 nm, no data were taken beyond 369 nm in the present  study,


since the UV-B portion of the spectrum was  the major region of concern


in the BACER program.

-------
                      -2                      -2
     Dividing the mW-ra   BUV by 3.06 (the mW'm   BUV of the Beltsville



control sunshine) provides the fraction of BUV measured at each location



relative to that of one Beltsville control sunshine.



Regression Analysis



     Linear regression analyses were performed on the weighted and unweighted



spectral data to obtain regression equations for predicting UV-B irrsdiance


     -2                     -2
(mW*m  ), BUV (weighted mW'm  ) in the 280-320 nm region, UV-B sun equivalents,


                                                    -2     21
and incident UV flux in the UV-B region in photons-m  " x 10   integrated



over a 6-hour day exposure.
                                —5—

-------
                         RESULTS AND DISCUSSION


Weighted and Unweighted Measurements of UV-B Irradiance


     Weighted and unweighted spectral measurements obtained at various


distances from a pair of FS-40 fluorescent sunlamps filtered with CA are


shown in Table 1.  In general, there was good agreement between the values


obtained for UV-B sun equivalents obtained on the Optronic radiometers and


those calculated from unweighted measurements obtained on the IRL UV


meter using the conversion factors provided by Thimijan et al. 1978.

     The UV-B sun equivalents obtained by summing values every nm from


280-320 nm (Table 1) or 250-329 nm (Table 2) on the automated spectroradio-


meter by means of computer calculation also agreed within 5 to 10% of


those obtained with the broad-band radiometers (Tables 1, 2).


     By using a CA filter over the FS-40 sunlamps, little UV radiation


in the 250-279 nm region was transmitted (Table 2).  The level of BUV


radiation transmitted in the 250-279 nm (UV-C); 280-330 nra and 250-330


nm regions under FS-40 lamps filtered with CA are shown in Table 3.  Since


UV-C radiation contributed virtually no measurable BUV, the total amount


of BUV obtained in the 250-330 nm region was approximately the same as


that obtained by summing the BUV values in the 280-330 nm region alone
                             *

(Table 3).
                                         *
     The total unweighted UV irradiance obtained at each 10 nm interval in


the 250-279 nm (UV-A), 280-320 nm (UV-B), 321-369 nm  (UV-C) and 250-369 nm

regions is shown in Table 4.   About 50% of the total UV irradiance


transmitted in the region of 250-369 nm was at 280-320 nm and 50% at


321-369 nm (Table 4).
                                   -6-

-------
Relationship Between  IRL-UV Meter Readings and UV-B Spectral Irradiance




     The actual and predicted relationship between broad-band radiometer




readings with an  IRL  UV meter and unweighted spectral irradiance in the




280-320 nm  (UV-B) region under  two FS-40 sunlamps filtered with CA is




shown in Table 5.  A  plot of these data (Fig. 1) and evaluation of the




correlation coefficient (r = 0.9995) indicate that the relationship between




UV spectral irradiance obtained on the UV spectroradiometer and that predicted




by the regression equation from the IRL meter readings agrees very well.




     The relationship between IRL UV meter reading and incident UV flux



                                   —2     21
at 280-320 nm measured in photons-m   x 10   integrated over a 6-hour




period is also shown  (Table 5).  Since most studies in the EAGER program on




simulation of ozone depletion were based on 6 hours of UV irradiation per




day, the calculations for incident flux are based on this duration of exposure,




rather than on the basis of seconds or minutes.  The correlation coefficient




(r) of 0.9976 indicates that there is good agreement between the calculated




and predicted incident UV flux in the 280-320 nm region (Table 5).   This




equation may, therefore, be used to describe the UV-B irradiance in terms




used by the photobiologist (see e.g., Seliger, 1978; Rupert, 1978;  Rupert




and Latarjet, 1978; Caldwell, 1972).

                            \

     The relationship between IRL meter reading and BUV radiation  in the




280-320 nm region is shown in Table 6.   The r values of 0.9992 obtained




indicates that the agreement between predicted and actual BUV is nearly




perfect.   The relationship between IRL meter readings and UV-B sun  equiva-




lents is  also shown (Table 6).   The r of 0.9991 obtained indicates  that




there is  excellent agreement between predicted and determined UV sun




equivalents.






                                   —7—

-------
     As rule of  thumb,  instantaneous meter readings  on  the  UV-B broad-


band radiometer  (IRL Meter) may be converted  to UV-B spectral irradiance


in photons-m~2 for a 6-hr exposure by multiplying  the reading by 10   .


Relationship Between UV-B Sun Equivalent and  UV-B  Spectral  Irradiance


     The relationship between UV-B sun equivalent  as measured with an


Optronic Model 725 broad-band radiometer (either the 0-10 scale or the


0-5 scale) and the unweighted UV spectral irradiance in  the 238-320 nm


region is shown  in Table 7.  The r's obtained, namely, 0.9996, 0.9995,


respectively, indicate  that the regression equations described  (Table


7) may be used to accurately estimate the total UV-B irradiance obtained


at any of the cooperating laboratories participating in  the BACER ter-


restrial effects program that were sent these instruments.


     Since all of the Optronics Model 725 radiometers were calibrated by


IRL under a pair of FS-40 fluorescent sunlamps filtered with 0.127 mm


(0.005 in.) CA aged 6 hours, it should be possible to obtain an intra-


laboratory and interlaboratory comparison of weighted and unweighted


spectral irradiance used at each pot location in any particular study by


use of these regression equations provided that no filters  were used over


the sensor.
                             *

     The relationship between UV-B sun equivalent as measured with an


Optronic Model 725 broad-band radiometer (either the 0-10 scale or the


0-5 scale) and the BUV radiation in the 280-320 nm region is shown in


Table 8.  The r values obtained, namely, 0.9998 and 0.9996, respectively,


indicate that the regression equations obtained may be accurately used to


estimate the level of BUV obtained at any location in the experimental


set-up.

-------
     The relationship between UV-B sun equivalent as measured with an




Optronic Model 725 broad-band radiometer  (either the 0-10 scale or the


                                                                      _2

0-5 scale) and the incident flux in the 280-320 nm region in photons-m




x 1021 per 6 hr day of UV irradiation is  shown in Tables 9 and 10.  The




r values obtained (Tables 9 and 10), namely, 0.9972 and 0.9974, respectively,




indicate that the regression equations obtained may be accurately used



                                              —?     21
to estimate the incident UV flux in photons-ra    x 10   received by a




plant during a single 6-hr period of UV exposure.




     Use of the regression equations described in this report should enable




the investigator to make accurate and rapid estimates of both weighted




and unweighted UV irradiance at any location in an experimental UV set-up,




provided that FS-40 fluorescent sunlamps  and 0.127 mm (0.005 in.)  cellulose




acetate filters  are used.  By doing so,  countless hours can be saved by




not having to make spectroradiometrie measurements at more than a few




selected locations.




     Thimijan et al. (1978) have described a method of calculating the




spectral power output under both filtered and unfiltered FS-40 lamps as




well as Westinghouse BZS lamps by summing values in 5 nm increments from




270 to 320 nm and adding the power output of the Hg lines at 253.6, 289.4,




296.7,  302.2,  and 313 nm.  This procedure^ however,  is more time-consuming




than the present method of using regression equations.
                                 -9-

-------
                              LITERATURE  CITED
     \



Ambler, J. E., D. T. Krizek,  and P.  Semeniuk.   1975.   Influence  of  UV-B


     radiation on early  seedling growth  and  translocation  of  ฐ-*Zn frOm



     cotyledons in cotton.  Physiol. Plant 34(3):177-181.


Anonymous.  1977. • United States investigations  to evaluate the  poten-



     tial threat of stratospheric ozone  diminution.  Presented at United


     Nations Environment Programme  (UNEP) International Meeting  on  the


     Ozone Layer.  Washington, D.C.  March 1-7,  1977.


Biggs, R. H.  1975.  Effects  on plants of increased UV-B radiation. Pp.


     62-65.  In Fourth Conference on CIAP.   U.S.  Dept. of  Transportation


     (T. M. Hard and A. J. Broderick, ed.) Washington, D.C.


Brandle, J. R., W. F. Campbell, W. B. Sisson, and M. M. Caldwell.   1977.


     Net photosynthesis, electron transport  capacity,  and  ultrastructure


     of Pisum sativum L. exposed to ultraviolet-B radiation.  Plant Physiol.



     60:165-169.


Caldwell, M. M.  1971.  Solar UV irradiation and  the growth and development


     of higher plants.  In_ A. C. Giese,  ed. Photophysiology 6:131-177.



     Acad. Press, New York.

                             \
Caldwell, M. M.  1972.  Biologically effective solar ultraviolet irradiation


     in the Arctic.  Arctic and Alpine Research 4(1):39-43.


Caldwell, M. M.  1977.  The effects of solar UV-B radiation (280-315 nm)


     on higher plants:  Implications of  stratospheric ozone reduction.


     Pp. 597-607.   In_ A. Castellani, ed.  Research in Photobiology.


     Plenum Press, New York.   726 p.
                                  -10-

-------
Cams, H. R., R. Thimijan,  and J. M. Clark.  Outline of irradiance distri-




     bution  of  fluorescent  lamps and combinations.. Paper 5.6.  Presented




     at Symposium  on Ultraviolet Radiation Measurements for Environmental




     Protection and Public  Safety.  National Bureau of Standards, June 8-9,




     1977.   Pp. 74-76.   In  Final Program and Abstracts.




Kostkowski,  H.  J.  and R. Saunders.  1977.  Second Quarterly BACER Report




     to the  Environmental Protection Agency.  National Bureau of Standards,




     Washington, D.C.




Krizek, D. T. (Chairman), IMOS Subcommittee on Biological and Climatic




     Effects Research.   1975a.  A proposed federal program to determine




     •the biological and  climatic effects of stratospheric ozone reduction.




     Federal Council for Science and Technology, Washington, D.C. Draft




     Report.




Krizek, D. T.   1975b.  Influence of ultraviolet radiation on germination




     and early  seedling growth.  Physiol. Plant 34(3):182-186.




Krizek, D. T.   1977a.  Biological and climatic effects of stratospheric




     ozone reduction:  A progress report on the BACER program.  Presented




     at Amer. Society for Photobiology Meetings.  San Juan,  Puerto Rico.




     May 15, 1977.




Krizek, D. T.   1977b.  Current UV measurement methodology and future needs




     in photobiological research.  Paper No.  5.2.  Presented at Symposium




     on Ultraviolet Radiation Measurements for Environmental Protection




     and Public Safety.   National Bureau of Standards.   June 8-9, 1977.




     Pp.  49-52.   In Final Program and Abstracts.




Nachtway,  D. S.   1975.   Climatic Impact Assessment Program (CIAP).




     Monograph 5,  Impacts of Climatic Change  on the Biosphere.  Part 1




     Ultraviolet Radiation Effects.   U.S. Department of Transportation.




     Washington, D.C.






                                  -11-

-------
 Norris, K. H.   1977.   Development  of  a  portable,  automated  UV-B spectro-

      radiometer.   Paper  No.  5.5.   Presented  at  Symposium  on Ultraviolet

      Radiation  Measurements  for Environmental Protection  and Public  Safety'.'

      National Bureau of Standards.  June 8-9,  1977.   Pp. 72-73.   In Final

      Program and Abstracts.

 Ormrod, D. P. and  D.  T.  Krizek.  1978.   Plant stress studies in controlled

      environments.  HortScience 13(4):453-456.

 Rowan, J. D. and K. H. Norris.  1978.   Instrumentation for  measuring

      irradiance in  the UV-B  region.   Final BACER  Report submitted to the

      Environmental  Protection Agency.

'Rupert, C. S.   1978.   Uniform terminology for radiations.   Pliotochem.

     Photobiol. 28:1.

 Rupert, C. S. and R.  Latarjet.  1978.   Toward a nomenclature  and dosimetric

      scheme applicable to all radiations.  Photochem. Photobiol. 28:3-5.

 Seliger, H. H.  1978.  Environmental photobiology.   Chapter  6.  Pp. 143-

     173.  Iii The Science of Photobiology (K. C.  Smith, ed.).   Plenum

     Press, New York.

 Sisson, W. B. and M. M. Caldwell.   1976.  Photosynthesis, dark  respiration,

     and growth of Rumex patientia L. exposed to ultraviolet  irradiance
                             v
      (288 to 315 nanometers) simulating a reduced atmospheric ozone column.

     Plant Physiol. 58:563-568.

Sisson, W. B. and M. M. Caldwell.   1977.  Atmospheric ozone depletion:

     Reduction of photosynthesis and growth of a sensitive higher plant

     exposed to enhanced UV-B radiation.  Jour.  Expt. Bot. 28:691-705.

Sisson, W. B. and M. M. Caldwell.   1975.  Lamp/filter systems for simula-

     tion of solar UV  irradiance under reduced atmospheric ozone.

     Photochem.   Photobiol.  21:453-456.


                                   -12-

-------
Skelly, J. M., M. F. George, H. E. Heggestad, and D. T. Krizek.  1978.




     Air pollution and radiation stresses.  Chapter 2.5.  In ASAE Mono-




     graph.  Modification of the Aerial Environments of Plants.  In press.




Thiraijan, R. W., H. R. Cams, and L. E. Campbell.  1978.  Radiation




     sources and related environmental control for biological and climatic




     effects UV research (BACER).   Final EAGER Report submitted to the




     Environmental Protection Agency.




Van, T. K. and L. A. Garrard. 1975.  Effect of UV-B radiation on net




     photosynthesis of some C^ and C, crop plants.  Proc.  Soil Crop.




     Sci. Soc. Fla. 35:1-3.




Van, T. K., L. A. Garrard and S. H. West.  1976.   Effects of UV-B radiation




     on net photosynthesis of some crop plants.   Crop Sci. 16:715-718.
                                 -13-

-------

JMJL Ol 01  X

-------
Weighted and J^k*eighted measurements of UV-B radiation obtained on November 8, 1977 in the laboratory under two Wj^fcing- *~r~~
house FS-40 ^^Prescent sunlamps filtered with 0.127 mm cellulose acetate (aged 6 hours) mounted in a single fixtul^ without
reflector and room lights off. Detector heads placed under the center of the fixture in a horizontal position.
Distance IRL
from Meter
sensor Reading
(cm) (107 scale)
a/ b_/

110 6.90
100 7.95
90 9.35
80 10 . 0 .
70 12.0
60 15.0
K 50 19.0
i
40 24.0
30 33.0
20 48.0
UV-B sun
Equiv.
calculated
from IRL meter
UV-B sun
equiv.
measured
on Optronic
Radiometer
UV~B sun
equiv.
measured
on Optronic
Radiometer
UV-B sun
equiv.
measured
on spectro-
radiometer
BUV mW • m 2
280-320nm
based on
spectro-
radiometer
UV-B spectral
irradiance
(280-32j)nm)
mW' m
c/ d/ ฃ/ f/ ฃ/ W
(0 to 5 scale) (0 to 10 scale)

0.8
0.9
1.1
1.2
1.5
1.9
2.3
3.0
4.1
5.9
i
0.9
1
1.1
1-2
, 1-5
1.7
2.1
2.6
3.3
4.4
off scale
—
0.8
1.0
1.1
1.4
1.6
2.0
2.5
3.2
4.3 .
6.1

0.7806
0.8861
1.0331
1.2317
1.4758
1.7811
2.2037
2.8514
3.7911
5.4484

2.3885
2.7116
3.1613
3.7689
4.5161
5.4503
6.7433
8.7252
. 11.6008
16.6722

206.7507
234.6552
274.8037
324.2766
392.0929
475.1478
591.2049
763.8646
1024.9800
1481.1394..
 a/  distance measured from outer wall of lamp to detector head
 b_/  measurements at 90,  100,  and 110 cm read at 10 ฐ scale and converted to 10'  scale
 cj  based on conversion  factor provided by R. Thimijar. of IRL meter reading x 0.123
 d/  set to a maximum of  5 UV-B sun equivalents, (15.3 mW-m  B.UV)
 ej  set to a maximum of  10 UV-B sun equivalents, (30.6 mW-m ^BUV)
, f/  weighted portion indicated for 280-320nm region alone                  _2
 ฃ/  BUV = biologically effective UV radiation from 280-320nm (weighted mW-m" )
     BUV of 3.06 mWm   =  one UV-B sun equivalent
 h/  actual unweighted flux obtained on an Optronic  spectroradiometer; UV-B irradiance measured every nm  from
     250-369nm but summed from 280-320nm

-------
Table 2.

UV sun equivalents obtained at various distances under two Westinghouse
FS-40 fluorescent sunlamps filtered with 0.127 mm (0.005 in.) cellulose acetate
(aged 6 hrs.).  Measurements taken with an Optronic UV spectroradiometer in
the laboratory with room lights off.
Distance
from
sensor
110
100
90
80
70
60
50
40
30
20
UV Sun
Equiv.
250-279 nm
.0057
.0066
.0075
.0092
.0110
.0136
.0172
.0215
.0288
.0431
UV Sun
Equiv.
280-329 nm
0.7806
0.8861
1.0331
1.2317
1.4758
1.7811
2.2037
2.8514
3.7911
5.4484
UV Sun
Equiv.
250-329 nm
0.7863
0.8927
1.0406
1.2409
1.4868
1.7947
2.2209
2.8729
3.8199
5.4915

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Table 3.

Biologically effectively (BUV) radiation in the 250-279nm, 280-330nm and
250-330nm region (mWra  )under 2 Westinghouse FS-40 fluorescent sunlamps
filtered with 0.127 mm (0.005 in.) cellulose acetate (aged 6 hrs.).
Values based on UV spectroradiometer measurements taken every nm from
250-369nm.
Distance'
from
sensor
(cm)
110
100
90
80
70
60
50
40
30
20
Total
BUV
250-279
weigh t-fi$
mW -m
.0177
.0202
.0229
.0283
.0335
.0415
.0526
.0659
.0882
.1319
Total
BUV
280-330
weighted
mW ' ni
2.3885
2.7116
3.1613
3.7689
4.5161
5.4503
6.7433
8.7252
11.6008
36.6722
Total
BUV
250-330
weighteji,
mW 'ra
2.4062
2.7318
3.1842
3.7972
4.5496
5.4918
6.7959
8.7911
11.6890
16.8041
                                         -17-

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Table 4.

Total UV irradiance  (unweighted mVJ-m") under  two Westinghouse FS-40
fluorescent sunlamps filtered with 0.127 mm  (0.005  in.) cellulose acetate
(aged 6 hours).  Measurements taken with an Optronic UV spectroradiometer
every nm from 250-369nm, and total irradiance determined  for the UV-A,
B, and C regions.
Distance
from
sensor
(cm)
110
100
90
80
70
60
50
40
30
20
Total
Irradiance
UV-C
250-279nm
mW 'ra
0.0223
0.0258
0.0294
0.0361
0.0428
0.0533
0.0668
0.0850
0.1127
0.1691
Total
Irradiance
UV-B
280-320nm
_2
mW -m
206.7507
234.6552
274.8037
324.2766
392.0929
475.1478
591.2049
763.8646
1,024.9800
1,481.1394
Total
Irradiance
UV-A
321-369nm
_2
mW -m
195.8107
226.1185
263.9468
314.7281
381.6198
467.3057
583.7436
760.5765
1,027.8479
1,489.0918
Total
Irradiance

250-369_i^n
mW • m
402.5837
460.7995
538.7799
639.0408
773.7555
942.5068
1,175.0173
1,524.5261
2,052.9406
2,970.4003
                                  -18-

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Table 5.  Actual and predicted relationship between IRL UV meter reading and unweighted UV spectral
          irradiance in the 280-320 nm (UV-B)  region.  YI = UV-B irradiance in mWm   and Y- = incide
          flux in the 280-320 nm region measured in photons-m~2xlO"  per 6 hour day.
UV-B Irradiance
in unweighted
roW -m~z
Distance
from
sensor
cm
110
100
90
80
70
60
50
40
30
20

IRL UV Meter Reading
(10 scale)

X
6.90
7.95
9.35
10.00
12.00
15.00
19.00
24.00
33.00
48.00

Instantaneous
Measurement

Yl
206.7507
234.6552
274.8037
324.2766
392.0929
475.1478
591.2049
763.8546
1024.9800
1481.1394
r = 0.995
Error of
Prediction
A
Yr*i
-9.9667
-14.6079
-17.8537
11.4719
17.2964
7.3636
-.5629
17.1073
-.7304
-9.5095

X = IRL UV meter reading
Incident UV-B
in photonsปm
Integrated for
6 hr day

Y2 '
6.7459
7.6564
8.9664
10.5306
' •<• 12.7934
**r 15.5033
19.2901
24.9237
30.7494
48.3272
r = 0.9976
X = IRL.UV meter
flux in
-2 in21
xlO
Error of
Prediction
A
Y2-y2
-.3446
-.4698
-.5407
.4323
.6723
.4230
.2642
.9658
-2.0861
.6957

reading
                                      (107 scale)
y  = predicted

^\
y, = 2.8457 + 30.9959X
                                                UV-B irradiance
    (10  scale)
A
y_ = predicted incident UV-B flux
     in photons-m~2 xlO21 per 6 hr day

vn = 0.2843 + 0.9864X

-------
I
ro
O
               table 6.  Actual and predicted relationship between IRL UV meter  reading  (10   scale)  and  weighted
                        UV spectral irradiance in the 280-320 nm  (UV-B) region.  Yn  = UV-B  sun  equivalent  and Y,
                        biologically effective UV (BUV) radiation in weighted mW*ra
Distance
from
sensor
cm
110
100
90
80
70
60
50
40
30
20


IRL UV Meter
Reading
(107 scale)
X
6.90
7.95
9.35
10.00
12.00
15.00
19.00
24.00
33.00
48.00
r
X
UV-B Sun
Determined
Yl
0.7806
0.8861
1.0331
1.2317
1.4758
1.7811
2.2037
2.8514
3.7911
5.4484
= 0.9991
Equivalent
Error of
Prediction
V*i
-.0478
-.0624
-.0755
.0487
.0640
.0261
-.0086
.0668
-.0231
-.0818

= IRL-jUV meter reading
Biologically Effective UV in
weighted mU-m~~
Actual
Y2
2.3885
2.7116
3.1613
3.7689
4.5161
5.4503
6.7433
8.7252
11.6008
16.6722
r = 0.9992
X = IRL UV meter reading
Error of
Prediction
Y2-y2
-.1491
-.1906
-.2272
.1547
.2073
.0996
.0034
.2488
-.0013
-.1394

'•
                                          (10  scale)
                                         = predicted UV-B sun
                                           equivalent
                                         = 0.3900 + 0.1144X
    (10  scale)
yป = predicted BUV (weighted mW-m~ )
y2 = 0.1412 + 0.3473X

-------
Table 7.  Actual and predicted relationship between UV-B sun equivalent as measured with an Optronic
          Model 725 broad band radiometer (0-10 scale and 0-5 scale) and unweighted UV spectral
          irradiance (mW -m  ) in the 280-320 nm (UV-B) region.
Distance
from
sensor
cm
110
100
90
80
70
60
50
40
30
20
UV-B Sun Equiv.
(0-10 scale)
Measured on
Optronic 725
Radiometer
Xl
0.8
1.0
1.1
1.4
1.6
2.0
2.5
*
3.2
4.3
6.1
UV-B Irradiance 2
in unweighted mW-m
Measured on
UV Spectro-
radiometer
Yl
206.7507
234.6552
274.8037
*ป
324.2766
392.0929
475.1478
591.2049
763.8546
1024.9800
1481.1394
Error of
Prediction
V*i
15.2720
-4.9999
11.0603
-11.7315
7.9083
-5.3898
-9.7739
-5.7420
-9.5873
12.9837
UV-B Sun Equiv.
(0-5 scale)
Measured on
Optronic 725
Radiometer
x2
0.9
1.1
1.2
1.5
1.7
2.1
2.6
3.3
4.4
off scale
UV-B Irradiance _ฃ
in unweighted mW- m
Measured on
UV Spectro-
radiometer
Y2
206.7507
234.6552
274.8037
324.2766
392.0929
. 475.1478
591.2049
763.8646
1024.9800
1481.1394
Error of
Prediction
Vy2
10.8111
-8.4680
8.0887
-13.2137
7.4191
-3.8931
-5.7949
1.7224
3.3283
— —
                                r = 0.9996
                                X = UV-B sun equivalent
                                y  = Predicted UV-B irradiance
                                y  =-1.2273 + 240.8825X
r = 0.9995
X->= UV-B sun equivalent
-'•
y? = Predicted UV-B.  irradiance
y2 = -16.3863 + 235.9177X

-------
NJ
            Table 8.  Actual and predicted relationship between UV-B sun equivalent on Optronic Model 725
                      radiometer (0-10 scale and 0-5 scale)  and biologically effective UV (BUV) radiation
                      in the 280-320 nm region.
Distance
from
sensor
cm
110
100
90
80
70
60
50
40
30
20


UV-B Sun Equiv.
(0-10 scale)
Measured on
Optronic 725
Radiometer
Xl
0.8
1.0
1.1
1.4
1.6
2.0
2.5
3.2
4.3
6.1


Biologically Effective
UV in weighted mW • m~2 UV-Sun Equiv.
(0-5 scale)
Measured


Yl
2.3885
2.7116
~ 3.1613
3.7639
4.5161
5.4503
6.7433
8.7252
11.6003
16.6722
r = 0.9998
X = UV-B sun
Error of
Prediction
„
Yryi
.1348
-.0821
.0976
-.1048
.1024
-.0434
-.1005
-.0086
-.1031
.1082

equivalent
Measured on
Optronic 725
Radiometer
X2
0.9
1.1
1.2
1.5
1.7
2.1
2.6
3.3
4.4
off scale

obtained
\ r- * • _•_ 	
Biologically Effective
UV in weighted mW -m
Determined Error of
Measured

Y2
2.3885
2.7116
3.1613
3.7689
4.5161
5.4503
6.7433
8.7252
11.6008
16.6722
r = 0.9996
X-3 UV-B sun
Prediction
A
Y -y
2 y2
.0976
-.1110
.0728
-.1172
.0933
-.0310
-.0674
.0534
.0045
— —

equivalent obtained
                                            on Optronic Model 725 radiometer
                                            (0 - 10 scale)
                                           = Predicted BUV in the 280-320nm
                                             region
                                           = .0936 + 2.700X
    (0-5 scale)
y  = Predicted BUV in the 280-320nm
     region
v^ = -0.1020 + 2.6587X

-------
I
NJ
V
                        Table 9.   Actual and  predicted  relationship  between UV-B sun equivalent as
                                  measured  with  an  Optronic  Model  725  broad band radiometer (0-10
                                  scale  and 0-5  scale)  and incident  flux in the 230-320 nm region
                                  measured  in photons-m  xl02l  integrated over a 6 hour day.
Incident UV-B flux in
photons-nT2 X1021 ner 6 hr dav

Distance
from
sensor
cm
110
100
90
80
70
60
50
40
30
20

UV-B Sun
Equiv.
Optronic 725
(0-10 scale)
X
0.8
1.0
1.1
1.4
1.6
2.0
* 2.5
3.2
4.3
6.1

Total Spectral Calculated Error of
irradiance
280-320 nm
mW * in

206.7507
234.6552
274.8037
324.2766
392.0929
475.1478
591.2049
763.8646
1024.9800
1481.1394



Y
6.7459
7.6564
8.9664
10.5806
12.7934
15.5033
19.2901
24.9237
30.7494
48.3272
r = 0.9972
Prediction
A
Y -y
.4523
-.1697
.3741
.3105
.3699
.0149
-.0296
.2403
-2.3626
1.4229

X = UV-B sun equivalent



y = predicted
incident UV flux at
                                                                        280-320 nm  in photons-nf
                                                                        xlO   per 6 hour day

                                                                    y = 0.1637 4-7.6624X

-------
I
ro
                       ~i.-abltr-.tfci.   ii~i.aal ki-vJ prfe-c^c-ted— ^-lati.,-.^-nipU,_-Jweew.	/-B L—J eqt__—lien,—
                                   measured with an Optronic Model 725 broad  band radiometer  (0-5
                                   scale) and incident  flux in the 280-320 nm region measured in
                                   photons-m~  xlO^l  integrated over a 6 hour day.


Distance
from
sensor
cm
110
100
90
80
70
60
50
40
30
20


UV-Sun
Equiv.
Optronic 725
(0-5 scale)
X
0.9
1.1
1.2
1.5
1x7
2.1
2.6
3.3
. 4.4
off scale


Total Spectral
irradiance
280-320 nm
mW • m

206.7507
234.6552
274.8037
324.2766
392.0929
475.1478
591.2049
763.8646
1024.9800
1481.1394

Incident UV-B"
photons -m xl
Calculated
Y
6.7459
7.6564
8.9664
10.5806
12.7934
15.5033
19.2901
24.9237
30.7494
48.3272

flux in
0 per 6 hr day
Error of
Prediction
Y - y
-.0365
-.3696
.0485
-.4728
.3164
.1790
.4066
1.0574
-.9470

                                                                    r

                                                                    X

                                                                    y
0.9974

UV-B sun equivalent

predicted  incident flux at 280-320 nm
in photons-m"2 x 10Z1 per 6 hour  day
                                                                    y =  0.3759 + 7.1183X

-------
                         FINAL REPORT
            MULTIPLE  EFFECTS OF UV-B IRRADIATION
                ON FUNGAL SPORE GERMINATION
                      Olga v0 H. Owens
                      Donald T. Krizek

                  Plant Stress Laboratory
                 Plant Physiology Institute
          Beltsville Agricultural Research Center
                 Beltsville, Maryland 20705
                       EPA-IAG-D6-0168
                       Project Officer:
                                                  i
                       R. J. McCracken
Agricultural Research, Science and Education Administration
                 U.S. Department of Agriculture
                   Washington, D.C. 20250
                       Prepared for
              Environmental Protection Agency
                       BACER Program.-
                    Washington, D.C. 20460

-------
                               CONTENTS
Abstract ....................................... . ...........




Table [[[   iv




Figures ... ................ . ....................... . ........    v




Acknowledgments ................................. . ..........  vii




     1.  Introduction .... ............... ........... . .......    1




     2 .  Materials and Methods .... .........................    2




              UV Source ... ........... . ......... . ...........    2




              Experimental Material .... ....................    3




     3.  Results .................. . ............ * ...........    5




              Growth of the Spores .........................    5




              Survival Curves ..............................    5




              Action Spectrum ..............................    6




              Reciprocity ..................................    7




              Character of 330 nm Inhibition ...............    7




              Delay, Outgrowth Rate and Photoreactivation ..    8




              Germ Tube Outgrowth in Relation to Sunshine




                and Ozone Reduction ...... . ....... ..... .....    9




     4.  Discussion ......... , ..............................   10





-------
                               ABSTRACT




     We investigated the influence of narrow-band UV irradiation in the




265-330 nm region on germination of fungal spores of Cladosporium




cucumerinum Ellis & Arth. using a xenon arc lamp and various filters.




Based on survival curves and action spectra data, we propose that there




are two active regions between 280 and 320 nm (UV-B) that might be




influenced by changes in the stratospheric ozone layer:  a short-wave




portion (265-295 nm) and a long-wave portion (300-330 nm).  Action spectrum




data obtained with narrow-band interference filters confirmed previous




reports of damage to DNA from UV irradiation at 265-295 nm UV and in




addition demonstrated significant inhibitory effects of UV irradiation




at 300-320 nm.  Further studies made of the 300-330 nm portion of the




spectrum using a combination of plastic and glass filters showed that the-




influence of UV irradiation in this region was primarily to produce a




non-photoreactivable delay in germ tube outgrowth.   The implications of




these findings are discussed in relation to the possible impact of strato-




spheric ozone reduction.
                                iii

-------
                                 TABLE


                                               -2       -1
Table 1.  Global downward energy fluence in J-m   •  5 nra   for a 6-hr



          period (assuming an average zenith angle of 30 )at three ozone



          concentrations at standard temperature and pressure.

-------
                             FIGURES



1,  Accumulated percent outgrowth of germ tubes of Cladosporium cucumerinura



    fungal spores following inhibition and placement on a water agar plate



    as described in the Materials and Methods.  The two symbols are



    replicate plates, and the vertical lines are one standard deviation.



    The curve was drawn by inspection.



2.  A, B, and C:  Survival curves for 5 nm half-band width UV exposures



    of Cladosporium cucumerinum fungal spores at the indicated central



    wavelengths.  Percent survival was determined at 22 hours and is



    the percent of control survival.  The fluences were obtained by



    varying the time.



3.  Action spectra for UV inhibition of fungal spore ge'rmination in



    Cladosporium cucumerinum plotted from data of Figure 2 (A, B, and C).


                                                     —9
    Action is expressed as reciprocal of the joules•m~z that gave 90%



    (o) and 37% (*) survival.  The vertical lines are the 95% confidence



    limits.



4.  Percent survival of Cladosporium cucumerinum fungal spores following


                                                         —1    ?
    exposure to UV irradiation at 265 nm given at 0.1 J*s   ซm   (o) and


            1   —9
    1.0 Jปs   *m   (•).  Counts were made as in Fig. 2.

                              t

5.  Percent survival of Cladosporium cucumerinum fungal spores following

                                          *

    exposure to a broad band UV source centering on 325 nm (inset)


                    12                —19
    given at 80 Jปs   -m   (o) and 800 Jซs -1 ซm~^ (a).   Counts were



    made as in Fig. 2.



6.  Survival curve for exposure of Cladosporium cucumerinum fungal  spores



    to a broad band UV source centering on 330 nm (inset) given at  400



    J*s~-*- •m~ ,  Counts were made at 22 hours as in Fig.  2.



7.  Accumulated percent outgrowth of Cladosporium cucumerinum fungal



    spores vs duration of exposure to broad band UV irradiation centering

-------
    at 330 nra.  The indicated values (360, 480, and 600) are kJ-nT2



    obtained by duration of exposure to 400 Jซs"~l *m~2 for 15, 20, and



    25 rain.,  respectively. Control spores received no UV irradiation.



8.  Time course for outgrowth of Cladosporium cucumerinum fungal spores



    showing comparative influence of short-wave and long-wave UV.


                                               _i    2   	   	
    A.  275 nm narrow-band UV (5 min.  0.57 J*s   *m  ;  SS^, ri).
                                            -1    7
    B.  330 nm broad-band UV (5 min. 520 Jซs   ซm  , 
-------
                                                                               ^ JIUซUB1 I—.
                           ACKNOWLEDGMENTS



     The authors gratefully acknowledge Mr. Karl H. Norris for his help




in the use of the spectroradiometer; Mr, Scott J. Ravitz for his




assistance in maintaining and supplying cultures; Mr. William A. Dungey




for help with the xenon arc lamp; Dr. Joseph H. Graham for suggesting




the use of C, cucumerinum; Mr. Richard H. Thimijan for advice on




statistical analysis of the data; and Drs. Sterling B. Hendricks,




Takuma Tanada, and Hugh Sisler for their critical review of the manuscript.




The research was supported by EPA Contract No. EPA-IAG-D6-0168.
                                 vii

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    Multiple  Effects  of  UV-B  Irradiation on Fungal Spore Germination


                               I/                     21
               . Olga v.H. Owens-  and Donald T. Krizek-
                              INTRODUCTION




     The possibility of  stratospheric ozone reduction has increased concern



about the potentially harmful effects of irradiation in the spectral region



where ozone absorption normally cuts off the sun's ultraviolet radiation.



This region (UV-B) is defined as the 280-320 nm region.



     The lethal and mutagenic effects of UV absorption at the shorter



wavelengths, 250-280 nm  (UV-C), are well-documented and are associated



with DNA and protein absorption.  Previous studies on the effects of UV



irradiation showed that  the 320-400 nm  (UV-A) region acts on a variety of



metabolites to produce lethal, inhibitory, and delaying effects  (l^ 2, 3,



6, 7, 15).  Between these two active regions is the UV-B region where there



are few major absorption peaks.  Because any effect observed at  the peak of



absorption of a photo-chemically active compound would be observed also in



the region of lesser absorption, it seems reasonable to expect that observed



effects of UV-A and UV-C irradiation would overlap in the UV-B region.
i'Formerly Plant Physiologist, Agricultural Research, Science and



  Education Administration.  Presently Associate Program Manager,



  Competitive Research Grants Office, USDA/SEA, 1300 Wilson Blvd.,



  Arlington, VA 22209.




—Plant Physiologist, Agricultural Research, Science and Education



  Administration.

-------
     Another major characteristic of the UV-B region is the 5-decade


decrease in the sun's irradiance from 320-280 nm caused by the sharply


increasing absorption coefficient of ozone.  It is evident that a reduction


in stratospheric ozone concentration could shift this sharp cut-off to


shorter wave-lengths, and increase UV irradiance on the short wave side


of the cut-off region.
                                          i

     In order to assess the biological effects of this increase in UV


irradiance, we need to estimate the effects at those wavelengths where


changes in the ozone concentration will have a major effect on the sun's


fluence at the earth's surface.  In this report we show that for germination


of non-proliferating fungal spores (conidia), the action of UV irradiance


from 265-295 nm on survival is closely correlated with nucleic acid


absorption.  From 300-330 nm the response resembles that reported for the


near UV region (8, 9) and presumably involves a delay in protein synthesis.


                          MATERIALS AND METHODS
          .     .••    -  .               3/        '         "
UV Source;  The UV source (Schoeffel)—  was a 2.5 kW high-pressure xenon


arc lamp equipped with collimating lenses, a 15 cm circulating cooled


water filter and a mirror at a 45  angle to produce a vertical beam.  To


provide narrow band irradiation we placed various filters in the parallel


beam: The first filter was a 1 cm deep, 1:1 mixture of saturated


solutions of NiSO -6H 0 and CSO •7H?0 in an open dish with parallel sides


and a flat quartz bottom.   In later experiments the mixture was diluted
                                                           i
3/
   Mention of a trademark, proprietary product, or vendor does not

   constitute a guarantee or warranty of the product by the U.S.  Department

   of Agriculture and does not imply its approval to the exclsuion of other

   products or vendors that may also be suitable.
                                   - 2 -

-------
 further with dis ti lied water that had no detectable effect on the



 transmission characteristics.  The dish was kept below 50ฐC with a



 cooling coil around the outside.  The second filter was either a 5 nm



 half-band width interference filter  (Corion) of the selected central


 wavelength or a combination of plastic and glass filters for the broader



 bands:  for 325 nm, 2 mm pyrex and 0.13 mm cellulose acetate; for 330 nm


 Corning Nos. 0-54 and 7-54.



     The beam was directed from the mirror through the NiSO.-CoSo.
                                                           44

 solution, then through the secondary filter onto a 15 rpm turntable



 containing the test organisms as described below.  The UV irradiance was


 measured with a pyroelectric radiometer (Molectron) that could be moved



 into the beam as needed.  From just below the mirror the beam was



 surrounded by a black box maintained at 21-23 C.  A tube surrounded the


 beam below the filters, and baffles prevented stray radiation from falling



 on the radiometer or the test organisms.


 Experimental Material;  The test organisms were conidiospores of the fungus,



 Cladosporium cucumerinum Ellis & Arth., a leaf pathogen of cucumbers.   We


 used this species because of its ease in cultivation and handling.  Its


 spores normally give 95-98% germination and under natural conditions are


 exposed in the imbibed state (from dew) to the sun's irradiance during their


 germination period on the leaf surface.


     Cultures of the fungus were maintained on potato-dextrose agar.   Spores


were harvested from 6-day-old colonies by addition of 10 ml of distilled


water to the colony and agitated to obtain a uniform suspension.   From the


 suspension  30 ul was dropped onto the  center of a water agar plate.  The

-------
water was absorbed by  the agar in about 20 minutes leaving an area of imbibed

spores, with a diameter of about 1 cm, in an even layer on the agar surface.

The spore area was one-third  the diameter of the shielded, collimated beam

from the xenon arc.  Following absorption of the water the covered plates

were held in darkness  at 40ฐC for 15-30 minutes until the start of the

experimental period.

     For exposure, the agar plate without a top was placed in the beam on

the turntable and exposed for a period of time that gave the specified photon.

fluence as determined  from the power readings with the radiometer.  Uncovered

matched control plates were placed in the same box but away from the beam

and shielded by mylar.  For longer exposure times, viz. 1-6 hours, covered

control plates were also included in the box.  There were no significant

differences in percent outgrowth between covered and uncovered controls.

     Following exposure, the plates were covered and incubated in darkness

(unless otherwise specified) at 22 C.   Germination of spores results in the

outgrowth of a germ tube.  The presence of the germ tube was used as the

indication of survival (percent survival or percent outgrowth).

     Observations of germ tube outgrowth were made at 150 x magnification

in a darkened room, and when necessary the microscope light source was
                              \
filtered through red and yellow colored plastic to prevent photoreactivating
                                          *
light from reaching the spores.  For each datum point all spores (about 100)
                                          2
in each of five random areas  (each 0.56 mm ) were counted and the proportion

with a distinct germ tube was recorded.   Data were expressed as  average

percent of spores with germ tube outgrowth ฑ the standard deviation.   In

most cases the outgrowth produced on the exposed plates was expressed as

percent of the outgrowth of the control plates.  For time course experiments,

the plates were removed at intervals for counts,  then returned to the incubator.

-------
      The method  used  for  spore handling had  several advantages:   (1) the



 spores were  located on  a  flat surface minimizing  scattering errors;  (2)



 microscopic  examination allowed  for elimination from  the counts of over-



 lapping  and  clustered spores; and  (3) no additional nutrients were



 necessary for  germ tube outgrowth.




                                 RESULTS



 Growth of the  Spores;   The population of fungal spores on the agar plate



 produced germ  tubes over  a period of time.   Figure 1  shows the accumulated



 percent  of spores with  germ tubes for replicate control plates determined



 at intervals over a period of 25 hours.  Outgrowth began after about 4 hours



 and reached  a  maximum after 20 hours.  The rate of accumulated outgrowth



 increased and  reached a maximum at about 9 hours, when 55-65% of the spores



 had germ tubes,  and then  decreased.  The accumulated  percent outgrowth



 superficially  resembled a cumulative normal  frequency distribution but



 deviated significantly  from normality in most cases.




 Survival Curves;  Figure  2 (A, B, and C) shows the influence of UV irradiance



 of the indicated 5 nm bandwidths on outgrowth of germ tubes of Cladosporium



 cucumerinum  fungal spores.  Percent of control outgrowth was determined

                             *

 after 22  hours of incubation as described under Materials and Methods.   The


                           -2
 dose, expressed as joules'm  , was obtained by varying the time of exposure.



 The curves for 265-295  nm demonstrate the lack of an exponential relationship



 between  percent outgrowth and dose at low doses.  The presence of the shoulder



 can be attributed to an active dark repair mechanism.  The curves for



 300-320  nm show only a  shallow linear relationship indicating that those



wavelengths were relatively ineffective.

-------
      Because we are primarily concerned with the effects of solar


 irradiation, we have concerned ourselves with the low dose parts of the


 curves,  located on the shoulders, corresponding to the sun's irradiance


 at each  wavelength examined.


      Demonstration of measurable decreases in germ tube outgrowth at


 310-320  nm required long exposure times of up to 6 hours.  We could not


 show  the lower part of the survival curves with the use of the narrow


 band  UV  filters.


 Action Spectrum:  Figure 3 shows two action spectra constructed from the


 data  in  Figure 2 (A, B, and C).   Action is expressed as the reciprocal of

            _2
 the joules-m  .  The upper curve shows the joules permitting 90% survival


which is on the shoulder of the survival curves.  We assumed that the


 survival curves were nearly linear to 70% and calculated a value for 90%


 survival from the regression equations developed from the individual points.


This  calculation introduced a small error and enlarged the 95% confidence


 interval (vertical lines) but allowed us to make a single action spectrum


 throughout the 265-320 nm region.   The lower curve of Figure 3 shows an


action spectrum constructed from the 1/e values (37% survival)  from those


 survival curves that could be extended to 37% survival.   These values

                             \
were  obtained from curves drawn by inspection.   The similarity of the two

                                         ซ
curves in the 265-295 nm region (the short-wave portion)  indicates that


the slope of the shoulder was proportional to the final  slope and suggests


that  the rate of dark repair was nearly proportional to  the rate of damage.


The 265-295 nm action spectra closely match published spectra (13,16)  and can


be attributed to damage to DNA.


     Values on the 300-320 action  spectrum are  too high  in relation to


265 nm to be attributable to DNA damage.   We conclude from these results


that  there are two active portions in the UV-B  region:   a short  wave portion

-------
 (265-295 rim) whose action corresponds  to DNA absorption and a long-wave



 portion  (300-330 nm) whose action corresponds  to an unknown component(s).



 Reciprocity;  Figure 4 shows  the effect of 265 nm irradiation at two  fluence


                       -1  -2
 rates:  0.1 and 1.0 J-s  ซm   .  For  the low rate a neutral density filter



was used and the duration of  exposure was increased 10-fold.  The figure



shows that at the low fluence rate the UV irradiation was less effective



 than at the high rate, indicating a  greater efficiency of  repair,   The ratio



of the fluences at the low and high  rates in producing a similar percent



survival was about 1.9.  These data  indicate that reciprocity was



incomplete.



     We could not measure reciprocity at 300-320 nm using the narrow band



interference filters because of the  long exposures that would be required



at low irradiances.  Therefore we used a broad band filter, centering on 325



nm, described under Materials and Methods, to provide a higher fluence



rate in this spectral region.  Figure 5 shows the influence of low (80 Jปs


 —2                  —1   ?
m  ) vs high (800 J's  -m  ) fluence rates on spore survival.   The inset shows



the transmission character of the filter determined spectrophotometrically.



Reciprocity was nearly complete, indicating that in the 325 nm portion of the



spectrum there was no detectable simultaneous repair mechanisms as that shown

                              t

for the 265 nm band.



Character of 330 nm Inhibition;  In the 325 nm experiment  described above,



the filters used allowed significant transmission below 300 nm.  We,  therefore,



used another filter combination (see Materials and Methods),  centering on



330 nm that gave <1% transmission at 300 nm and above 370  nm (inset,  Fig. 6).



     Figure 6 is a survival curve obtained with the 330 nm broad band filter.



As for exposures at 265 nm, the points represent counts at 22  hours  after

-------
 exposure.  We noticed, however,  that  several hours later additional  spores



 had  germinated—an  effect not  observed  at any of  the other wavelengths,


                                              -1  -2
     The influence  of  330 irradiation (AGO J-s  -m  ) on percent outgrowth



 was  determined at intervals over a period of time for control and irradiated



 spores  (Fig. 7).  Three  fluences were chosen to coincide, respectively, with



 three points on the survival curve shown in Fig.  6: (1) on the shoulder (360


     -2                                      -2
 kJ-rn ); (2) where  the curve bends (480 kJ-m  ); and (3) on the steep part


                      _2
 of the curve (600 kJ-m   ).  The  figure  shows that broad band UV irradiation



 centered at 330 nm delayed the start  and depressed the rate of outgrowth.



 Thus, the data  in Fig.  7 indicate that the "survival curve" of Fig. 6 is



 an indication of both a  delayed  and a depressed rate of outgrowth; the shape



 of the curve depended on the time at which spores were counted,



 Delay, Outgrowth Rate, and Photoreactivation;  We compared the effect of



 narrow-band irradiation  at 275 nm (representing the short-wave portion), to



 the  effect of broad-band 330 nm  irradiation, (representing the long-wave



 portion).  Both exposures were for 5 min.   The fluence rates were 0.57


   -2  -1            -2  -1
J-m  -s   and 520 J-m  -s  , respectively.  Both fluences were on the



 shoulders of the percent survival curves.   Following exposure, replicate



plates were incubated in darkness or under a bank of 1500 raA cool white
                             \


 fluorescent lamps filtered with  0.127 mm (0.005 in)  Mylar (short-wave UV
                                          *


cut-off, 3% T at 320 nm).  Controls received no UV exposure.   Percent



outgroxtfth,  shown in Fig. 8,  was  determined at the intervals indicated in



Fig.  7.



     Figure 8 shows that the incubation light inhibited the rate of outgrowth



of the controls, but did not delay onset of outgrowth.   The fluorescent



lamps used for incubation contained,  in addition to  the visible radiation,


                                 -1  -2                       -1  -2
UV irradiation at 365 nm (95 mJ-s  -m  ) and at 334  nm (9 mJ-s  -m  ).

-------
 This  irradiation  may  have  produced  a  cumulative  inhibitory  effect during  the



 incubation  period.  Figure 8B  shows that  exposure  to broad  band  330 nm



 followed by either  dark  or light  incubation  significantly delayed onset of



 outgrowth in comparison  with the  controls.   After  the delay the  rates of  out-



 growth  in dark  equalled  those  in  the  light.  Delay in outgrowth  of spores,



 incubated in either light  or dark,  was greater from the 275-  (Fig. 8A) than



 from  the 330-nm (Fig.  8B)  treatment.  With spores  treated at  330 nm, light



 during  incubation shortened the delay and increased the rate  of  outgrowth to



 a value equal to  that  for  the  light-incubated control.  The results of this



 experiment  and  previous  ones showed that  the effects of the short-wave and



 long-wave portions  of  the  UV region studied were similar in that both delayed



 the start of outgrowth of  spores.   Effects differed between the  long-wave and



 short-wave  portions.   With the long-wave portion the delay  in the start of



 outgrowth was not affected by  light incubation and  UV irradiation had little



 effect  on the rate  of  outgrowth except at high doses (Fig. 7:).



 Germ Tube Outgrowth in Relation to  Sunshine and Ozone Reduction:  By use  of the



 regression  equations developed from data in Fig. 2, we determined the effective-



 ness of each 5 nm wavelength band of  solar irradiation in inhibiting germ tube



 outgrowth.  The data of  Shettle et  al. (11) for solar irradiance were used as



 a basis for  the calculations.v  We used values for dowm^ard  global flux, 30



 zenith  angle and,  ozone  concentrations of. 0.32 atnrcm, 0.28 atm-cm,  0.16 atm-cm.



Assuming that the fungal spores would be exposed to a maximum of 6 hours of



 sun in June at the  Washington,  B.C.  latitude, average zenith angle 30ฐ,


                            —2     —1
we converted the data  to J-m   .5 nm   for 6 hours.   These values are



 shown in Table 1.    Figure  9 shows the calculated percent inhibition that



would be expected for  the  irradiance values at the  three ozone concentrations



of Table 1.   The inhibition assumes no photoreactivation and does not distinguish.

-------
between lethality and  temporary delay of spore outgrowth.  At 0.32 atnrcm,




an approximation of  "normal" ozone, i.e. no reduction, the sun's irradiance




throughout  the 300-320 nm region is sufficient to produce a 5% inhibition




at each wavelength,  a  result also noted by Jagger (5) for the near UV region.




A reduction in stratospheric ozone concentration to 0.28 attn'cm would not




significantly inhibit  germ tube outgrowth, but a reduction to 0.16 atra-cm,




a 50% depletion would produce a significant inhibition of nearly 20% at




295-300 nm.  The inhibition at 295 nm was somewhat under-estimated because



the fluence value was slightly beyond the survival curve shoulder (Fig. 2, B).




The effectiveness of the 295-300 nm region in inhibiting germ tube outgrowth




of Cladosporium spores agrees with the analysis of Setlow (10) and approxi-



mates that of Elkind et al. (2) for effectiveness of normal sunlight in




skin cancer production.





                               DISCUSSION



     The fungal spore produces the germ tube after a period of germination




that is primarily a period of protein synthesis.  In our study we used lack




of appearance of the germ tube as a measure of damage to the protein-




synthesizing system of the spore.   Most fungal spores have a complete




system for protein synthesis in which enzymes increase in activity and amount




during the termination period (12).  Protein synthesis and germ tube outgrowth
                                          *


are inhibited by cycloheximide indicating involvement of a ribosomal system.



New RNA synthesis occurs, but the extent of the requirement for new RNA has




not been established.  DNA replication does not occur until the time of




outgrowth or after (4,13), but our results indicate that DNA must function at



some stage of the germination sequence.
                                  10

-------
     We propose that there are two active regions between 280 and 320 nm.




Our results suggest that the short-wave portion primarily affects DNA and




the long-wave portion is primarily related to effects of near UV noted by




other investigators.  Because the DNA-absorbing part indicates the presence




of both a dark repair mechanism and photoreactivation, a moderately




increased irradiance from the sun might be ineffective in producing an




immediate inhibition.  Enhanced UV irradiance from the sun, however, might




retard the rate of growth, observable even at normal sunlight, that would




not be repaired in either darkness or light.   We have shown that  the




critical region for major effects, whether lethal or delaying, is at 295-




300 nm, a region that should be thoroughly analyzed.
                                   11

-------
                          Literature Cited





1.  Doyle, R. J. and H. E. Kubitschek.  1976.  Near ultraviolet  light



    inactivation of an energy-independent membrane transport  system in



    Saccharomyces cerevisiae.  Photochera. Photobiol.  24:291-293.



2.  Elkind, M. M., Antun Han, and Chin-Mei Chang-Liu.  1978.   'Sunlight'-



    induced mammalian cell killing:  A comparative study of ultraviolet



    and near-ultraviolet inactivation.  Photochera. Photobiol.  27:709-715.



3.  Fong, F., J. Peters, C. Pauling, and R. L. Heath.  1975.   Two mechanisms


    of near-ultraviolet lethality in Saccharomyces cerevisiae;  A respiratory



    capacity-dependent and irreversible inactivation.  Biochem. Biophys. Acta



    387:451-460.



4.  Hollomon, D. W.  1973.  Protein synthesis during germination of



    Peronospora tabacina conidia: An examination of the events involved



    in the initiation of germination.  J. Gen. Microbiol. 78:1-13.



5.  Jagger, J.  1975.  Inhibition by sunlight of the growth of Escherichia



    coli B/r.  Photochera.  Photobiol. 22:67-70.



6.  Jagger, J.  1976.  Effects of near-ultraviolet radiation on microorganisms.



    Photochem. Photobiol.  23:451-454.



7.  Koch, A. L., R. J.  Doyle, and H. E. Kubitschek.  1976.   Inactivation of



    membrane transport  in Escherichia coli by near-ultraviolet light.
                                         *


    J. Bacteriol. 126:140-146.



8.  Rambhadran, T.  V.  1975.   The effects of near-ultraviolet and violet



    radiations (313-405 nm) on DNA,  RNA,  and protein synthesis in E,.  coli



    B/r:  implications for growth delay.  Photochem. Photobiol.



    22:117-123.                                                        •
                                    12

-------
 9.  Ramabhadran,  T.  V.  and J. Jagger.   1975.   Evidence  against  DNA as




     the  target  for  334  nm-induced  growth  delay in  Escherichia coli.




     Photochem.  Photobiol. 21:227-233.




10.  Setlow, R.  B.   1974.  The wavelengths in  sunlight effective in




     producing skin  cancer: A theoretical  analysis.   Proc.  Nat.  Acad. Sci.




     U.S. 71:3363-3366.




11.  Shettle, E. P.,  M.  L. Nack, and A.  E.  S.  Green.  1975.  Multiple




     scattering  and  influence of clouds, haze,  and  smog  on  the middle UV




     reaching the  ground.  JEH Nachtwey,  D.S. (ed.).   Impacts of  Climatic




     Change on the Biosphere, CIAP Monograph 5,  Part  1 - Ultraviolet




     Radiation Effects DOT-TST-75-55. pp 2-38  to 2-49.




12.  Sussman, A. S.  and  H. A. Douthit.   1973.   Dormancy  in  microbial




     spores.  Ann. Rev.  Plant Physiol. 24:311-352.




13.  Tyrrell, R. M.   1978.  Solar dosimetry with repair  deficient bacterial




     spores: Action  spectra, photoproduct  measurements and  a comparison




     with other biological systems.  Photochem.  Photobiol.  27:571-579.




14.  Van Etten, J. L., L. D. Dunkle, and R. H.  Knight.   1976. "Nucleic acids




     and fungal spore germination" in Weber, D.  J. and W. M. Hess (eds.)




     The Fungal Spore, Form and Function,  Wiley, N.Y.  p. 243-299.




15.  Webb, R. B.  1977.  Lethal and mutagenic  effects of near-ultraviolet




     radiation,  In Photochem. Photobiol.  Reviews, Vol.   2 (K. C.  Smith,




     ed.)  P.'Ierium Publishing Corp., N.Y.                '.




16.  Webb, R. B. and M.  S. Brown.  1976.   Sensitivity of strains of




     Escherichia coli differing in repair  capability to  far UV, near UV,




     and visible radiations.   Photochem. Photobiol.  24:425-432.
                                    13

-------
Table 1.  Global downward energy fluence in J'm~2ซ5nin   for a 6-hour
          period (assuming an average zenith angle of 30ฐ) at three
          ozone concentrations at standard temperature and pressure.
          Values calculated from Shettle et al.  (1975).
Wavelength
5 nm band
320
310
300
295
290
280

0.32 atra-cm
3.44 x 104
1.19 x 104
5.62 x 102
1.76 x 101
3.31 x 10~2
2.27 x 10-16
Ozone Concentration
0.28 atm-cm
3.59 x 104
1.37 x 104
9.08 x 102
4.22 x 101
1.67 x 10"1
5.99 x 1Q-14

0.16 atm'cia
4.07 x 104
2.10 x 104
3.81 x 103
5.92 x 102
2.21 x 101
1.09 x 10~6
                               14

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 100
                          10        !5
                          TIME, MRS.
25
Figure 1.   Accumulated percent outgrowth of germ tubes  in  Cladosporium
                                                    ;
           cucumerinum fungal spores following inhibition  and  placement

           on a water agar plate as described in the Materials and

           Methods.  The  two symbols are replicate plates,  and the

           vertical  lines are one standard deviation.  The curve was

           drawn by  inspection.
                              15

-------
          100
          30
        w  10
        (-
        z
        Ul
        o
        CC
        w
        a  3
           I
             -  265
100
            0246

              .M-2 (XIO-2)
   0   10   20

    J-M-2 (X IO'2)
                           FLUENCE
         100
          80
        CC.

        
-------
       10
         -I
       10
         -2
    5
    •
    CO
    O
       IO
           -3
         "3
    ง  ,0-4


    O
    UJ
    oc
       10
         -5
       10
         -6
           260     280     300     320    340

                      WAVELENGTH, NM.
Figure 3.   Action spectra  for UV inhibition of fungal spore


           germination  in  Cladosporium cucumerinum plotted


           from data  of Figure 2 (A, B, and C).   Action is

                                                 -2
           expressed  as reciprocal of the joules-m   that


           gave 90% (o)  and 37% (o) survival.  The vertical


           lines are  the 95% confidence limits.
                             17

-------
   100
   90
   80

   70

   6O
UJ
UJ
Q.
   50
   40
   30
               A.
               r
                                   I	I    I
      0      50     100   150     200   250
                   FLUENCE, J-M-2
Figure 4.  Percent survival of  Cladosporium cucumerinum fungal
          spores following exposure to UV irradiation at 265
                                -2
          nm given at 0.1 J-s   -m   (o) and 1.0 J-s
          Counts were made as  in Fig.  2.
                                                 ~2
                                                m   (ป) .
                        18

-------
       100
        90
        80

        70
     CO
     h-
     550
     o
     UJ
     Q.
       40
       30
%T
 60

 40

 20
                 280    320    360
           0          100        200      300
                  FLUENCE, KJ-NT2
Figure 5.  Percent survival  of Cladosporium cucumerinum fungal
          spores following  exposure to  a broad band UV source
                                                 -*1  —2
          centering on 325  nm (inset) given at 80 Jซs  ซm   (o)
                    -1  -2
          and 800 J-s  -m   (e).   Counts were made as in Fig.  2.
                            19

-------
     100

      80



      60




      40
   CE

   3

   CO
      10
Z  8
Ui

ฃ  ^
bJ
0.
             %T


             40



             30



             20




             10
               300   320   340    360

                         X.NM.
                ZOO      400     600


                        FLUENCE, KJ-M'2
800
                                                2400
Figure 6.   Survival curve for exposure  of  Cladosporium cucumerinum



           fungal spores to a broad  band UV  source  centering on 330


                                     —1   —2
           nm (inset)  given at 400 J-s  -m  .  Counts were made at



           22 hours as in Fig.  2.
                               20

-------
too
              10    15    20    25    30    35    40    46    55
                          TIME, HRS.
Figure 7.   Accumulated percent outgrowth of Cladosporium cucumeririum



           fungal spores vs duration of exposure to broad band UV



           irradiation centering at 330 nm.  The indicated values


                                        -2
           (360, 480, and 600), are kJ-m   obtained by duration of


                              -1  -2
           exposure to 400 J-s  -m   for 15, 20, and 25 min.,
                                                               •t


           respectively.   Control spores received no UV irradiation.
                                21

-------
                         10      15     20     25
                               15

                        TIME, MRS.
20     25
Figure 8.   Time course for outgrowth of Cladosporium cucumerinum



           fungal spores showing comparative influence of short-



           wave and long-wave UV.  A. 275 nm narrow band UV  (5 min.



           057 J-s"1-™  ; ga, O).  B. 330 nm broad band UV  (5 min. 520


               1  —2
           J'S~ -m  ; <ฃ*ป m <ฃ>).   Controls (no UV exposure:   •, o).

                                                                 *

           Plates were incubated in dark (closed symbols) or in



           light, (open symbols).
                                22

-------
   H
   5
   O

   O
< ID
-J O
< UJ
O O
        85
   a:
   UJ
   G-   75
           280
300
320
      Figure 9.  Influence of stratospheric  ozone reduction on percent


                germ tube outgrowth of Cladosporium cucumerinum fungal


                spores in the UV-B region.  Values were calculated from


                the data of Fig. 2 and Table 2.  The values assigned to


                the curves represent "normal" ozone concentration, 0.32


                atm-cm; 12% reduction, 0.28 atm-cm; and 50% reduction


                0.16 atm-cm.
                                    23

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                       FINAL REPORT
  RESPONSE OF  SELECTED VEGETABLE AND AGRONOMIC CROPS TO
  INCREASED UV-B IRRADIATION UNDER GREENHOUSE CONDITIONS
                      John E. Ambler
                     Randy A. Rowland
                      Nancy K. Maher
                 Plant Stress Laboratory
               Plant Physiology Institute
         Beltsville Agricultural Research Center
                Beltsville, Maryland 20705
                     EPA-IAG-D6-0168
                     Project Officer:

                     R. J. McCracken
Agricultural Research, Science and Education Administration
                U.S. Department of Agriculture
                  Washington, D. C. 20250
                       Prepared for
              Environmental Protection Agency
                       BACER Program
                    Washington, D.  C.  20460

-------
                               ABSTRACT


      Biomass measurements for alfalfa, rice, and wheat in greenhouse


 experiments showed no reduction in growth of enhanced UV-B treated plants


 as compared with  the Mylar—  control.  None of these species showed chlorotic

                                                                     _o
 tissue.  However, the 'Poinsett1 cucumber cultivar under 13 to 15 mWra"


 BUV of UV-B showed chlorosis of about 11 percent of the total leaf tissue,


 whereas, 'Ashley* showed chlorosis of about 1 percent of tissue on a dry


 weight basis.  Leaf area and weight of both cultivars showed about a


 7 percent reduction in response to enhanced UV-B as compared to the Mylar


 control.


      For each species studied, there seemed to be a shade effect due to


 the experimental design of the set-up.  Biomass yields were higher from


 plants at the perimeters than at the centers of the experimental areas.
—   Mention of a trademark name or a proprietary product does not constitute


    a guarantee or warranty of the product by the USDA and does not imply


    its approval to the exclusion of other products that may also be available.

-------
                              INTRODUCTION

     Recent  greenhouse and growth chamber studies conducted  in  the Plant

 Stress Laboratory  (PSL)  (1,  3,  5, 6,  9) have demonstrated  the inhibitory

 effects of high levels of biologically effective UV  (BUY)  radiation on

 plant growth and development  of cotton and selected vegetable species.

     We conducted  our experiments to  extend these studies  to other

 plant species including alfalfa, cucumber, rice, and wheat.  We examined

 the effects  of UV-B on vegetative growth and biomass production in alfalfa,

 cucumber, rice, and wheat and on grain production in wheat.

                        MATERIALS AND METHODS

     The UV-B enhancement facilities  were developed in cooperation with

 the Agricultural Equipment Laboratory (AEL), Beltsville Agricultural

 Research Center (BARC).  Enhancement  studies were conducted according to

 the guidelines established for  the BACER program (2, 4).   Two lamp fixtures

 provided UV  radiation, each containing two Westinghouse FS-40 fluorescent

 sunlamps filtered with 5 mil Mylar (UV-A) or 5 mil cellulose acetate (UV-A,

 -B).  The UV lamps were kept 62 cm from the plant canopy,  and the lamp

 fixtures were 60 cm apart.  Filters were aged for 6 hours  before use and

were changed twice weekly (4).  We measured UV at every pot location at the
                             \
 start of the experiments and at selected points periodically thereafter

with either  an Optronic Laboratories, Inc.,  Model 725, UV-B Radiometer or

an Instrumentation Research Laboratory (IRL) UV-B Radiometer (7, 8).

Radiometer readings were verified by spectral irradiance determinations

 (250-369 nm) with an automated spectroradiometer (7, 8) at selected locations

in the experimental irradiation areas.
                                                   _o
     Weighted irradiance levels are reported as mWm   BUV, the biologically

effective UV derived from the AE9 weighting function, and unweighted

-------
                 -2
 irradiance as Wra   obtained by summing the measured or calculated values


                                                 -9                     -2
 at each nanometer from 280-320 nm.  Dividing mWm   BUY by 3.06 (the mWm



 BUV of the Beltsville control sunshine) provides the fraction of BUV received



 by each plant relative to that of one control sunshine (10).



      Where UV irradiation was obtained by filtering the FS-40 lamps through



 cellulose acetate (CA), BUV was limited to the UV-B region (280-320 nm).



      For details concerning average control sunshine,  spectral character-



 istics of UV fluorescent lamps and filters, and the weighting function, see



 the BACER final reports of the AEL and IRL, BARC (8, 10).



      Experimental plants receiving UV-B were combined  into groups averaging



 7 mWnT2 BUV (3.1 Wm~2), 10 mWnT2 BUV (4.5 Wm~2), 13 mWm"2 BUV (5.8 Wm~2)


           _2            _9
vand 15 mWm   BUV (6.7 Wm  ) for comparison of the results.



 Cultural Procedure (General)



      Plants were seeded in 12.5-cm white plastic pots  containing a commercial



 peat-vermiculite mix, and they were thinned on emergence  as follows:   alfalfa,



 5 plants;  cucumber,  1 plant; rice, 8 plants;  and wheat, 8 plants  per  pot.



      Plants were grown in a glass greenhouse with and  without supplemental



 UVB during the summer-fall 1977.   They were irradiated daily from emergence



 for 6 hours from 1000 to 1600 hours.



      Plant material  was dried* in a forced-draft oven at 70ฐ C,  weighed and


                                                                         _2
 combined into  the four groups receiving the average 7,  10, 13,  or 15  inWm



 BUV under  the  CA filter and compared to matched set of control  plants placed  in



 like groups for similar numbered positions  grown under Mylar filters.



                                  RESULTS



 I.   UV-B effects on  alfalfa (Medicago  sativa  L.)



      Procedure:   Alfalfa cv.  'Williamsburg* was seeded on August  8, 1977.



 Biomass was harvested by cutting  the plants  5.1 cm  above  the  soil level,



 6 weeks, 9 weeks,  and 12 weeks  from seeding.

-------
     Results:  The results showed no effect from enhanced UV-B  (Table 12).

Table 12.  Effects of UV-B on biomass  (g) of alfalfa*
7mWm~2 BUV lOmWm"2 BUY 13tnWm~2 BUY
Harvest - wks. Harvest - wks. Harvest - wks.
Filter 6 9 12 6 9 12 6 9 12
Mylar 5.2 2.7 1.6 3.4 1.8 1.4 4.0 2.2 1.5
CA 6.4 3.0 2.2 4.9 2.1 1.2 4.5 1.9 1.4
15mWm~2 BUV
Harvest - wks.
6 9 12
3.2 1.7 1.4
3.8 1.9 1.6
* Mean weight 10 plants.
Discussion:  A shade effect was noted—bioraass yield was largely influenced
by pot position.  Plants grown directly under light fixtures produced less
growth than plants not directly under the fixture.
II.  UV-B effects on cucumber (Cucumis sativus L.)
     Procedure:  Pots were seeded alternately with 'Ashley1 or 'Poinsett*
cultivars on November 28, 1977,  and thinned to one plant per pot on
emergence.  Above-ground plant parts, excised at the cotyledonary node were
harvested 4 weeks after seeding.  Leaf areas were measured and selected
plant sections were placed in a 1:1:18 FAA solution for morphological
examination.
                            *
     Results:  The effects of increased UV-B on cucumber are shown in
                                        *
Table 13.

-------
 Table  13.   Effect of UV-B on  leaf weight and  leaf area  in cucumber
mWra"2 BUV

UV-B* 7
irradiation
10
13
15

UV-B* 7
irradiation
10
13
15
CA
'Ashley1
.343
.244
.235
.209

CA
'Ashley1
194
129
132
114
. Dry weight (g)
filter
'Poinsett1
.345
.295
.244
.199
Leaf
filter
'Poinsett1
200
161
132
119
Mylar
'Ashley'
3.67
3.23
3.02
3.06
area
Mylar
'Ashley'
203
191
170
176
filter
'Poinsett'
4.34
3.84
3.40
4.06

filter
'Poinsett'
242
221
193
216
* Number of samples for 7, 10, 13, and 15 mWm   BUV  were 12, 16, 22, and 6,


  respectively.


     Discussion:  Dry weights.  Three effects were noted.  A shade effect


from light fixtures, enhanced UV-B effect and a chlorotic leaf-tissue effect.


Yield of control plants (Mylar) directly under light fixtures was about


90 percent of the yield of plants not directly under light fixtures.  This


was due to a shading effect.


     Leaf dry weight of plants grown under the CA filters, as compared with


plants at like positions grown under the Mylar filters, were reduced 9, 8,


7, and 5 percent for 'Poinsett' and 9, 8, 8, and 7 percent for 'Ashley1,
                                                                     i

respectively.

-------
                                                                     _2
     Chlorotic leaf tissue  in  the  'Poinsett' cultivar under 13-15 mWm


UV-B made up about 11 percent  of the leaf tissue on a dry weight basis.


In  'Ashley' for similar BUY levels chlorotic leaf tissue comprised about


1 percent ,of the total leaf tissue on a dry weight basis.


    Plants grown under Mylar filters (control) appeared green and normal


for all pot positions.


    Leaf Area


    Two effects were noted, a  shade effect and an enhanced UV-B effect.


Leaf area of control plants grown directly under light fixtures was about


90 percent of the values of plants not grown directly under light fixtures.


Leaf areas of plants grown  under the CA filters, as compared with plants in


like positions under Mylar  filters, were reduced by 17, 27, 32, and 45 percent


for 'Poinsett' and 4, 32, 22, and 35 percent for 'Ashley1,  respectively, with


increasing UV-B demonstrating varietal differences within species.


III.  UV-B effects on rice  (Oryza sativa L.)


    Procedure:  Rice was seeded in soil enclosed by a plastic bag within


a waxed container on August 8,  1977.  Plants were thinned to eight  plants


per pot and flooded 2 weeks after seeding.   Flooded conditions were


maintained throughout the experiment.   Plants were harvested on December 27,


1977,  19 weeks from seeding. *


    Results:   The results of increased UV-B in rice are shown in Table 14.


Table 14.  Effect of UV-B on biomass (g) of rice*
Filter
Mylar
CA
7mWm~2 BUV
21.2
22.3
lOmWm"2 BUV
14.3
19.0
13mWm 2 BUV
15.7
15.5
15mWm~2 BUV
10.7
10.8
* Mean weight per pot.

-------
     Discussion:  One effect was noted, a shade effect which directly




affected the plants growing in the center of the quadrant.  Plants on the




outer perimeter were not affected by shade and thus were greater in height




and growth.  The inner plants were shaded by light fixtures and also shaded




by plants external to their pot positions.




IV.  UV-B effects on wheat (Triticum aestivum L.)




     Procedure:  Wheat cv. 'Pacific Triple Dwarf was seeded in the summer




of 1977 and harvested 12 weeks after seedling emergence.




     Results:  The effects of increased UV-B on vegetative growth and grain




development were negligible (Table 15).  Plant growth appeared to be green




and normal under both Mylar and CA filters.




Table 15.  Effect of UV-B on growth and development in Pacific Triple Dwarf




           wheat from emergence to age 12 weeks.
Straw
dry weight (g)
per 10 plants
Grain, No. seeds
per 10 plants
Grain weight (g)
per 100 seed
Filter
CA
Mylar
CA
Mylar
CA
Mylar
7mWm"~2 BUV
7.72
8.51
161
209
2.73
* 2.29
lOmton"2 BUV
6.49
7.35
137
161
2.72
2.37
13mWnT2 BUV
7.11
7.04
132
181
2.56
2.32
_2
15raWm BUV
7.27
7.45
140
161
2.69
2.46

-------
                           LITERATURE CITED




1.  Ambler, J. E., D. T. Krizek, and P. Seraeniuk. . 1975.  Influence of UV-B




    radiation on the early seedling growth and translocation of   Zn from




    cotyledons in cotton.  Physiol. Plant 34:177-181.




2.  Cams, H. R., R. Thimijan, and J. M. Clark.  1977.  Outline of irradiance




    distribution of UV fluorescent lamps and combinations.  Symposium on




    Ultraviolet Radiation Measurements for Environmental Protection and




    Public Safety.  June 8-9, 1977.  NBS, Gaithersburg, MD (Abstr.).




3.  Krizek, D..T.  1975.  Influence of ultraviolet radiation on germination




    and early seedling growth.  Physiol. Plant 34:182-186.




4.  Krizek, D. T.  1977.  Current UV measurement methodology and future




    needs in photobiological research.  Symposium on Ultraviolet Radiation  .




    Measurements for Environmental Protection and Public Safety.  June 8-9,




    1977, NBS, Gaithersburg, MD (Abstr.).




5.  Krizek, D. T.  1978.  Response of selected vegetables and agronomic crops




    to increased UV-B irradiation under greenhouse and growth chamber conditions.




    Final BACER Report submitted to the Environmental Protection Agency.




6.  Krizek, D. T.  1978.  Differential sensitivity of two cultivars of




    Cucumis sativus L. to increased UV-B irradiance.   Plant Physiol.  Suppl.




    61(4):92.   (Abstr.).     *




7.  Norris, K. H.  1977.  Development of a portable,  automated UV-B




    spectroradiometer.  Symposium on Ultraviolet Radiation Measurements for




    Environmental Protection and Public Safety.  June 8-9, 1977.  NBS,




    Gaithersburg, MD (Abstr.).




8.  Rowan, J.  D.  and K.  H.  Norris.   1978.   Instrumentation for measuring




    irradiance in the UV-B region.   Final BACER Report submitted to the




    Environmental Protection Agency.

-------
 9.  Skelly, J. M., M. F. George, H. E. Heggestad, and D. T. Krizek.  1978.




     Air pollution and radiation stresses.  In ASAE Monograph.  Modification




     of the Aerial Environment of Plants, Chapt. 2.5.




10.  Thimijan, R. W.,  H. R. Cams, and L. E. Campbell.  1978.  Radiation




     sources and related environmental control for biological and climatic




     effects UV research.  Final BACER Report submitted to Environmental




     Protection Agency.

-------
                       FINAL REPORT
  RESPONSE OF SELECTED VEGETABLE AND AGRONOMIC CROPS TO
    INCREASED UV-B IRRADIATION UNDER FIELD CONDITIONS
                      John E. Ambler
                     Randy A. Rowland
                      Nancy K. Maher
                 Plant Stress Laboratory
               Plant Physiology Institute
         'Beltsville Agricultural Research Center
                Beltsville, Maryland 20705
                     EPA-IAG-D6-0168
                     Project Officer:

                     R. J. McCracken
Agricultural Research, Science and Education Administration
                U.S. Department of Agriculture
                  Washington, D. C. 20250
                       Prepared for
              Environmental Protection Agency
                       BACER Program
                    Washington, D.  C.  20460

-------
                               ABSTRACT                       :




     Enhanced UV-B radiation under field conditions on various economic




crops conducted at the Beltsville Agricultural Research Center ranged




from ambient up to eight times ambient.  The enhanced radiation was




achieved with either unfiltered BZS-CLG or FS-40 Westinghouse—  sunlamps.




    All crops were affected less by the enhanced radiation than similar




crops grown under greenhouse and growth chamber conditions.




    Although variable, these results supported results obtained else-




where in this BACER Program which indicate that higher plants have a high




light energy-requiring system of photorepair or photoprotection.   With




all crops investigated, additional research is needed to establish the




levels of UV-B required to injure plants in the field.
—  Mention of a trademark name or a proprietary product does not constitute




   a guarantee or warranty of the product by the USDA and does not imply




   its approval to the exclusion of other products that may also be available.

-------
                             INTRODUCTION




    Growth  chamber and greenhouse experiments have indicated that several




crop plants displayed reduced growth and chlorosis when subjected to




enhanced levels of UV-B  (1, 2, 3, 4).  The field experiments reported




here were designed to examine the responses of selected crops to enhanced




UV under field conditions.




                        MATERIALS AND METHODS




    The field selected for these studies was located on the south farm of




the Beltsville Agricultural Research Center, Beltsville, Maryland.  The




plots were silt loam, characteristically consisting of recently deposited




materials washed from acid crystallite rock of the capitol Piedmont.




These soils also have a concentration of fine mica, which contributes




to poor drainage.  The area was rototilled in early spring of 1977 and




fertilized with a 10-10-10 fertilizer at a rate of 500 pounds/acre 3 weeks




before planting.




    The total area was divided into six plots,  which were supplied with




separate UV-enhancement assemblies.   The lamp assemblies, constructed




by the Agricultural Equipment Laboratory, were designed so that height




adjustment could be easily maintained by a simple pulley system (Fig.  1).




A height of 1.6m was mainta&ied above the plant canopy during the course




of the experiment.                        •                               :




    For the first experiment,  lamp banks were designed to obtain a two




dimensional gradient  of UV irradiation—one parallel to a lamp assembly,




the other at right  angles to the lamp assembly.   Each assembly consisted




of lamp fixtures  placed end to end as follows:   A two-lamp fixture at  the




high UV-B end;  then two single lamp  fixtures  placed end to end;  a 33-cm

-------
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   .    WwgiSySijsSSJSsSEaa^     S-KfiSi      :-.
                                                                                                                          - *   ,x."
                               •:•                           '  '              -:. !  ••<&  >:

                            -    -      ^                        p       1
                                                       •" '    •           . **       1     -",
                                                     "r:
                                                            ^•-^    •! •* v>
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                                                    ,             ...                 .       ,,.
                                                                            .st1            • -•--

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       Figure 1
                                                                                              r

-------
 space  and another single lamp; a 65-cm  space and a  final  lamp  that  was
 taped  to reduce the UV-B irradiance by  one half.  This positioning  of the
 lamps  provided a uniform gradient of supplemental UV within  the  experimental
 plot area.  Each plot consisted of four 11.0-m rows parallel to  the UV-B
 gradient.  Two rows were 0.5 m from the center, line of the lamp  assembly,
 the other two were 1.5 m from the assembly center line.   Supplemental
 UV radiation was provided by unfiltered Westinghouse BZS-CLG 40  watt
 fluorescent lamps.  Exposure time from  emergence to harvest  was  6 hours/day
 from 1100 to 1700 hours.  Table 1 shows the irradiance levels  obtained
 for each meter of row length.
                                                  -2
    Weighted irradiance levels are reported as mWm   BUV, the  biologically
 effective UV derived from the AZ9 weighting function, and unweighted
                 —2
 irradiance as mWm   obtained by summing the measured or calculated  values
                                                 7                     —2
 at each nanometer from 280-320 nm.   Dividing mWm * BUV by 3.06 (the mWm
 BUV of the Beltsville control sunshine) provides the fraction  of BUV
 received by each plant relative to that of one control sunshine  (6).
     Two different crops were chosen for each plot to minimize shading
 effects, and they were grown in alternate 0.5- and 1.5-ra rows.   The paired
 crops were as follows:  squash (Curcurbita maxima (L.) cv. 'Early Prolific
 Straightneck') and bean (Phaseolus vulgaris (L.) 'Contender Bush'); sweet
 corn (Zea mays (L.)  cv.  'Golden Cross Bantuin')  and sorghum (Sorghum
 biocolor (L.) Moench cv. 'R-720');  and soybean (Glycine max  (L.) Merr.
 cv. 'Amsoy-71') and  sugar beet (Beta vulgaris (L.) cv.  '7322-0').  Also
 included were guard  rows of pea (Vigna unguiculata (L.) Walp. cv. 'Cow')
 on either side of the 1.5-m rows.   Each plot of  paired crops was duplicated.
All plants were sown in  May unless  otherwise specified.
    A second experiment  was undertaken in mid-summer using unfiltered
Westinghouse FS-40 UV fluorescent  lamps.  A similar  gradient assembly was

-------
Table 1.  Spectroradioraeter measurements at 1-m intervals along the 11-m
          rows for BZS-CLG sunlamps unweighted (mWm~J and weighted
          BUY) and fraction of control sunshine above ambient.
Row
position
(m)

__
mWm

-2
mWra BUV
Control
sunshine
(fraction)
0.5-m Row
0
1
2
3
4
5
6
7
8
9
10
11
9.113
96.131
138.105
•• 117.339
94.996
79.977
63.131
42.018
28.319
7.562
1.768
.662
.146
1.669
2.498
2.131
1.736
1.450
1.137
.898
.518
.124
.026
.009
.05
.55
.82
.70
.57
.47
.37
.29
.17
.04
.01
.00
1.5-m Row
0
1
2
3
4
5
6
7
8
9
10
11
12.109
45.701
65.431
76.497
66.966
53.034
32.011
29.146
19.644
5.246 *
1.227
.459
.193
.790
1.175
1.553
1.146
.956
.547
.491
.283
.068
.014
.004
.06
.26
.38
.51
.47
.31
.18
.16
.09
.02
.00
.00

-------
Table 2.  Spectroradioraeter measurements at 1-m intervals along the 14-m
          rows for FS-40 sunlamps unweighted (mWm  ) and weighted
          BUV) and fraction of control sunshine above ambient.
Row
position
(m)

o
mWm *

mWm~2BUV
Control
sunshine
(fraction)
0.5-m Row
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
88.672
. 450.287
709.471
607.306
512.496
421.542
347.719
232.890
163.677
60.898
17.505
8.025
4.263
1.583
.432
3.227 ,
17.594
28.736
24.280
19.879
17.035
14.271
9.473
6.763
2.333
.636
.292
.155
.058
.015
1.05
5.75
9,39
7.93
6.50
5.57
4.66
3.09
2.20
.76
.21
.09
.05
.02
.00
1.5-m Row
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
80.56
409.287
310.778
266.025
251.797
207.110
105.920
93.942
80.403
29.915
13.132
6.020
3.198
1.188
.324
3.182
7.566
12.357
11.889
9.734
5.132
4.299
3.617
* 3.296
1.857
.478 •
.193
.137
.041
.010
1.03
2.47
4.04
3.89
3.18
1.68
1.40
1.18
1.08
.61
.16
.06
,.05,
..01
iOO

-------
 constructed with twice as many'lamps, which substantially increased  the



 supplemental UV irradiation.  Rows were 14 ra long.  Table 2 shows  the



 irradiance levels obtained for each meter of row length in the plot  area.



    Radiation was measured with a single monochrometer spectroradiometer


 described in the Instrumentation Research Laboratory final report  (5).


 Data were obtained at 1-nm intervals from 250 to 369 nm.  Experimental


 results were subjected to analyses of variance.


                        RESULTS AND DISCUSSION


    Table 3 gives the temperature and percipitation means for the  duration


 of the experiments.  There were no unexpected temperature or rainfall


 extremes, but variability from row to row and within rows was considerable


 in all experiments, which may account for the lack of statistical  significance


 in many of the parameters measured.  Because of this variability,  Probability



 Values (P) of 0.3 and lower are shown for all measured responses.


 BZS-CLG lamp assemblies used in the first experiment plus sunshine provided

                                                                 _o
 total average irradiance levels as follows; 3.1, 3.7, and 4.2 mWm   BUV


 for the 1.5-m row and 3.2, 4.2, and 5.1 mWm~2 BUV for the 0.5-m row.  These


 irradiances provided approximately 1.0, 1.2, and 1.4 times the Beltsville


 control sunshine for the 1.5-m row and 1, 1.4,  and 1.7 times for the


 0.5-m row.  The results obtained using these lamps are discussed below on


 a crop by crop basis:                    .


    Squash.  Squash was selected as a subject species because its related


 species,  the 'Poinsett'  cucumber,  was one of the most sensitive crops in


 Beltsville greenhouse and growth chamber experiments (4).   Table 4 shows


 the effects of UV enhancement on dry weight of  tops, fresh weight of fruits,


number of fruits,  and number of male and female flowers.   Only the reductions


 in fruit  weights and number were significant (P = 0.2 to 0.3).  The dry


weight of tops also decreased with each increase in UV-B irradiance, but

-------
Table 3.  Temperature and Precipitation Measurements, 1977
Mean high
temperature
(ฐC)
May
June
July
August
September
October
November
25.4
26.9
31.7
30.7
27.2
18.7
12.8
Mean low
temperature
(ฐC)
11.8
14.8
19.5
19.6
16.3
7.3
5.3
Precipitation
(mm)
37.1
60.2
112.5*
42. -9
33.8*
133.9
104.9
* Includes supplemental irrigation

-------
Table 4.  Means of squash parameters and  standard  error for the 0.5-m rows and 1.5-m rows and means and
          ranges of weighted mWra~2 for the three levels of biologically effective UV above ambient UV.
Row
Control
0.5
1.5
Low
Dry
weight
of
Range Mean tops
(mWm~2 BUV) (mWrn~2 BUY) (g)

m .009- .518
m .004- .193


.165
.070


585.75
464.89

Fresh
weight
of
Standard fruits
Error (g)

567.82
433.02

Fruits
Standard
Error (No.)

55.4
110.3


18.0
11.0

Flowers
Standard
Error (No.)

1.4 15.
1.9 6.


62
75

Standard
Error

3.5
2.5

Flowers
Standard
(No.) Error

4.63
2.38


2.1
0.8

enhancement
0.5
1.5
m .898-1.450
m .491- .956
1.162
.665
514.63,
357.99
20.6 436.54
44.6 253.95
126.6
84.7
14.3
7.0
1.7 14.
1.1 6.
67
17
3.3
2.7
4.67
1.67
1.0
0.8
High
enchancement
0.5
1.5
0.5
1.5
m 1.669-2.498
m .790-1.553
m
m
2.009
1.166
P =
P -
485.16 .
.396.53
N.S.
N.S. .
37.6 418.59
20.0 258.65
0.3
N.S.
45.5
25.7


13.1
7.9
0.2
0.3
1.7 14.
0.9 6.
N,
N.
50
25
S.
S.
2.2
4.7


6.38
2.50
N.S.
N.S.
2.8
0.8



-------
not  significantly.  The results suggested that higher  irradiance levels

may  adversely affect plant growth, and continued investigation under

field conditions are warranted.

     Bean.  Garden beans were unaffected by UV-B enhancement.  The crop

was  not visibly damaged during the course of the experiment  (Table 5).

     Soybeans.  Although weight of seed and plant height differed significantly

(P = 0.2 and 0.3, respectively) no consistent trends were observed (Table 6).

As with beans, the plants were not visibly injured but the results warrant

continued investigations.

     Sugar beets.  This was the most sensitive species tested in the field

experiments.  All parameters measured except sucrose content at the lower

irradiance levels were significantly affected (Table 7).  Each UV-B

increment reduced dry weight of tops and fresh weight of roots.  In contrast,

sugar content increased with increased UV-B at the higher irradiance levels,

probably as a result of lower metabolic activity in leaves which provided .

higher carbohydrate concentrations for transport to the roots.

    These data, however, suggest that even in field experiments small

increases of present ambient UV-B may injure this crop.

    Sorghum.  The higher irradiance levels reduced fresh weight of tops

(P = 0.3) (Table 8).  Because the probability level was so low and because
                             \
plant height was not significantly affected, further research is needed
                                         *
to confirm possible UV-B effects.

    Sweet corn.  The fresh weight  of ears,  the number of ears, and the

plant height were reduced (P = 0.2 to 0.3)  (Table 9).  As with the crops

other than sugar beets, the level  of probability was low.   However,  since

three out of the four parameters measured were significant,  continued

evaluation of UV-B effects under field conditions seems warranted.

-------
Table 5.  Mean of bean parameters and standard error for the 0.5-m rows and 1.5-m
          rows and means and ranges of weighted raKm~2 for the three different  •
          levels of biologically effective UV above ambient UV.
Row
                       Dry
                       weight
                       of
   Range       Means    tops
(mWnf2 BUY)  (mWnT2  BUV) (g)
          Fresh
          weight
          of
Standard  Fruits  Standard
  Error    (g)      Error
Fruits
      Standard
(No.)   Error
Control
0.5 n
1.5 m
Low
enhancement
0,5 m
•1.5 m
High
enhancement
0.5 m
1.5 m

.009- .518
.004- .193


.898-1,450
.491- .956


1.669-2.498
.790-1.533

.165
.070


1.162
.665


2.009
1.166

94.33
98.55


97.29
104.91


84.00
102.25

8.11
4.41


15.70
2.30


6.04
3.00

719.0
759.4


730.1
818.7


634.2
789.0

6.53
6.69


4.28
4.00


3.12
2.92

15.9
15.5


17.0
17.1


12.8
17.9

1.33
2.44


.61
' .40


2.11
.74
Differences were not significant at the 0.3 level according to the P test.

-------
Table 6.   Means of soybean parameters  and standard  error  for  the  0.5-m  rows  and
          the 1.5-m rows and means  and ranges  of  weighted mWm  for  the three
          different levels of biologically effective UV above ambient.
Row
Control
0.5 m
1.5 m
Low
enhancement
0.5 m
1.5 m
High
enhancement
0.5 m
1.5 m
0.5 m
1.5 m
Range
(raWm"^ BUV)

.009- .518
.004- .193


.898-1.450
.491- .956

1.669-2.498
.790-1.533


Mean
(mWnT2 BUV)

.165
.070


1.162
.665

2.009
1.166
P =
P =
Weight
of seed
(g)

275.88
366.88


267.73
289.30

277.15
303.63
N.S.
0.2
Standard
Error

9.40
36.52


13.2
13.3

15.5
12.7


Height
(cm)

476.98
620.65


470.73
648.57

541.43
518.08
0.3
N.S.
Standard
Error

22.2
53.6


41.5
77.2

6.1
41.1


                                         10

-------
Table 7.  Means of sugar beet parameters and standard error for the 0.5-m rows and
          the 1.5-ra rows and means and ranges of weighted raWnT^ for the three
          different levels of biologically effective UV above ambient.
            Range
Row
                 BUY)
          Dry              Fresh
          weight           weight
          of               of
Means     tops   Standard  roots
  r2 BUV) (g)      Error    (g)
          Root
Standard  sucrose  Standard
  Error    (%)       Error
Control
0.5 m
1.5 m
Low
enhancement
0.5 m
1.5 ra
High
enhancement
0.5 m
1.5 ra
0.5 m
1.5 m

.009- .518
.004- .193


.898-1.450
.491- .956


1.669-2.498
.790-1.533



.165
.070


1.162
.665


2.009
1.166
P =
P = -

470.13
540.33


295.26
428.21


243.14
388.37
0.05
0.3

54.3
67.8


26.7
48.3


22.2
39.8



5705.78
5600.00


3720.27
3844.97


3059.40
3997.55
0.1
0.2

874.7
689.8


482.9
341.5


391.2
518.1



13.98
14.73


16.90
14.43


16.35
15.05
0.05
N.S.

0.5
0.2


0.5
0.0
• •

0.5
0.4


                                         11

-------
Table 8.  Means of sorghum parameters  and  standard  error  for  the  0.5-m rows  and  the
          1.5-m rows and means and  ranges  of weighted mMn  for the  three  different
          levels of biologically effective UV above ambient.
                                          Fresh
                                          weight
                                          of

Rows
Control
0.5 m
1.5 m
Low
enhancement
0.5 m
1.5 m
High
enhancement
0.5 m
1.5 m
0.5 m
1.5 m
Range
(mWm~^ BUY)

.009- .518
.004- .193


.898-1.450
.491- .956


1.669-2.498
.790-1.533


Mean
(mWnT2 BUV)

.165
.070


1.162
.665


2.009
1.166
P =
P =
tops
(8)

2526.68
2243.23


2162.23
2136.63


2144.05
2163.80
0.3
N.S.
Standard
Error

245.9
109.9


7.3
155.5


38.9
100.3


Height
(cm)

1130.15
852.53


1110.30
866.23


1191.40
838.40
N.S.
N.S.
Standard
Error

47.1
55.1


53.5
76.1


79.1
59.1


                                         12

-------
Table 9.   Means  of  sweet corn parameters and standard error for the 0.5-m rows and the 1.5-m rows and means and
          ranges of weighted mWnf^ for the three different levels of biologically effective UV above ambient.


Row
Control
0.5 m
1.5 m
Low
enhancement
0.5 m
1.5 m
High
enhancement
0.5 m
1.5 m
0.5 ra
1.5 m

Range


(mWnr2 BUV)

.009- .
.004- .


.898-1.
.491- .


1.669-2.
.79 -1.



518
193


450
956


498
533



Means
(mWm~2 BUV)

.165
.070


1.162
.665

ป
2.009
1.166
P =
P =
Dry
weight
of
tops
(8)

140.06
185.00
*•

117.19
196.46


116.63
142.76
N.S.
N.S.

Standard
Error

14.3
35.0


13.2
17.8


11.0
21.9


Fresh
weight
. of
ears
(g)

109.40
137.55


83.42
119.91


60.97
111.16
0.2
N.S.

Standard
Error

14.9
14.5


18.4
17.8


8.0
13.7



Height
(cm)

140.40
144.35


146.73
177.83


128.95
147.15
N.S.
0.3

Standard
Error

28.8
102.1


13.3
274.4


170.6
18.9


Ears

(No.)

4.75
5.75


3.33
5.00


2.75
4.38
0.2
0.3

Standard
Error

.7
.6


.8
.9


.4
.5



-------
    For the second experiment, the FS-40 lamp assemblies provided




significantly higher total irradiances than those on the first experiment,




as well as different spectral characteristics.  Irradiances levels averaged




3.8, 5.0, and 11.9 mWnT2 BUY for the 1.5-ra row and 3.9, 7.9, and 23.9 raWm"2




BUV for the 0.5-m row.  These irradiances provided approximately 1.2,




1.6, and 3.9 times for the 1.5-m row and 1.3, 2.6, and 7.8 times for the




0.5-m row of the control Beltsville sunshine.




    Squash.  This species was quite resistant to these high irradiance  .




levels.  Dry weight of tops and fresh weight of fruit decreased significantly




only at the highest irradiances,  and then only at P = 0.2 or 0.3 (Table 10).




    Broccoli.  Broccoli was much more sensitive to UV than squash,  providing




P values of 0.05 and 0.01 at the higher irradiance levels (Table 11).




However, irradiance levels two to eight times the value of the Beltsville




control sunshine were required to provide this level of significance.




    We conclude from these field experiments that plants are considerably




more resistant to injury from enhanced UV in a field environment,  with its




higher visible light energy,  than they are when grown in a greenhouse or




a growth chamber.  Whether resistance increases enough to preclude UV




damage at projected UV-B increases,  however,  cannot be determined  from




our results.                  ^
                                   14

-------
Table 10.  Means of squash parameters and standard error for the 0.5-ra rows and
           1.5-m rows,  means and ranges of weighted mWm   for the three different
           levels of biologically effective UV above ambient provided by unfiltered
           FS-40 lamps.
                                     Dry           Fresh
                                     weight         weight
                                     of            of               Fruits
Row
Control
0.5
1.5
Low
Range Means tops Standard fruits Standard
(mWm BUV) (mWm~2 BUV) (g) Error (g) Error (No.)

m
m


.106- 3.
.088- 3.


227
182


.805
.743


444.
381.


85
10


53.0
54.7


460.95
396.83


62.0 15.
64.0 15.


6
5

Standard
Error

1.8
2.8

enhancement
0.5
1.5
High
m
m

.636- 9.
.478- 3.

473
296

4.801
1.887

358.
384.

95
09

45.5
38.9

342.41
302.58

22.3 14.
69.2 11.

6
5

2.6
1.6

enhancement
0.5
• 1.5
0.5
1.5
m 14
m 4
m
m
.271-28.
.229-12.


736
257


20.840
8.797
P =
P =
337.
362.
0.
N.
27
64
3
S.
30.3
55.4


304.69
314.89
0.2
N.S.
35.9 14.
38.6 14.
N.
N.
2
1
S.
S.
1.3
1.6


                                       15

-------
Table 11.  Means of broccoli parameters and standard error for the 0.5-m row and
           the 1.5-m rows,  means and ranges of weighted mWm   for the three
           different levels of biologically effective UV above ambient provided
           by unfiltered FS-40 lamps.
Row

Range
(mWm~2 BUY)
Dry
weight
of
Means tops Standard
(mWnT2 BUY) (g) Error
Fresh
weight
of Fruits
Fruits Standard Standard
(g) Error (No.) Error
Control
0.
1.
Low
5
5

m .106- 3.227
m .098- 3.182

.805
.784

844.48
977.25

79.4
111.9

794.
729.

82 49.1
16 155.4

3.20
2.50

.3
.3

enhancement
0.
1.
5
5
ra .636- 9.473
m .478- 3.296
4.801
1.887
617.33
849.15
45.3
61.2
605.
505.
90 43.9
28 59.6
3.75
2.50
.2
.4
High
enhancement
0.
1.
0.
1.
5
5
5
5
m 14.271-28.736
m 4.299-12.257
m
m
20.840
8.797
P =
P =*
602.70
781.16
0.05
0.3
31.7
21.7


438.
642.
0.
N.
96 53.4
34 51.2
01
S.
2.30
2.20
0.05
N.S.
.3
.3


                                        16

-------
                           LITERATURE CITED




1.  Ambler, J. E., D. T. Krizek, and P. Semeniuk.  1975.  Influence of UV-B




    radiation on the early seedling growth and translocation of   Zn from




    cotyledons in cotton.  Physiol. Plant 34:177-181.




2.  Krizek, D. T.  1975.  Influence of ultraviolet radiation on germination




    and early seedling growth.  Physiol. Plant 34:182-186.




3.  Krizek, D. T.  1978.  Response of selected vegetables and agronomic crops




    to increased UV-B irradiation under greenhouse and growth chamber conditions.




    Final BACER Report submitted to the Environmental Protection Agency.




4.  Krizek, D. T.  1978.  Differential sensitivity of two cultivars of




    Cucumis sativus L. to increased UV-B irradiance.   Plant Physiol. Suppl.




    61(4):92.   (Abstr.).




5.  Rowan, J.  D.  and K.  H. Norris.  1978.   Instrumentation for measuring




    irradiance in the UV-B region.  Final BACER Report submitted to the




    Environmental Protection Agency.




6.  Thimijan,  R.  W., H.  R. Cams, and L. E.  Campbell.  1978.  Radiation




    .sources and related  environmental control for biological and climatic




    effects UV research.  Final BACER Report submitted to Environmental




    Protection Agency.
                                   17

-------
                        FINAL REPORT
  EFFECTS OF UV-B RADIATION ON PLANT MEMBRANE PERMEABILITY,
              RESPIRATION AND OXYGEN EVOLUTION
                        P. C. Jackson
                  Plant Stress Laboratory
                 Plant Physiology Institute
          Beltsville Agricultural Research Center
                 Beltsville, Maryland 20705
                      EPA-IAG-D6-0168


                      Project Officer:

                      R. J. McCracken

Agricultural Research, Science  and Education Administration
               U. S. Department of Agriculture
                  Washington, D.C. 20250
                      Prepared for
                  Environment Protection Agency
                      BACER Program
                   Washington, D.C. 20460

-------
                                  ABSTRACT




     Poinsett cucumber plants were grown in a growth chamber under UV-A


                         _n

and under UV-A + 8-24 mWm~  UV-B (2.8-7.9 Sun equiv.) for 6 hours a day




midway in the light period.  Leaves of plants under UV-A + UV-B were




smaller, weighed less, and took up ions, respired and evolved 02 at slower




rates than leaves of plants under UV-A alone.  The pattern of appearance




of these effects varied among leaves and with duration of treatment.  The




effects of UV-B were much the same whether treatment was started as soon



as the plants emerged or on the eighth day of plant growth.  When rates


     •4*       —
of Na  and Cl  uptake, respiration and Oo evolution are expressed on the




basis of amounts per leaf instead of amounts per gram, the inhibitory




effects of UV-B are greater because of the lower weights of the leaves




under UV-B.   There is no evidence of an increase in ion permeability in




these plants under conditions of this study.  The data are more indicative




of a decrease in permeability insofar as the ion uptake rates were inhibited




and somewhat more K+ was retained by the plants under UV-B.  Effects of




respiration, Oo evolution and ion uptake generally preceded appearance of




visual symptoms (chlorotic spots).   Thus measurements of either ion uptake



or respiration could provide an early assay for plant sensitivity to UV-B.

-------
                               INTRODUCTION

      Recent awareness that the use of chlorofluorocarbons in aerosol spray

cans  and as refrigerants may decrease the protective layer of ozone in  the

stratosphere sufficiently to cause ?n increase in ultraviolet radiation in

the region of 280-320 nm (UV-B) raises the question of the effect of increased

UV-B  radiation on agricultural crops.  Although effects of ultraviolet

radiation in the regions of 100-200 nm (UV-C) and 315-400 nm (UV-A) on  plants

are well known, there is a dearth of information on effects of UV-B on  plants.

The work presented here was undertaken to provide information on early

effects of UV-B radiation on plants and to assess whether measurements  of

respiration, oxygen evolution and permeability to ions can serve as assays

for UV-B effects before symptoms of toxicity are visible.

                    MATERIALS AND METHODS

      Seeds of cucumber (Cucumis sativus var. Poinsett) (a cultivar known to .be

sensitive to UV-B radiation)  were germinated on moist paper towels at 30ฐC.

On the second day, each seedling was planted in a 10-cm pot of moistened

vermiculite and placed in a grovrth chamber in the presence or absence of UV-B

radiation (Fig.  1).   UV-B radiation was from four Westinghouse—  FS-40 sunlamps

suspended 30 cm above the tops of the plants and covered either with 5-mil
                              \
Mylar film (UV-A control) or  with cellulose acetate at a thickness of 5 mil
 'Mention of a trademark, proprietary product,  or vendor does not constitute

  a guarantee or warranty of the product by the U.S.  Department of Agriculture

  and does not imply its approval to the exclusion of other products or

  vendors that may also be suitable.

-------
  (CA-5) or 10 mil (CA-10).   The intensity of the UV-B irradiance as measured



 by a Norris Spectroradiometer is shown in Table 1.   The plants were maintained



 on a 16-hour light period at 30ฐC with f> hours of UV-B exposure midway during



 the light period, and an 8-hour dark period at 25ฐC.   They were watered daily



 with a 1/5 Johnson solution and the pots were rearranged daily to minimize



 effects of position.   Samples of whole leaves, leaf  sections,  or cotyledons



 were taken immediately after UV-B treatment.



     Oxygen uptake was measured in the dark and oxygen evolution was measured



 in the light, in water at 25ฐC by means of oxygen electrodes.   Permeability



 was assayed by measuring fluxes of K , Na , Cl , Ca   ,  and Mg   .  Samples were


                                      -2           -A
 maintained in aerated solutions of 10   M NaCl + 10    M CaSO,  at pH 5.5 for



 various periods from  1 to 6 hours.   Then they were removed,  rinsed twice with



 demineralized water,  blotted gently and weighed.   The samples  were then ashed



 at 480ฐC for 1 hour.   The ashed samples were  dissolved  in  1  N  HNO- + 10%



 CH-COOH and aliquots  of these solutions were  taken for  analyses of ion content.


   +      +                                        2+        2+
 Na  and K  were determined  by flame photometry,  Ca    and Mg    were determined



 by atomic absorption  and Cl  was measured by  potentiometric  titration.




. .                  .            RESULTS




 Week-Old Cucumber Plants

                               \

      First leaves of  plants grown for 7  days  before UV-B treatment was  initiated



 showed  little or no difference from controls  (under Mylar) in  leaf size or



 weight  during the subsequent 2 weeks  under  UV-B  (Table  2).   On about  the sixth



 day,  small chlorotic  areas  appeared on the  edges  of the second and third leaves



 of  the  plants under CA-5 filters.   Leaves of  plants under  CA-10 filters had few



 or  no chlorotic  spots.   Examination of these  areas under a microscope  revealed

-------
 that they were not only devoid  of  chlorophyll  but  almost  devoid  of  chloroplasts,



 starch and other structures  as  well.   No  necrotic  areas or  oxidized phenols


                                                      /

 were apparent in these  leaves when they were examined after removal of



 chlorophyll.



      The  second and third  leaves of control plants  emerged  about  the third day



.of  UV-B treatment,  but  emergence and expansion of  these leaves under UV-B was



 delayed initially as shown by relatively  low weights  at 6 days in Table  2.



 The weights caught  up somewhat  when the control leaves reached full develop-



 ment however.   Cotyledons  tended to decrease in weight sooner under UV-B.  The



 UV-B irradiance had no  significant effect for  the first 10  days on  K concen-


                                                                           -2
 trations  of the first and  second leaves whether they  were in water  or in 10



 M NaCl.   However,  K  concentrations were significantly higher in these leaves



 at  14 days and at  6 days in  the third  leaves.



      Rates of  Na  uptake by  first  leaves under  UV-B generally were  somewhat



 stimulated initially (Table  3)  but were not effected  after  2 days.   Although'



 Na   uptake by  second leaves  was inhibited at 6  days,  soon after emergence, UV-B



 had no effect  thereafter and had no effect on Na  uptake by third leaves.  Both



 Na"*~ and Cl~ uptake  by cotyledons under CA-5 was  inhibited from the  first day



 of  treatment,  but UV-B  at  the lower intensity had no  consistent effect.  Rates



 of  Cl~ uptake  were  more sensitive  to UV-B exposure, especially in leaves under
                               s


 CA-5,  the  higher UV-B intensity.   Inhibition was observed as early  as 6 days in


                                                                            2+
 second leaves  and in all leaves by the fourteenth day.  Concentrations of Ca


       9+                                                    '
 and  Mg  were  affected  little by UV-B exposure  (Table 4).   First leaves had


         2+2+
 higher Ca   and  Mg    concentrations at 14 days  than control leaves and third


                    2+
 leaves had  lower Ca   concentrations at 6 days.  Rates of oxygen uptake and



 evolution  were  also  stimulated  during the first days of UV-B treatment and



 inhibited  thereafter (Fig. 2).  The inhibitory  effects were generally greater



 under  CA-5.




                                   4

-------
 Two-Day-Old  Cucumber Plants



      Leaves  of plants exposed  to UV-B from the time of epicotyl emergence




 (2 days)  generally respired and evolved' 0  at somewhat inhibited rates during




 the  first  few days after leaf  emergence, but thereafter, the rates were about




.the  same  as  or slightly higher than those of control leaves on a nmole/min-g




 fresh wt.  basis  (Fig. 3).  Leaves of these UV-B-treated plants, however,




 weighed less than control leaves (Table 5) and were 30-40% smaller, so that




 respiration  and  0™ evolution on a nmole/plant basis were inhibited.  Leaves of




 plants under UV-B for 8-10 days also took up Na+ and Cl~ from 10~2 M NaCl at




 slower rates than control leaves (Table 6).  Rates of uptake by first and




 second leaves were less inhibited by the fifteenth day, suggesting some degree




 of photorepair.  Ion uptake rates by cotyledons were inhibited from the first




 and  showed no evidence of photorepair.  Since weights of leaves in these experi-




ments were less also under UV-B, particularly at the higher UV-B intensity




 (CA-5), Na  and Cl~ uptake rates were likewise inhibited on a micro-equiv/




 plant basis.  There was no consistent effect of UV-B on K  concentrations




 (Table 5).  The concentrations in the cotyledons and leaves under UV-B were




sometimes higher than those in control leaves;  consequently, leakiness in the



                                                                  94-       2+
 leaves under UV-B treatment was not evident.   Concentrations of Ca^" and Mg




in leaves under UV-B were generally lower than concentrations in control


                                                     Ot        o I

leaves.   This probably resulted from inhibi'tion of Ca   and Mg~  uptake from




the nutrient solution used to water the plants and not  from leakiness.  The



  74-       2+
Ca   and Mg   concentrations of control leaves increased with age,  but concen-




trations of these ions in UV-B-treated leaves increased very little until about




the fifteenth day.

-------
                              CONCLUSION




     In general, leaves of Poinsett cucumber plants exposed to UV-B radiation




 in  these experiments were smaller, weighed less, and took up ions, respired,




 and evolved oxygen at slower rates than leaves of control plants under UV-A.




 The magnitude of the effects reflected the intensity of the UV-B radiation,




 but did not reflect the age of the plants.  Effects of UV-B were much the same




 whether treatment began on the second day or on the eighth day of plant growth.




 Weights of the leaves growing under UV-B were lower in both groups of plants,




 so  that expression of the rates of ion uptake, respiration and 0- evolution




 and ion contents on the basis of equivalents per plant instead of equivalents




 per gram would greatly intensify the inhibitory effects.  Some degree of photo-




 repair was suggested insofar as the effects were sometimes less as time




 proceeded.                                                            .




     There is no evidence of an increase in ion permeability in these studies.




The data are more indicative of a permeability decrease since ion uptake rates




were inhibited and K  was not lost.  Effects on ion uptake, respiration and Oo




evolution generally preceded the appearance of visual symptoms.  The onset of




 inhibition of Na  and Cl  uptake occurred about the same time,  regardless of




 the leaf development, which suggests that the UV-B affected the whole plant.




Generally ion uptake was affected at about the same time as respiration and 0^




evolution.   This and similarities in the patterns of the effects suggests that




ion uptake rates reflect respiratory rates.   Thus measurements  of either




respiration or ion uptake could provide an early assay for plant sensitivity




to UV-B radiation.

-------
Table 1.  UV-B Irradiances in the Growth Chamber.
1 t
'Filter '
Mylar
CA 10 Mil.
CA 5 Mil.
•
Biologically Effective UV '
i
(mWm~2 ) i
0.265
10.954 - 8.604
24.131 -16.733
UV-B Sun
0.09
3.58
7.89
Equivalents

- 2.81
- 5.47
Plants were maintained at 30 cm from the UV source.  Pots were




rearranged 5x/wk.  The higher figures are the irradiance when the




filters were new and the lower figures are the irradiance a week




later, when the filters were replaced.

-------
 Table 2.  Effects of UV-B radiation on K  concentration and weights of




           cucumber leaves
1 I
r i
.'UV-B '
'Treatment '
Days
Cotyledons
1
2
6
Leaf 1
1
2
6
10
14
Leaf 2
6
10
14
Leaf 3
6
10
.14
t
+ ,
K Concentration
Mylar
neq/g
50
56
61
91
91
73
62
57
85
66
63
62
81
73
CA-10
% My
108
107
115
112
103
97
100
117**
92
106 .
119**
138**
96 %
107
CA-5 '
lar
103
112
116
108
97
103
108
112**
93
115
111**
152**
100
113
Mylar
8
0.55
0.60
0.63
0.39
0.62
1.39
1.40
1.35
2.35
2.73
3.24
1.18
3.70
4.46
t
Weight '
CA-10
%
91
108
72
79
102
90
93
120
72*
91
102
103
87
101
CA-5 '
Mylar
108
92
77
91
93
81
101
119
63*
91
88
49*
64
83
Plants were 7 days old when UV-B irradiance was started. The * and **




indicate significant differences from controls (Mylar) at the 5% and 1%




levels, respectively.

-------
Table  3.  Effect of UV-B radiation on rates of Na  and Cl  uptake by
                                              _o
          cucumber leaves from solutions of.10 "M NaCl, pH 5.5-
1 t
'UV-B
'Treatment
Days
Cotyledons
1
2
6
Leaf 1
1
2
6
10
14
Leaf 2
6
10
14
Leaf 3
6
10
14
Na Uptake Rates
Mylar
neq/min-g
31
18
35
30
42
36
41
44
45
61
54
51
51
53
CA-10
%
115
55
111
103
117
116
127
97
.78
93
82
99
86
95
CA-5
Mylar
78
53
65
121
136
108
88
82
76
82
88
95
* 101
93
t i
' Cl~ Uptake Rates '
1 Mylar
neq/min-g
19
9
22
18
27
20
19
24
28
21
32
18
23
' '*• 25
CA-10
%
102
100
104
77
103
119
107
67
86
95
63
112
85
73
CA-5 '
Mylar
68
72
79
122
99
122
91
71
72
77
61
115
63
59
The plants were 7 days old when UV-B irradiance was started.

-------
                                        2+       2+
 Table  4.   Effect of  UV-B  radiation on Ca   and Mg   concentration of



           cucumber leaves.
1
'UV-B
'Treatment
Days
Cotyledons
1
2
6
Leaf 1
1
2
6
10
14
Leaf 2
6
10
14
Leaf 3
6
10
14
i
2+
' Ca Concentration
' Mylar
neq/g

103
110
190

. 77
83
151
165
140

59
170
187

47
75
141
CA-10
% Mylar

130
85
137

90
88
88
120
160**

81
90
120

45**
110
110
CA-5


113
138
133

75
88
95
134
184**

81
87
105

35**
90
88
i
2+
MS
1 Mylar
neq/g

85
85
133

36
45
84
110
84

56
105
98

47
73
83
i
Concentration '
CA-10
% Mylar

115
86
114

102
106
89
114
126

82
91
105

92
97
106
CA-5 '


100
118
121

86
93
96
98
144*

85
85
104

89
90
88
Plants were 7 days old when UV-B irradiance was started.  The * and **


 indicate significant differences  from controls  (Mylar)  at the 5% and 1%



 levels,  respectively.

-------
                                                                                .OUUUIL-.
Table  5.   Effects  of  UV-B radiation on K+ concentration and weights of




           cucumber leaves
1
UV-B '
Treatment '
Days
Cotyledons
1
2
3
4
5
8
10
Leaf 1
5
8
10
12
15
Leaf 2
8
10
12
15
Leaf 3
10
12
15
i
K"*" Concentration '
Mylar
neq/g
61
69
61
76
62
60
51
88
72
71
66
52
91
70
88
51
99
109
57
CA-10
%
137**
102
102
81
98
119
121**
103
141**
117**
83
99
127*
124
75
111
117
75
95
CA-5 '
Mylar
105
100
88
81
108
117
149*
116 .
132*
123**
79
114
106
113
73
121*
123
81
112
Mylar
g
0.196
0.236
0.412
0.458
0.515
0.565
0.492
0.385
0.710
0.812
0.942
0.975
0.762
1.690
2.G65
2.170
0.685
1.137
2.848
i
Weight '
CA-10
%
93
116
61*
98
98
97
115
68
79**
94
89
97
68**
78*
62*
93
83
88
71*
CA-5 '
Mylar
98
110
62*
93
83**
79**
95
53*
63**
80**
72**
72**
48**
62**
51**
49**
51**
52*
59**
Plants were 2 days old when UV-B irradiance was started.   The * and **




•indicate significant differences from  controls  (Mylar)  at the %5 and 1%




level, respectively.

-------
Table  6.   Effect  of  UV-B  radiation on  rates  of  Na+ and Cl~ uptake from




           10~2 M  NaCl (pH  5.5) by  leaves of  cucumber seedlings^
1 	
*UV-3
'Treatment
Days
Cotyledons
1
2
3
4
5
8
10
Leaf 1
5
8
10
12
15
Leaf 2
8
10
12
15
Leaf 3
10
12
15

I
' Na+ Uptake
' Mylar
neq/min-g
49
40
35
33
24
28
18
36
47
50
24
38
50
57
75
103
32
118
132

CA-10
% M
90
66
91
83
100
76
96
132
84
82
88
84
73
89
74
94
108
52
95

Rates
CA-5
ylar
75
58
67
66
84
82
77
104
77
75
77
' -
69
%S7
68
66
80
37
72

t
1 Cl~ Uptake Rates
' Mylar
neq/min-g
9.2
11.7
9.2
9.6
12.5
14 .2
12.3
16
20
18
17
20
17
26
24.
36
13
20
52

i
i
CA-10 CA-5 '
7, Mylar
100
-
86
78
73
82
81
91
105
113
110
81
64
84
88
91
98
88
57


100
79
77
72
97
50
74
109
103
84
64
94
62
78
91
91
72
90
50
4
Plants were 2 days old when UV-B irradiance was started..

-------
                                          2+        2+
 Table  7.   Effects  of  UV-B  radiation  on  Ca  and Mg    concentrations  of

           cucumber leaves.
1 1
'UV-B '
'Treatment '
Days
Cotyledons
1
2
3
4
5
8
10
Leaf 1
5
8
10
12
15
Leaf 2
8
10
12
15
20
Leaf 3
10
12
15
20
„ 2+
Ca
Mylar
neq/g
65
135
106
175
123
168
228
203
123
172
260
250
68
122
296
197
316
81
127
169
231
Concentration
CA-10
% Mylar
101
91
94
92
98
107
91
103
87
97
79
81
90 *
• 73**
71
81
106
CA-5

93
88
88
80*
94
95
95
99
84
90
61
83
72*
78**
51*
92
90
98 106
84
91
114
80
97
90
• M,2+
' Mylar
neq/g
76
89
92
104
109
131
164
55
71
97
127
123
51
. 67
87
134
115
52
58
88
75
Concentration
CA-10
% Mylar
96
93
91
100
103
103
94
84
107
99
92
100
73**
80*
96
t
89:
107
93
90
89
111
i
CA-5 '

93
95
92
90
112
100
87
71**
97
93
49*
77
77*
78**
77
86
82
85
96
100
83
Plants were 2 days old when UV-B irradiance was started.  The * and **
indicate significant differences from controls (Mylar)  at the 5% and 1%
levels, respectively.

-------
                             FIGURE LEGENDS




Fig. 1.  Diagram of  the growth chamber showing levels of visible light.




         Positions of  the UV lamps are indicated by dashed lines.  Sheets




         of methylmethacrylate, shown by solid lines, divided the chamber




         into 3 sections for the UV-B treatments.  The treatments are




         indicated at  the top of the diagram by descriptions of the filters




         which covered the UV lamps.  Initially, there were about 20 pots




         of plants located in the center of each section.




.Fig. 2.  Effects of UV-B radiation on rates of Qฃ uptake and evolution by




         leaves of cucumber plants.  The rates are expressed as percentages




         of the control rates  (nmoles/min-g fresh wt.).  Solid lines are




         for treatment under CA-10 filtered UV-B and dashed lines for CA-5




         filtered UV-B.  The plants were 7 days old "when UV-B irradiance




         began.




Fig. 3.  Effects of UV-B radiation on rates of (^ uptake (Fig. 3a)  and




         evolution (Fig.  3b) by leaves of cucumber plants.   The rates are




         expressed as percen^<ฐes of the control rates  (nmoles/min-g fresh




         wt.).  The plants were 2 days old when UV-B irradiance began.

-------
     o  9
e
5 mil CA    10 mil  CA
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1



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300

330
305
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310

330
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310
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310
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                                              to
                                              •H

-------
   O2 UPTAKE
O2 EVOLUTION

80
inn
IUU
80
t-
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6
Gฃ
z
O
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120
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IUU
80
60
THIRD LEAF
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           DAYS OF UV-B TREATMENT
                  Fig. 2

-------
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-------
                            FINAL REPORT
   PHYSIOLOGICAL AND BIOCHEMICAL EFFECTS ON UV RADIATION:
CHANGES IN ANTHOCYAN'IN PIGMENTATION IN COLEUS BLUMEI BENTH,
                           A.  L. Fleming
                     Plant Stress Laboratory
                    Plant Physiology Institute
             Beltsville Agricultural Research Center
                    Beltsville,  Maryland 20705
                        EPA-IAG-D6-0168
                        Project Officer:

                        R.  J.  McCracken
   Agricultural Research,  Science and Education Administration
                  U.S.  Department of Agriculture
                    Washington, D.C.  20250
                        Prepared for
              Enviromental Protection Agency
                        BACER Program
                     Washington, D.C.  20460

-------
                                  ABSTRACT




     Rooted cuttings of  Coleus  blumei  Benth.  were  exposed  to  enhanced UV




radiation  [50  to 800%  increase  in biologically  effective UV (BUV)  radiation]




for 1 to 48 hours.  The  UV radiation was provided  by  Westinghouse  FS-40




fluorescent sunlamps,  unfiltered  (UV-A, B,  C) or filtered  with 5,  10 or




20 mil cellulose acetate  (UV-A, B) or  5 mil Mylar  (UV-A).   Exposure to high




levels of unfiltered UV  radiation (700 to 800%  increase in BUV)  resulted




in a significant decrease in  the concentration  of  anthocyanin extracted




from the leaves with methanol and HC1.  Degradation of the pigment occur-




red after 12 hours of  exposure  and was intensified with an increase in




duration of exposure up  to 24 hours.  Exposure  to  a 100% increase  in BUV




under 5 mil cellulose  acetate caused glazing  of the leaf surfaces,  distortion




of the leaf margins, and inhibition of leaf expansion.  At this  UV level, a




significant decrease in  the anthocyanin content of leaves  occurred  after




36 hours of exposure (6  days at 6 hours/day).




     A leaf injury index was developed to provide  a visual evaluation of




the extent of UV injury.  This  index was useful in rating  the severity of




plant responses to UV  treatments at increases in BUV  of less  than  100%.




Spectrophotometric analysis of methanol-extractable materials in coleus




leaves indicated that  the components absorbing at  280, 330, 412-434,  and




525 nm decreased with UV treatment,  whereas those  absorbing at 415-425 and




650-660 nm increased.

-------
     Recent  studies  (1,  7,  8,  9,  12,  13)  have  demonstrated  that exposure




to UV radiation  can  alter  levels  of anthocyanin  and  associated plant




pigments.  Wellmann  (12, 13) showed that  UV  irradiation induced flavonoid




synthesis and phenylalanine ammonia-lyase (PAL)  activity in parsley seedlings.




Ambler et al. (1)  induced  red  pigmentation in  cotton seedlings by exposing




them to UV-B radiation.  Semeniuk (8)  reported that  the leaves of 'Supreme




Annette' poinsettia  formed  a purple red anthocyanin  when exposed to a 100




percent or greater increase in biologically  effective UV (BUV) radiation.




In contrast, high  levels of UV-B  radiation significantly decreased the




anthocyanin  content  of 15  Coleus  cultivars (8).  Time-lapse photography




of Coleus blumei Benth. plants (unpublished  data,  this laboratory) revealed




that the anthocyanin pigment broke down within 24  hours of  exposure




to broad-band UV radiation  from unfiltered Westinghouse FS-AO  sunlamps.




The extent of the  color change was a  function  of the physiological age of




the leaves and the total UV irradiance.   In  addition,  no degradation of the




pigment was  noted  in portions  of  leaves that were  shaded by the plant canopy.




     In subsequent experiments, the UV radiation of  FS-40 lamps was filtered




with cellulose acetate (CA) to approximate the natural UV spectrum (11).   The




spectral cutoff and  total UV irradiance were controlled by  varying the filter




thickness.                                             '     .    '        >      •
                                           *



     The purpose of  this study was to describe the time course of the




anthocyanin  pigment  changes in coleus and to obtain  an indication of the




metabolic effects of broad  band UV radiation.
"Mention of a trademark, proprietary product, or vendor  does  not  constitute




a guarantee or warranty of the product by the USDA and does not imply  its




approval to the exclusion of other products or vendors that may also be




suitable.

-------
                            MATERIALS AND METHODS


     UV-B enhancement  facilities were developed  in  cooperation  with  the


Agricultural Equipment Laboratory  (AEL), Beltsville Agricultural  Research


Center  (BARC).  Enhancement studies were conducted  in accordance  with


the guidelines established for  the BACER program (2, 3).   UV  radiation


was provided by one or more Westinghouse FS-40 fluorescent sunlamps



unfiltered or filtered with 6-hour-aged 5 mil Mylar (UV-A)  or 5,  10, or


20 mil cellulose acetate  (UV-A, B).


     UV-B irradiance levels were determined with either an Optronic


Laboratories, Inc. Model  725 UV-B Radiometer or  an  Instrumentation


Research Laboratory (IRL) UV-B  Radiometer (4, 6).   Radiometer readings were


verified by spectral irradiance determinations (250-369 nm) with  an  auto-


mated spectroradiometer (4, 6)  at selected locations in the experimental


irradiation areas.

                                                    _2
     Weighted.irradiance  levels are reported as  mVm BUV,  the biologically


effective UV derived from the AT9 weighting function, and  unweighted

                 -2
irradiance as mWm   obtained by summing the measured or calculated values at


                                             —2                    —2
each nanometer from 280-320 nm.  Dividing mWm  BUV  by 3.06  (the mWm  BUV


of control sunshine) provides the fraction of BUV received  by each plant


relative to that of one control sunshine.


     When UV irradiation was obtained by filtering  the FS-40  lamps through


cellulose acetate, BUV was limited to the UV-B region (280-320  nm).


     For details concerning average control sunshine, spectral  character-


istics of UV fluorescent lamps  and filters, and  the weighting function, see


the BACER final reports of the  AEL and IRL, BARC (6, 11).


     The first experiment (Expt. 1) was conducted with unfiltered UV


radiation to determine the change of anthocyanin in the leaves of coleus

-------
 with time.  The subsequent experiments (Expts. 2 and 3) were conducted with


 filtered UV radiation to determine the effect of UV radiation on the spectra


 of methanol-extractable substances in coleus leaves, and to assess the


 reliability of the anthocyanin content as an indicator of UV damage.


 EXPERIMENT 1:  EFFECT OF UV EXPOSURE ON ANTHOCYANIN PIGMENT OF COLEUS LEAVES


      Six-week-old coleus cuttings were exposed to broad-band UV radiation


 in a laboratory maintained at 26 + 2ฐC.  The light sources were one unfiltered


 FS-40 sunlamp and three 40 watt cool-white fluorescent lamps, positioned

                                                            -2 —1
 20 cm above the plants to provide approximately 150-200 yEm  s   of


 photosynthetically active radiation (PAR).  Treatments consisted of


 continuous exposures of 1, 2, 4, 8, 12, and 24 hours.  A UV control was run


 concurrently under a section of the same lamp bank fitted with a plexiglass


 filter;  this filter has a cutoff at approximately 340 nm.  Plants (54 cm


 in height) were trimmed to two pairs of leaves at the 4th and 5th nodes.


 The stem was excised above the 5th node.   Each pair of leaves was analyzed


 as a separate sample.


      Following UV exposure fresh weights were determined and the leaves were


 frozen at dry ice temperatures.  After 15 minutes, the samples were thawed


.and cut  into strips in preparation for extraction (5).  Following extraction
                                 \

 with 100 ml of a solution of methanol containing HC1 (99:1), the anthocyanin
                                             *

 content  was measured at 525 nm with a Gilford spectrophotometer.  Pigment


 concentration was reported as absorbance/gin fresh weight.


      To  determine the effect of shading,  several leaves of a separate set of


 plants were partially shielded by cardboard masks.  The change in anthocyanin


 was observed and recorded photographically.


      Because of the large variation in the initial concentration of anthocyanin


 the experiment was repeated with changes in the sampling and analytical methods,

-------
 Instead  of  using  entire leaves as analytical samples,  leaf disks (11 mm


 diameter) were  taken  from each leaf  half.   Samples were taken at 0,  12, or 24


 hours.   These  treatments were randomized  within each leaf pair and replicated


 three  times.


     Three  leaf disks from each leaf were placed in the barrel of a  plastic


 syringe  and extracted for 1 hour with 99:1 methanol-HCl.   The disks  were


 rinsed twice at 20-minute intervals  with  5 ml portions of the extracting sol-


 ution.   The concentration of anthocyanin  was determined spectrophotometrically


 at 525 nm.  The results were reported as  absorbance/3  leaf disks.


 EXPERIMENT  2:  EVALUATION OF UV DOSE - RESPONSE FOR COLEUS


     In  an  effort to  further define  the UV dose-response  relationships  in


 coleus,  4-week-old coleus cuttings were exposed to broad-band UV radiation


 produced by two FS-40 lamps,  with appropriate plastic  filters.   The  filters,


 5 mil Mylar, 5 mil CA,  10 mil CA, and 20  mil CA were used.   The spectral


 cutoff characteristics  of these filters are illustrated in Fig.  7.


     Plants were grown  on a 16 hour  photoperiod at ca  28/25ฐ  day/night


 temperature.  PAR was provided by four 1500 ma cool white fluorescent


 lamps.  Plants were exposed to UV irradiation during the  last 6 hours of


 the photoperiod for 3,  6,  and  8 days  at UV irradiances and  BUV  levels shown

                                *
 in Table 1.  UV measurements  were taken from 250-369 nm with  an Optronic


Laboratories Model 725  UV-B spectroradiometer (4,6).


     After 3, 6, and  8  days of treatment,  the second set  of  leaves below the


apex was extracted with methanol-HCl  (99:1).   The  anthocyanin concentration


in each leaf was measured  spectrophotometrically at  525 nm  and  reported  as


absorbance/gm fresh weight.   Plants were scored  for  visual  leaf  injury  based


on leaf color,  shape and  size.                                         •

-------
EXPERIMENT  3.   DETERMINATION  OF  SPECTRAL  CHANGES  OF METHANOL-EXTRACTABLE




                SUBSTANCES  IN  COLEUS LEAVES.




     Following  exposure  to  unfiltered and  filtered UV  radiation,  coleus leaves




were extracted  with methanol-HCl  (99:1).   The  resulting  solution  was  concen-




trated by flash evaporation to less than  5 ml  and diluted  to  10 ml with the




extracting  solution.  UV and  visible absorption spectra  were  determined




with a Perkin-Elmer UV-Vis  spectrophotoraeter.




     In order to classify  the methanol-extractable constituents of coleus




leaves, other samples were  extracted with  methanol, concentrated  by flash




evaporation, and chromatographed on paper  (5,  10).  Following development  with




butanol:acetic  acid:water  (BAW, 4:1:5,  top layer) the  chromatographs  were




viewed under a  UV lamp.  Fluorescent spots were eluted with methanol-HCl  (99:1)




and diluted to  2 mil.  The  UV and visible  spectra were determined as  indicated




above.




                            RESULTS AND  DISCUSSION-




EXPERIMENT 1:   EFFECT OF UV EXPOSURE ON ANTHOCYANIN PIGMENT OF COLEUS LEAVES




     The exposure of coleus to unfiltered  UV radiation resulted in epidermal




damage, glazing and reduction in the anthocyanin pigment content.  This damage




was primarly confined to the upper leaf surfaces and initially involved only




those tissues exposed to the radiation.  The effect of shading is shown in Fig.  1.




The extractable anthocyanin of coleus leaves exposed to  continuous UV radiation




(1 FS-40 + 3 cool white fluorescent lamps  at 16 cm) for  1-24 hours decreased with




increasing time of UV exposure (Table 2).  The trend in  pigment reduction  was




most striking between 16 and 24 hours.  However, due to  considerable variability




in initial pigment intensity, the differences were not statistically  significant




at the 5% level by the F test.  When improved sampling techniques were used,

-------
 the  anthocyanin  concentration was  found  to be significantly decreased




 by exposure  to unfiltered  UV radiation for 12 hours (Table 3).




 EXPERIMENT  2:  EVALUATION  OF UV  DOSE - RESPONSE STUDIES




     The  leaf injury  in coleus,  produced by exposure to UV radiation, can be




 easily  observed  (7,8).   In previous  experiments with unfiltered UV radiation




 (a 700-900%  increase  in BUV) exposure for 12 hours produced a rapid disap-




 pearance  of  the  anthocyanin pigment  without concomitant changes in leaf




 shape.  However,  exposure  to FS-AO lamps filtered  with 5 mil CA (42 hours




 under a 300-500% increase  in BUV)  caused distortion of leaf margins (Figs.




 2, 3).  The  expanding apical leaves  assumed a "sickle shape," resulting




 from decreased development of  the.leaf half nearest to the UV source.




 Although  this symptom did  not  preclude further leaf expansion,  the curva-




 ture remained until senescence,  even with a subsequent reduction in UV




 exposure.  Both  symptoms were  used in developing a leaf injury  index (Table




 4).  Using these parameters,  injury  produced by the UV treatments over an 8-




 day period ranged from  slight  injury,  with the 10  mil CA filter, to intense




 injury with  the  5 mil CA filter.   The range of UV  injury from filtered




 treatments is shown in  Fig.  4.




     The  spectral transmission curves (Fig.  7) indicated that 5 mil Mylar




 had an effective cutoff at approximately 310^-315 nm;  the spectral cutoff




 for the CA filters ranged  from approximately 286-292,  depending upon the




 thickness of the  filter.




     The  contrast in UV injury between the plants  grown under Mylar (Fig. 5)




 and those grown  under 5 mil  CA (Fig.  6)  was  striking.   Symptoms of UV injury




 under 5 mil CA included marginal distortion,  glazing  of the surfaces and




 pigment changes  in the  apical  leaves;  under  5 mil  Mylar,  no visible changes




were observed.

-------
      Statistical  analysis  (Table 5)  indicated that the anthocyanin content




 of  plants  irradiated  under  5 mil CA  decreased significantly with increases




 in  the  UV  dose.




 EXPERIMENT 3:  DETERMINATION OF  SPECTRAL CHANGES  OF METHANOL-EXTRACTABLE




               SUBSTANCES IN COLEUS  LEAVES




      The visible-  spectra of  methanol-extractable  constituents  of coleus




 leaves  were modified  by exposure to  UV  radiation.   The extracts  from unirradiated




 control plants and  those filtered with  Mylar,  produced strong  absorbance peaks in




 the 412-434, 520-535,  and the 640-660 nm regions.   UV  treatment  was  characterized




 by a  reduction in the  absorbance at  the 520-535 peak and  increases at 412-434




 and 640-660 nm.  The  contrast between the unirradiated control plants and those




 irradiated  for 24 hours under unfiltered UV  is shown in Fig. 7.




      Similar results  occurred for a  6-hour unfiltered  treatment  when the




 plants  were more highly pigmented.   The visible spectrum  for greenhouse




 grown coleus is shown  in Fig.  8.  Strong absorbance bands  occurred at 420




 and 525 nm, with a minor peak at  657 nm.  The  spectrum for  the 6 hour




 unfiltered  UV treatment was  characterized by  reduced absorbance  at 525 nm




 and substantial increases at  425  and 657  nm  (Fig.  9).   This pattern  can




 be used to  demonstrate the effect of filtered  treatments  over  time, shown




 in Experiment 3.  Exposures  of plants to  3,  6, and  8 days  of UV  under




 FS-40 lamps + 5 mil CA were  associated  with  progressive increases  in the




 absorbance  at 425 and  657 nm  and  relative decreases at  525 nm  (Fig.  10).




     The UV spectrum of the  extracts from control plants  shows absorbance




peaks at 275-295 and 320-340  nm.  Twenty-four  hour  exposure to unfiltered




UV radiation reduced the absorbance  in  both regions  (Fig.  11).   The  effect




of UV radiation can be visualized by calculation of absorbance ratios.




Table 6 contains these data  for a 6-hour unfiltered treatment  and  the




corresponding unirradiated control.




                                     7

-------
      Paper  chromatography and  subsequent spectroscopy were used to determine

 the methanol-extractable  components  of  coleus leaves responsible for the

 observed  spectral  characteristics.   A summary of  the spectral characteristics

 of these  bands  is  given in Table  7.

      Band 7 produced  a large,  but poorly defined  peak in the region of a 210-

 230 run and  sharp'maxima at 415 and 657  run.   UV treatments increased the absor-

 bance at  415 and  657  nm (Figs.  12 and 13).   Band  6 contributed a small peak

 at 290 nm and a large peak at  330 nm, which decreased with intensity of UV

 treatment (Figs. 14 and 15).   Band 4 (Figs.  16 and 17)  absorbed at 270 and 330

 and appeared as a  large UV-absorbing area on the  chromatograph.  The spectrum of

 band  2 showed strong  peaks at  280 and 520-535 (Figs.  18 and 19).  UV treatment

 was particularly effective in  eliminating this band.

     With the exception of band 2, which appeared to  be a derivative of cyanidin

 (11), identification  of the compounds involved in this  separation has not been made,

     The destructive  effect of UV radiation on coleus leaf constituents, which

 absorb in this  region was  expected.   However phenols,  such as cinnamic or ferulic

 acids, did  not  increase measurably due  to UV treatment.                    .

                                   SUMMARY

     Short-term exposure  (12 to 16 hours) of 3-week-old coleus plants to high
                               \
 levels of UV radiation (an increase  in  BUV  of 100% or more)  produced by

unfiltered  FS-40 fluorescent sunlamps resulted in a significant decrease in

 the concentration  of  anthocyanin that could  be extracted from the leaves.

Although the concentration of  the pigment in unexposed  plants was variable

and depended on other environmental  conditions, such  as temperature,  light

 intensity,  and  nutrition  of the plants,  the  specific  decrease due to UV

 treatment was observed by  evaluation of  the  changes which  occur in portions

-------
of  the  same  leaf  and  in  the  opposite  leaves  of  the same  plant.  Significant




degradation  of  the  anthocyanin  pigment  occurred after  12 hours  of  exposure




and was  intensified with an  increase  in exposure up to 24 hours.   Complete




degradation  of  the  pigment occurred within  36  to 48 hours,  depending on




the experimental  material.   The quantitative relationship between  the




concentration of  the  pigment and  the  UV dose,  observed at a high level  of




exposure  (700 to  900% increase  in BUV),  did  not apply  proportionally to a




lower level  of  UV radiation  (less than  a 100%  increase in BUV)  produced with




CA filters.  Exposure to a 100  percent  increase in BUV caused distortion of




the leaf margins, and an inhibition of  leaf  expansion,  as well  as  some




degradation  of  anthocyanin.




     The visual leaf  injury  index appears to be a more useful indicator of




injury than  does  the  measured anthocyanin content of coleus leaves at




levels of exposure  constituting less  than a  100 percent  increase in BUV.




However, the anthocyanin content determined  after a 24-hour UV  exposure




to a 300 to  500 percent  increase in BUV  was  more reliable as an indicator




of UV dosage than was the index.  The effect of UV radiation on the




methanol-extractable  constituents of  coleus  leaves was particularly dramatic.




The UV-absorbing  components  of  the leaves were  substantially decreased  by




UV exposure.  In  the  visible range, the  strong  absorbance at 525 (anthocyanin)




was decreased by  maximuraal UV exposure,  whereas the peaks at 410-420 and




657 were increased.   Semeniuk (7) reported large increases  in absorption




of extracts from  coleus  leaves  examined  at 268,  286, and  328 nm.   This




relationship was  not  resolved by this study.  The reason  for increased




absorbance at 410-420  and 657 nm following UV exposure was  not  determined.

-------
     Although the underlying mechanisms of UV injury  in coleus  remain  to




be explained, the plant responses to enhanced UV radiation observed




(glazing of leaf surfaces, deformation of expanding leaves and  specific




decreases in anthocyanin pigmentation) may serve as useful predictors  of




UV damage in higher plant.
                                    10

-------
                             LITERATURE CITED




1.  Ambler, J.  E., D. T. Krizek and P. Semeniuk.  1975.  Influence of UV-B




    radiation on  the early  seedling growth and  translocation of   Zn from




    cotyledons  in cotton.   Physiol. Plant 34:177-181.




2.  Cams, H. R., R. Thimijan, and J. M. Clark.  1977.  Outline of irradiance




    distribution of UV fluorescent lamps and combinations.  Symposium on




    Ultraviolet Radiation Measurements for Environmental Protection and Public




    Safety.  June 8-9, 1977.  Nat'l Bureau of Standards Gaithersburg, MD  (Abstract)




3.  Krizek, D. T.  1977.  Current UV measurement methodology and future needs




    in photobiological research.  Symposium on Ultraviolet Radiation Measurements




    for Environmental Protection and Public Safety.  June 8-9, 1977.




    Nat'l Bureau of Standards Gaithersburg, MD  (Abstract).




4.  Norris, K. H.  1977.  Development of a portable, automated UV-B spectroradio-




    meter.  Symposium on Ultraviolet Radiation Measurements for Environmental




    Protection and Public Safety.  June 8-9, 1977.  Nat'l Bureau of




    Standards, Gaithersburg, MD (Abstract).




5.  Robinson, Trevor, 1975.  Flavonoids and Related Compounds.  Chap. 9, p. 190-




    223.  In_ The Organic Constituents of Higher Plants. Third Ed. p. 347.




    Cordus Press, New Amherst, Mass.




6.  Rowan, J. D. and K.  H. Norris.  1978.  Instrumentation for measuring




    irradiance in the UV-B region.  Final BACER Report submitted to the




    Environmental Protection Agency.




7.  Semeniuk, P.  1977.   Response of Coleus blumei Benth.  to enhanced UV-B




    radiation.  HortScience 12 (4) Section 2:395.  Abstract.




8.  Semeniuk, P.  1978.   Biological effects of ultraviolet radiation on plant




    growth and development in florist and nursery crops. Final BACER




    Report submitted to  the Environmental Protection Agency.
                                    11

-------
 9.  Skelly, J. M., M. F. George, H. E. Heggestad, and D. T. Krizek.  1978.




     Air pollution and radiation stresses.  Chapter 2.5. I_n ASAE Monograph.




     Modification of the Aerial Environment of Plants.




10.  Swain, T.  1976.  Flavonoids. p. 166-205.  In. T. W. Goodwin, (ed.), Chemistry




     and Biochemistry of Plant Pigments. Vol 2, Academic Press, NY.




11.  Thimijan, R. W., H. R. Cams, and L. E. Campbell.  1978.  Radiation sources




     and related environmental control for biological and climatic effects




     UV research.  Final BACER Report submitted to Environmental Protection




     Agency.                                               .




12.  Wellmann, E.  1974.  Regulation of flavonoid synthesis by ultraviolet light




     and phytochrome in cell cultures of parsley. Ber. Deutsh. Bot.  Ges. 87:267-273.




13.  Wellmann, E.  1975.  UV dose-dependent induction of enzymes related to




 ;    flavonoid biosynthesis in cell suspension cultures of parsley.   FEES




     Letters.  51:105-107.                                                   '
                                      12

-------
Table 1.  Weighted and unweighted UV measurements  of  UV  irradiance
          under FS-40 fluorescent sunlaraps  covered with various
          filters.

Filter
5 mil Mylar
20 mil CA
10 mil CA
5 mil CA
UV-B
Irradiance
mWm
129.7
982.0
1494.5
2161.4
BUV2
,^,-2
mWm
0.23
4.52
9.44
18.15
"Tieasurements were taken with a spectroradiometer  12.7  cm  above
 the canopy

2
 A base line level of biologically effective UV  (BUV) radiation was
 equivalent to 3.06 mWrc   for a standard Beltsville  sun.
                                     13

-------
Table 2.  Concentration of anthocyanin in Coleus  blumei  Benth.  leaves  as  a
          function of UV exposure and node position.   Basal  node  =  1.
          (Absorbance/g fresh wt. of leaf tissue).  One  unfiltered  FS-40
          lamp placed 16 cm above the top of  the  plants.
Time of
Exposure
(Hours)
1
2
4
8
16
24
Average anthocyanin concentration
(Abs/g fresh wt . ) 525 nm

Node
4
.499
.352
.298
.600
.385
.451
-UV
Node
5
.501
.470
.330
.386
.445
.518
+UV
Average
.500
.411
.314
.493
.415
.485
Node
4
.560
.384
.370
.575
.394
.083
Node
5
.509
.403
.405
.696
.292
.007
Average
.535
.394
.388
.636
.343
.045
                                        14

-------
Table 3.  Change in anthocyanin concentration of Coleus blumei  Benth.  leaves
          with duration of UV exposure under one unfiltered FS-40  lamp at
          30 cm.  (Absorbance per 3-leaf disks, each 11 mm diameter.)
Time of
Exposure
0
12
24
Anthocyanin
Node 5
.506a*
.264 b
.117 c
Concentration
Abs/3 leaf disks
Node 4
.492a
.269b
.215 b
Average
.499
.267
.166
*Mean followed by a common letter are not significantly different at
 the 5% level according to Duncan's multiple range test.
                                       15

-------
Table 4.  Effect of UV  exposure  on visual  leaf  injury index.
Duration                         Injury  index
of Exposure*
Days*
3
6
8
Weighted mWm
% Increase in
Mylar 5 mil CA
0 +
0 ++
0 -H-f
0.23 18.15
BUV - 493
10 mil CA 20 mil
0
o
•+ 0
9.44 4.
209 48
CA



52
* 6 hours/day

Key:     0 = no injury
         + = moderate  (slight leaf distortion  with  negligible  pigment  loss)
        •H- = severe  (leaf distortion with  •  50%  pigment  loss)
       -H-f = intense  (leaf distortion with   50% pigment loss)
                                  16

-------
Table 5.  Anthocyanin concentration in Coleus blumei  Benth.  leaves  as  a
          function of UV exposure.
Duration
of Exposure**
Days



3
6
8
-2
Weighted mWm
%
Increase in BUY
Anthocyanin
Mylar
1.66 ab**
1.28 bcde
1.37 abed
Concentration (Abs/g fresh
5
1.
1.
0.
0.23 18.
- 493

mil CA
50 ab
04 cde
84 e
15

10 mil CA
1.
1.
1.
9.
209
27 bcde
51 ab
49 abc
44

weight)
20 mil
1
1
1
4
48
.74 a
CA

.43 abc
.47 ab
.52




* 6 hours/day

**Means followed by a common letter are not significantly different at
  the 5% level according to Duncan's multiple range test.
                                      17

-------
Table 6.  Absorbance/g fresh weight  for  leaf  extracts  from unfiltered
          UV treatments  (FS-40 at  30 cm).
                 Absorbance/gm at
Treatment        Wavelength  (nm)
            330   •     420       525
      Absorbance Ratio

320/420    420/525   330/525
Control    0.472      0.052     0.068

Unfiltered 0.351      0.100     0.14
    UV
  9.07       0.77      6.94

  3.51       7.14     25.0
                                       18

-------
Table 7.  Chromatographic separation of methanol-extractable components
          from the leaves of Coleus blumei Benth.

Band
1
2
3
4
5
6
7

Rf
(x 100)
10.5-19.2
24.5-33.3
35.1
36.0-57.9
59.6
61.4-94.7
94.7 .

Color
Visible
Pink
Red
x
X
X
X
Yellow-green

UV
Rose
Rose
x
dark brown
(absorbing)
x
bright blue
yellow-brown

Absorption maxima
Visible UV
* *
520-535 280
* *
* 270,
* *
* 280,
415, 657 210,
(run)



330

330
230
* No observed maximum


  Colorless
                                      19

-------
                              LEGENDS TO FIGURES

Figure  1.    Influence  of  shading  Coleus. blumei Benth.  leaf from UY radiation.

Figure  2.    Abnormal growth of  upper Coleus blumei Benth.  leaves exposed to   ;

             UV-B  (2 FS-40 lamps filtered vdLth 5 rail cellulose acetate and

             mounted at 30 cm).  Left to right:  Plants irradiated 6 hour/day
                ป
             for 6, 9,  and 12 days,  respectively.

Figure  3.  Response of expanded leaves  of Coleus blumei Benth.  to UV-B.

             Left  to right:   Plants  irradiated for 6,  9, and 12  days respectively

             under 2 FS-40 fluorescent sunlamps filtered with 5  mil cellulose

             acetate lamps mounted 30 cm above the plants.

Figure  4.    Effect of  UV  radiation  on 4-week-old  Coleus blumei  Benth cuttings

             irradiated 6  hour/day for 8 days.   Filter  treatments (1-r)  5 mil

             Mylar, 5 rail  CA,  10 mil CA,  and 20 mil CA.

Figure  5.    Four-week-old Coleus blumei Benth.  cutting  irradiated 6 hour/day

             for 8 days under  2  FS-40 lamps  filtered with 5 mil  Mylar.

Figure  6.    Four-week-old Coleus blumei Benth.  cutting  irradiated 6 hour/day

             for 8 days under  2  FS-40 lamps  filtered with 5 mil  CA at a  493%

             increase in BUV.

Figure  7.    UV spectral irradiance  obtained  under  2 FS-40  fluorescent sunlamps

             filtered with 5 mil CA  (setup  1),  10  mil CA (setup  2),  20 mil CA

             (setup 3)  and 5 mil Mylar (setup  4) at a distance of  cm above the

             canopy.                                         •'

Figure 8.   Visible absorption  spectrum  of  methanol-extractable  constituents

            of Coleus  blumei Benth.  leaves.  Plants were grown  in a greenhouse

            without supplemental UV  treatment.

Figure 9.   Visible absorption  spectrum  of methanol-extractable  constituents

            of Coleus  blumei Benth.   leaves  after  6 hours of  exposure to

            unfiltered  FS-40 fluorescent sunlamps.

-------
Figure 10.  Visible  absorption  spectra  of  methanol-extractable constituents



            of Coleus  blumei  Benth.  leaves irradiated 6 hour/day for 3,  6,  and



            8 days under  FS-40  lamps  filtered  with 5 mil CA.



Figure 11.  Comparative UV  absorption spectra  of  methanol-extractable components



            of Coleus  blumei  Benth.  leaves left unirradiated  (bottom curve)



            .or exposed to unfiltered  FS-40 fluorescent sunlamps for  6 hours



            (top curve).



Figure 12.  Visible  absorption  spectrum of band 7 obtained  from extract  of



            Coleus blumei Benth.  leaves following 6 hours of  UV irradiation



            under unfiltered  FS-40 lamps.



Figure 13.  Visible.absorption  spectrum of band 7 obtained  from extract  of



            Coleus blumei Benth.  leaves taken  from an unirradiated control  plant.



Figure 14.  UV absorption spectrum of band 6 obtained from  extract of Coleus



            blumei Benth. leaves  taken  from unirradiated control plant.



Figure 15.  UV absorption spectrum of band 6 obtained from  extract of Coleus



            blumei Benth. leaves  exposed for 6 days to 2 FS-40 lamps + 5 mil



            CA at 30 cm above the canopy.



Figure 16.  UV absorption spectrum of band 4 obtained from  extract of •Coleus



            blumei Benth. leaves  taken  from unirradiated control plant.



Figure 17.  UV absorption spectrum of band 4 obtained from  extract of Coleus
                                             *


            blumei Benth. leaves  following 6 hour  of  UV irradiation  under



            unfiltered FS-40  lamps.



Figure 18.  Visible absorption  spectrum of  band 2 obtained  from extract  of



            Coleus blumei Benth.  leaves taken from unirradiated control  plant.



Figure 19.  UV absorption spectrum of band  2 obtained from  extract of Coleus



            blumei Benth  leaves taken from unirradiated  control plants.
                                        21

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                     FINAL REPORT
  EFFECTS OF UV-B RADIATION ON PHOTOSYNTHESIS AND GROWTH
              OF SELECTED AGRICULTURAL CROPS
                    J. H. Bennett
               Plant Stress Laboratory
              Plant Physiology Institute
       Beltsville Agricultural Research Center
              Beltsville, Maryland 20705
                   EPA-IAG-D6-0168
                   Project Officer:

                   R. J. McCracken
Federal Research, Science and Education Administration
            U.S. Department of Agriculture
              Washington, D.C. 20250
                     Prepared for
           Environmental Protection Agency
                     BACER Program
                Washington, D.C.  20460

-------
                              ABSTRACT





     Selected  snap bean,  soybean, clover, cotton, cucumber and wheat



varieties were exposed to UV-B radiation over 2  to 6 week periods  (6 •



or 24 hr/day)  under greenhouse and growth chamber conditions.  Biologically



effective UV-B irradiances based on an experimentally determined action



spectrum ranged from 1-8  Sun Equivalents (SE).   Carbon dioxide exchange



rates (CER), plant biomass production, stomatal  diffusion and transpiration



were determined.  UV-B effects on CER and foliar diffusivities were correlated



with the amount of visible injury induced (i.e., chlorosis, leaf and petiole



pigmentation,  leaf stipple).  In the absence of  visible injury, CER, leaf



conductances,  and biomass production were not measurably depressed in



experimental plants given the extended UV-B exposures.



     The plant  species differed markedly in their susceptibilities to



UV-B radiation.  Greenhouse-grown snap beans and soybeans sustained high



levels before  they were injured (in excess of 125 hr exposures—3 weeks,



6 hr/day—to more than 4 SE).  Wheat and clover were not injured by the



maximum UV-B exposures tested (3-4 week treatments at 2 SE).   Poinsett



cucumber developed marginal chlorosis when irradiated for 1-3 weeks at



1-2 Sun Equivalents.  Cotton petioles and midveins at the leaf bases
                                          *


became (red) pigmented in the 4 SE trials.   Snap bean plants grown under



low-light conditions in the growth chamber were more sensitive to UV-B



injury than when grown in the greenhouse.

-------
                              INTRODUCTION


     Ultraviolet B  (280-320 nra) irradiation corresponding to enhanced


levels reaching the earth's surface due to projected stratospheric ozone


(03) destruction by halocarbon emissions has been reported to suppress


photosynthesis in certain agricultural plant species (1-4).  Some data


indicate that vegetation grown and exposed to UV-B under low photo-


synthetically active radiation (400-700 nra) regimes may be more sensitive


than when irradiated in bright sunlight (4).  Photorepair mechanisms may

be important in mitigating the plant damage.


     Experimental plants showing UV-B depressed carbon dioxide exchange


rates (CER) produced less plant biomass when irradiated over an extended


period of time.  Brandle et al. (1) observed chloroplast structural damage


in UV-B injured leaves and correlated it with reduced Photosystem II activity.


This was proposed as a causal factor in depressing photosynthesis and


growth.   They further concluded from their studies that CER suppression was


not caused by stomatal closure induced by UV-B damage to the leaf epidermis.


Stomatal resistance to gas exchange might increase without specific injury


to the epidermal cells, however,  as mesophyll and chloroplast disruption can


result in stomata closure as a consequence of higher C02 concentrations

                              \     .
within the leaves (as well as other factors).


     Little information has been published concerning visible injury

on UV-B irradiated leaves showing reduced photosynthesis and growth.


It was not possible to effectively assess the extent to which growth

reduction correlated with tissue damage and to integrate this with CER


suppression.

     Considering the potential importance of the reports referred to above


on impending governmental regulatory actions dealing with the environmental

-------
 impact  of  chlorofluorocarbon use  In  the United  States,  this  research was

 conducted  as  part  of a Federal interagency  cooperative  project  to  corroborate

 earlier findings and to expand the available  information  on  UV-B radiation

 effects on agricultural plants.   Research reported  here present data on

 UV-B exposures  and the exposure ranges required to  measurably depress

 C(>2  assimilation in selected crop plants.   Special  attention was given to

 integrating the results with incipient visible  injury.  UV-B effects on gas

 diffusion  through  the upper  and lower leaf  surfaces under typical  growth

 chamber and greenhouse conditions were also investigated.

      Soybean, cotton,  wheat,  clover, cucumber,  and  two  snap  bean varieties

 were tested.

                         METHODS AND MATERIALS

      Experimental  plants, with the exception of cucumber,  were  grown

 and  irradiated  with UV-B under (i) common growth chamber  conditions

 or  (ii)  in a  glass greenhouse.  The plants were cultured  in  15-cm  dia.

 clay pots  containing a sand-silt  (1:3) soil mix.  They were  fertilized

 weekly  with Peter'si'  fertilizer  containing micronutrients.  The (heated

 or wet-pad, fan cooled)  greenhouse was equipped with a high-volume

 charcoal air  filtration system to prevent plant injury  from  oxidant  air

 pollution  (5).  Cucumbers were grown and irradiated in fiberglass  green-

 houses  in  12.5-cm  dia.  pots  filled with a peat-vermiculite mix  (1:1).

 They were watered  daily with  1/4  strength Hoagland's solution during the

 first 3  weeks and  with 1/2 strength Hoagland's  solution thereafter.
—' Mention of a trademark or a proprietary product does not constitute a
   guarantee or warranty of the product by the USDA and does not imply
   its approval to the exclusion of other products that may also be
  . suitable.

-------
The cucumber plants tested were part of another U.S. Department of

Agriculture  (USDA) study  (6) concerned with UV-B injury and growth

reduction in the sensitive Poinsett variety and more tolerant Ashley

variety.

     Growth chamber (Controlled Environments, Inc., Model PCW 36)

conditions are shown in Figure 1.  The plants were given 12-hr photo-

periods  (0800-2000) utilizing cool white fluorescent + incandescent

lighting.  Greenhouse plants received solar radiation.  Photosynthetically

Active Radiation (PAR) was monitored with a LI-COR 190-S Quantum Sensor.

     UV-B exposures were carried out using standardized procedures (7)

developed for USDA UV-B studies employing FS-40 sunlamps with 5 rail

cellulose acetate (CA) filters.  Mylar filters were used for the controls.

Spectral irradiances (250-370 nm range) were measured with an Optronic

Laboratories, Inc., Model 725 spectroradiometer developed by the

Instruments Research Laboratory (IRL), SEA, USDA (8).  Spectroradiometer

data were obtained for every nanometer over this range.  Routine broadband

UV-B measurements were made with an IRL UV-B Radiometer.  Plants were

irradiated for 6 hours per day (between 1000-1600) or, in some companion

experiments, for 24 hours per day.
                              \
     Figure 2 shows a typical UV-B spectral irradiance curve for the CA-
                                           •
filtered FS-40 sunlamp system obtained at a distance providing a total
                                                          _2
irradiance for the 280-320 nm wavelength range of 1079 mWm  .   The

plant injury Action Spectrum, empirically determined for cucumber and

certain other plants (9),  and the weighted biologically effective

ultraviolet irradiance (BUV) are also plotted.  For the FS-40  sunlamp

system the summed BUV over the 280-320 nm region, called the Action

Integral ZAป  represented approximately 1% of the total UV-B irradiance

-------
             f\
 (cf.  10  mWm   in Figure  2).   This varies  somewhat  as  the  CA  transmission

 changes  with exposure  time  (solarizes).   UV-B  spectral  transmission  of

 the CA filters was  routinely  monitored with  a  Beckman DB  UV-visible

 recording  spectrophotometer.   Spectroradiometer  data  were also obtained

 for different  exposure times.  The CA filters  were changed every 4 days.

      The unweighted and  BUV-weighted irradiance  curves  in Figure 2

 represent  UV-B exposures for  Figure 1 experiments.

      Paired (control vs  UV-irradiated) intact  leaves  or whole plants

 stratified according to  age,  position, stage of  development, and condition,

 were  used  in each CER  trial.   The paired  foliar  subjects  were examined

 simultaneously under identical conditions in matched  Physiological

 Activity and Diagnostic  Chambers (PhAcDC) which  permitted C02 and water

 vapor exchange rates and leaf  and air temperatures  to be  continuously

 monitored  during  the experimental runs (10,11).  The  PhAcDC  tests

 regulate and standardize physical parameters that  enter into the leaf

 energy balance equation  [ie.,  Radiant Energy (input)  = Reradiation +_

 Convection Hh Evapotranspiration ฑ Metabolic  Energy  (net photosynthesis

 or respiration )].  PhAcDC cuvettes were equipped with internal mini-

 systems  for  humidity and wind  control.   PAR was  derived from Quartzline
                               \
 lamps filtered through a 10-cm 1^0 heat filter.  Dual PhAcDC experiments

                                                  9   —1
were conducted at PAR intensities (900 H^ 100 yE  nT^sec  ) that gave maximum,

but light-unstressed, steady-state apparent photosynthesis (Pmax) rates.

Chamber  temperature, relative humidity,  and wind conditions were:  27 Hh

2ฐC, 60 + 5% and  0.5 + 0.1 m sec"1,  respectively.   Soil temperatures were

25 + 2ฐC.
                                                                      4
     Leaf,  air, and soil temperatures were monitored with an 11-channel

YSI Telethermometer (Probe types:  T2600,  T2631).  Leaf temperature

-------
 calibrations  and  spot  checking were made with a Mikron  15  IR noncontact


 Thermometer.  Wind  speeds were determined with Hastings RF-1 and AB-27


 Air Meters  (Probes: N-7B and  S-27).  Soil moisture and  pH x^ere determined


 with a Bouyoucos  Soil  Moisture Meter and the Kelway  Soil pH/Moisture


 Tester, Model HB-2.


     Each paired  run required approximately 4 hours  in  the dual-PhAcDC


 systems for complete examination and diagnosis including steady-state


 Pmax rates, dark  respiration  rates, and leaf diffusive  resistances.


 Relative CER  data given in Tables 1 and 2 were calculated from steady-


 state Pmax rates."  Foliar diffusive resistances were computed from


 evapotranspiration  rates and  data taken with a Lambda Diffusion Porometer


 (10,11).  Plant biomass data  were determined from leaf  area measurements


 and fresh and dry weights.  The experiments were conducted during


 January-September 1977.


                        RESULTS AND DISCUSSION


 Leaf diffusive resistance;  Effects of UV-B and experimental conditions


     Results  of experiments to investigate the influence of exposure


 conditions on gas diffusion through the stomata of UV-B  irradiated plants


 are shown in  Figure 1.  Two snap bean varieties (Phaseolus vulgaris cvs.
                              \

 Bush Blue Lake 290 and Astro) grown and exposed to UV-B radiation in


 the growth chamber and greenhouse are compared.   An inset gives their


 relative plant biomasses at harvest.                           :


     Ratios for the upper ru vs lower r-^ leaf surface diffusive resistances


 to transpired water vapor are plotted as functions of the total leaf


resistance R.   Foliar diffusive resistances reflect the leaf health and


stress physiology—responding to the moisture balance,  phytotoxic agents,


aging, mesophyll C0ฃ levels, and a number of other factors.  The adaxial


and abaxial surfaces were compared since it was  postulated that UV-B

-------
 irradiation could cause more  epidermal injury to the upper  exposed



 surface leading  to an early effect on gas permeation through  this surface.



 Leaf diffusion was more restricted in growth chamber plants than greenhouse-



 grown plants.  [Growth chamber plants:  R = 3-10 sec cnT^;  greenhouse plants;



 R = 1-3 sec cm""*.]   Stomatal  opening and development, light dependent


                                                                           _2
 processes, were  undoubtedly suppressed under the lower PAR  levels (270 pE m



 sec~l) of the growth chamber.  Consequently, relative photosynthetic rates


 would be restricted by diffusion limitations as well as by  the lower PAR


 available for light-harvesting chloroplast reactions.  PhAcDC studies

                                                                          _2
 indicated that snap bean leaves required PAR intensities of about 800 yE m


 sec"  for maximum photosynthesis.



     Diffusive resistances given in Fig.  1 are mean values  for all


 (<3/4 expanded)  first, second and third trifoliates sampled biweekly


 over the 4-week  UV-B irradiation period.   The data were taken during mid-


morning to noon—on sunny days in the greenhouse—on well-watered plants.


 Lower surfaces of greenhouse and chamber-grown leaves exhibiting moderate



 diffusive resistances (i.e., 2-5 sec cm"*) were about twice as permeable


as the upper surfaces for both UV-B exposed and control plants.   The ru/ri


ratios increased rapidly as higher or lower total diffusive resistances
                              *

were measured.  The arrayed data do not indicate that diffusion through


the upper surfaces of UV-B irradiated plants was significantly altered


relative to that of the lower surface; though,  there may be a slight


tendency for increased ru/ri ratios in UV-B exposed plants.


     After several weeks in the chamber some UV-B injury was observed on


Bush Blue Lake 290 (BBL 290) bean leaves.   [UV-B injury symptoms:   Red


pigmentation of the petioles and leaves with slight leaf stipple,  i.e.,


scattered small "flecks" of necrotic cells.]  Two to three weeks  after

-------
 losing  their cotyledons BBL  290, which  is more sensitive to a number of


 known environmental  stresses  than Astro (10,11), showed a gradual loss


 of vigor  in both UV-B exposed and control plants.  As the foliage became


 stressed  with  time in the chamber (perhaps by inadequate photosynthesis),


 leaf diffusive resistances increased accordingly.  The Astro variety


 withstood the  growth chamber  conditions better than BBL 290 and was also


 less sensitive to UV-B injury.  The plants were removed after 4 weeks in


 the growth chamber and returned to the  greenhouse to check for recovery.


 At harvest (2  weeks  later), UV irradiated BBL 290 plants had less biomass


 than the  controls (Fig. 1 inset).  Astro plants exposed to UV-B radiation


 in the  chamber did not differ statistically from the controls.


     The  healthy, vigorous greenhouse plants grew larger than the chamber


 plants  (cf:  C/G, Fig. 1 inset).  Foliar resistances remained low throughout


 the experimental period.  At  harvest neither BBL 290 nor Astro plants grown


 and irradiated with UV-B in the greenhouse differed in biomass from their


 Mylar controls.  No visible UV-B injury occurred on the greenhouse plants.


     The  results tend to corroborate previous reports that plants under


 low PAR regimes may be injured more by UV-B irradiation than plants in


 bright  light.  The more sensitive BBL 290 snap bean variety, furthermore,


 grew less vigorously in the low PAR chamber environment than the Astro


 cultivar.  Preliminary trials with Pennseott clover showed this clover


variety to grow well in the growth chamber.  The chamber-tolerant clover

                                                     _2
was not injured by comparable BUV exposures (E^lOmWin  ,  6 hrs/day) during


a 4-week trial.


UV-B effects on CER,  plant injury,  and growth


     Table I summarizes the results of CER experiments conducted during


the spring and summer of 1977 on six crop plants exposed  to increasing

-------
UV-B doses.   Table  2  gives an abridged array showing:  visible injury


index ratings for the PhAcDC investigated leaves; comparative leaf

conductances  for evapotranspired water vapor (See footnote, Table 2); and

relative plant biomasses of the irradiated plants given as percent of

control.  The experimental plants were cultured and irradiated with specified


UV-B doses in the greenhouse and transferred with their paired controls to

leaf or whole plant dual-PhAcDC systems for examination and characterization.

The CER trials were restricted to UV-B exposures that produced no more

than 20% leaf injury  (Injury Index II).  Data for snap bean, soybean,

cotton, cucumber, and clover were taken on intact leaves or trifoliates.

Wheat and additional  clover data were obtained from whole plant studies.

Equivalent numbers of leaves on the controls and UV-B exposed plants were

used.  The data were  normalized on a weight basis.

     UV-B exposed leaves showing no visible injury at the time of testing

did not statistically  differ from their paired controls in CER or foliar

conductances, nor was  plant biomass reduced by the extended exposures.

CER values were lowered roughly in proportion to the amount of visibly

damaged leaf  tissue.   Snap bean leaves with an Injury Index I rating

(1-10% injury) showed mean CER values 8% below those of the controls.

Soybean and cucumber  leaves assigned to the Injury Index II class (10-

20% injury) gave CER values averaging 14% and 16% below the controls.

     High UV-B radiation levels were required to injure greenhouse grown

snap bean and soybean plants—in excess of 125 hrs exposure (3 weeks,
                                 fy
6 hrs/day) to more than 12.5 mWru   of biologically effective UV-B radiation.

Cotton was marked by this dosage.   Poinsett cucumber was the most sensitive

plant tested.  Wheat and clover were not injured by the highest doses  given

in those particular experiments.   Visible symptoms of injury for cucumber

                                   8

-------
and cotton were well-defined chlorosis along the leaf margins  (cucumber)

and red pigmentation of the petioles and juncture with the leaf base  (cotton),

Snap bean injury symptoms were described previously.  UV-B injured soybean

leaves showed increased pigmentation or bronzing of the leaves with scattered

necrotic stipple.
                        -?                                     -2
     Dividing the Z^(mWm  ) in Table 1 and Figure 1 by 3.06 mWm   gives

plant BUV exposures relative to one control sunshine equivalent SE.  One

SE is the weighted BUV integrated over the UV-B region for the control

sunshine used at the Beltsville Agricultural Research Center (6,8,9).
        _ 9
Five mWm  , the minimum Z^ included in Table 1, represents 1.6 SE, or a

60% increase above 1 SE.  One hundred hours' daytime exposure to 1.6 and

2.1 SE was insufficient to cause injury to any of the soil-grown greenhouse

plants tested.  Gregg cotton developed basal leaf and petiolar pigmentation

in the 4 SE exposure tests, but no cellular necrosis or statistically

significant reduction in CER.  The very high UV-B levels required to

visibly injure greenhouse snap bean and soybean plants, 8 SE, combined with

nighttime irradiation (the plants were irradiated 300 hrs, 24 hrs/day) are

exceptionally adverse treatments and will not be discussed further.  The

data are included to show the range of exposures given.
                             %
     Poinsett cucumber leaves showed some chlorosis when subjected to

ca. 1.2 SE.  [See Krizek (6) for experimental data.]  The sensitivity of

this cucumber variety varied slightly with season,  increasing in the fall
                                                           t-
and winter and decreasing in the spring and summer.  The seasonal effect

was attributed largely to photorepair in plants grown under the higher PAR.

PhAcDC examined Poinsett leaves exposed to 1.6 and  2.1 SE showed mean

Injury Index ratings of I and II based on the amount of marginal chlorosis

-------
observed  (cf. Table 2).  Nonchlorotic portions of the leaves appeared to




be healthy and functional.  Predicted biomass (dry weight) loss, based on




data generated from the larger population from which the plants were drawn




(48 plants), indicated that 1.6 and 2.1 SE exposures would be expected to




cause 3% and 6% reduction in plant dry weights, respectively (6).  This was




less than the mean leaf chlorosis and reduction in CER observed.




     Increased BUV corresponding to 1.6 - 2.1 control sun equivalents




represent values greater than the 1.4 SE predicted for the maximum




stratospheric ozone depletion due to chlorofluorocarbon emissions.




Experimental data generated from this study give no evidence that the snap




bean, soybean, wheat, clover, or cotton varieties tested should be




measurably damaged by the proposed UV-B increases.  The potential exists




for injury to Poinsett cucumber plants but anticipated growth reductions




of the magnitudes expected would be difficult to statistically detect in  .




nature even when some observable chlorotic injury might result.
                                     10

-------
                           LITERATURE CITED



1.  Brandle, J. R., W. F. Campbell, W. B. Sisson, and M. M. Caldwell.

        1977.  Net photosynthesis, electron transport capacity, and ultra-

        structure of Pisum sativum L. exposed to ultraviolet-B radiation.

        Plant Physiol. 60:165-169.

2.  Sisson, W. B. and M. M. Caldwell.  1976.  Photosynthesis, dark

        respiration, and growth of Rumex patientia L. exposed to ultraviolet

        irradiance (288 to 315 nanometers) simulating a reduced atmospheric

        ozone column.  Plant Physiol. 58:563-568.

3.  Van, T. K. and L. A. Garrard.  1975.  Effect of UV-B radiation on net

        photosynthesis of some C- and C, crop plants.  Proc. Soil and Crop

        Sci. Soc. of Florida 35:1-3.

A.  Van, T. K., L. A. Garrard, and S. H. West.  1976.  Effects of UV-B

        radiation on net photosynthesis of some crop plants.  Crop Sci.

        16:715-718.

5.  Heggestad, H. E.   1973.  Photochemical air pollution injury to potatoes

        in the Atlantic coastal states.   Amer. Potato J. 50:315-328.

6.  Krizek, Donald T.  1978.   Differential sensitivity of two cultivars of

        cucumber (Cucumis sativa L.)  to increased UV-B irradiance:  I. Dose-
                             *
        response studies.  BACER Final Report, U.S. Dept. of Agr., 33 pp.

7.  Krizek, D. T.  1977.  Current UV measurement methodology and future

        needs in photobiological research.  In Symposium on Ultraviolet

        Radiation Measurements for Environmental Protection and Safety.

        pp. 49-52.  Held at National Bureau of Standards, Gaithersburg, Md.

        June 8-9, 1977.
                                      11

-------
 8.  Norris, K. H.  1977.  Development of a portable, automated UV-B




         spectroradiometer.  In Symposium on Ultraviolet Radiation Measure-




         ments for Environmental Protection and Public Safety,  pp. 72-72.




         Held at National Bureau of Standards, Gaithersburg, Md.  June 8-9,




         1977.




 9.  Cams, H. R., R. Thimijan, and J. M. Clark.  1977.  Outline of irradiance




         distribution of UV fluorescent lamps and combinations.  In Symposium




         on Ultraviolet Radiation Measurements for Environmental Protection




         and Public Safety,  pp. 74-76.  Held at National Bureau of




         Standards, Gaithersburg, Md.  June 8-9, 1977.




10.  Bennett, J. H.  1977.  Ozone and leaf physiology.  In Proceedings of the




         Fourth Annual PGRWG Meetings, Hot Springs, Ark.   pp. 323-330.




11.  Bennett, J. H.  1978.  Foliar exchange of air pollutants and




         physiological gases.  In S. Krupa, W. W. Heck and S. N. Linson (eds.)




         "Handbook on methodology for the assessment of air pollutant




         effects on vegetation", (Chapter 10), Proc. Specialty Conference




         of the Air Pollut. Contr. Assoc.  Held minneapolis, Minn.,




         April 19-21, 1978.



                             \
                                     12

-------
7 _

6 _

5_

4 _

3 _

2 _

1
O Astro  (Control)
• Astro  (UV-B)

O BBL 290  (Control)
• BBL 290  (UV-B)
                                 CHAMBER
                                                    X
                                           co  CQ--'
                                           *"-*    •
                                      R  range
                                         1—i—t
                                                             10
               GREENHOUSE
          o
           R range
                               Relative  Plant Biomass at Harvest
                              UV /Control:
                         Chamber
                         Greenhouse

                       C(Control).
                       G(Control)'
                                                (7. Control)
                                            BBL 290    Astro
                                              89*       95
                                              96       102
                                                69*
                                                        88*
                              Significant at
                                                  level
             -i—i—i—i—ง—
                          R Csec/cmJ
                                                           10
| Experimental Conditions
PAR Light (uE nT2 sec"1)
BUV HA (mW m-2)
Temp (ฐC) [day/night]
R H (7.)
t
Experimental Period: Jan
Growth Chamber
270 ฑ30
10ฑ2.5
[23/16]
35-55
- Mar, 1977.
Greenhouse
1300 (sunny day)
10ฑ2.5
[20-30/15-20]
Usual Range: 30-60

Figure 1.    Relative foliar resistances  to  transpired water vapor
diffusion through  the upper r  and lower r.  leaf surfaces of UV-B
irradiated and  control plants plotted as functions of total leaf
resistance R(sec/cm).  The data compare  plants exposed under growth
chamber and greenhouse conditions.  Experimental conditions and
relative plant  biomass at harvest (8 weeks  of age) are summarized
in block inserts.
                                 13

-------
IU  rT

"   1
2
^^  fO
3   'B
    09
    .u
    4J
    CO
CC  s
d

                        280
300
320
                          WAVELENGTH (run)
      Figure  2.   UV-B  irradiance curve  for Figure  1  experiments produced
      by the  FS-40  sunlamp systems filtered through 5 mil cellulose acetate
      (exposed  6  hours).   Also shown are the Action Spectrum for UV injury
      to cucumber (and  other)  plants (9),  and the Biologically-harmful UV
      Action  IntegralZ.  (inset).  The Action Integral represents the integ-
      rated product of  the Action Spectrum Y times  the Irradiance I as a
      function  of wavelength.
                                      14

-------
 Table  1.  Summary of relative photosynthetlc rates (nee carbon dioxide exchange rates, CER)* for UV-B
           irradiated and control plants [n • 5-8 replications per treatment).
              UV-B Exposure

   (Expt'l. Period: Apr.-Sept. 1977J
                            Relative CKR

                           (Z of Control)
  Exposure Range      UV-B                         Snap beans  Soybean    Clover      Wheat    Cotton   Cucumber
  IA (mWatts m-2)   Exposure  Exposure   CER Test   "BBL 290"   "York"  "Pennscott"   "Monon"  "Cregg"  "Polnsett"
    (Cf.  Fig. 2)    (Hours)    Class     (Weeks)
   5.0*0.5(6 hr/day)  100*20

   ซ.5ฑ0.5(    "   )    "

  12.5ซ.5(    "   )  125ฑ25

   25*5  (24 hr/day)  300150
2-3

3-4

4-6
                                                        I
  MEANS INSIDE BOUNDARY  NOT SIGNIFICANTLY DIFFERENT      I

105         99       103       107          [     84*

 97        102                          95   |

 92*        86*
  *CER for Intact leaves  calculated  as ug C02  dm"  mln"1;  Wheat and clover normalized on weight basis.
   One control sun equivalent  (SE) equals 3.06 mWatts or2  based on action spectrum given.
  *Meซns Significant  at  P QJ  level
Table 2.  Summary Table showing relative net carbon dioxide exchange rates for UV-B exposed and  control
          plants arrayed to Include leaf Injury indices, leaf conductance ratios  for transpired  water
          vapor, arid relative plant blomass.
Flint/Variety
Snap bean
"Bush Blue
Lake 290"
Soybean
"York-
Clover
"Pennscott"
Wheat
"Monon"
Cotton
"Cregg"
Cucumber
"Polnsett"
UV-B
Exposure
Class
[See Table 1]
C
D
C
D
B
B
C
A
B
Plant
Blomass
(7. Control)
105
94
96
92
101
99
93
97C
94C
Relative
CER
(T. Control)
97
92*
102
86*
% 103
107
95
94
84*
Ratio8
LUV/LC
1.0
0.9
1.1
0.9
1.1
*
1.0
1.0
0.9
Leaf
Injury
Indexb
0
I
0
II
0
0
Tr
I
II
Symptoms of Injury

No visible Injury
Leaf pigmentation; stipple
No visible Injury
Leaf bronzing; stipple
No visible Injury
No visible Injury
!


Pigmentation of petiole and
base of leaf
Marginal chlorosis of
ii . n ii
leaf
II
           Ratio of leaf conductance L (reciprocal of leaf resistance)  for UV-B exposed  and  control  plants.
           Conductance roefflcients linearly relate gas exchange rates  with concentration gradients.

blnjury Index Scale:  0 (no visible injury); Tr ซ17. injury); I (1-107.);  II (10-207.)
cPlant btomass (dry weight) based on regression data generated from 48  cucumber plants
                                                      15

-------
                                                  ffi.au
                                     .  ซ??
          BIOLOGICAL AND CLIMATIC. EFFECTS RESEARCH
              TERRESTRIAL NON-HUMAN ORGANISMS
                   EXECUTIVE SUMMARY
                      EPA-IAG-D6-016S
                      Project Officer:

                      R. J. ^IcCracken
Agricultural  Research, Science and Education  Administration
                U.S. Department of Agriculture
                  Washington, B.C. 20250'
                       Prepared lor
              Environiiiental Protection Agency
                       BACER Program
                    Vfashington, D.C. 20460

-------
                            EXECUTIVE SUMMARY




     Tlits final report describes the research undertaken by the U.S. Department




of A'.'r tculture during the period of October 1, 1976, to February 28, .1978, as




;i parr of the Biological and Climatic Effects Research (BACER) Program




conducted under interagency agreement with the Environmental Protection Agency.




Tin- objective of this research was to assess the biological impact of in-




c>.v.risecl radiation in the UV-B region (280-320 nui) reaching the earth's surface




on ;i)\r {.culturally important and native terrestrial plants and animals that




ini>;ht; result from stratospheric ozone reduction caused by inadvertent release




of ciilorofluoromcthanes (CFM's).          .                           -     -




     The organizations reporting and participating in this research arc




li:;ted below:




     U.S. Department of Agriculture




          U.S.. Forest Service,  Fort Collins, Colorado




          Science and Education Administration




               Arizona-New Mexico Area,  Las Cruces,  New Mexico




               Beltsville Agricultural Research Center, Beltsville, Maryland




                    Agricultural Equipment Laboratory




                    Chemical and Biophysical Control Laboratory




                    Florist and Nursery  Crops Laboratory




                    Instrumentation Research' Laboratory-




                    Organic Chemical Synthesis Laboratory




                    Plant Stress Laboratory




               Florida-Antilles Area, Gainesville, Florida




               National Animal Disease Center, Ames, Iowa




               Northern Regional Research Center, Peoria, Illinois

-------
               Federal Research Contracts


                 University of Florida,  Fruit Crops Department,  Gainesville,


                   Florida


                 Colorado State University,  Department of Horticulture, Fort


                   Collins, Colorado


     Major accomplishments resulting from the U.S.  Department of Agriculture


liACER Program are as follows:


     1.  An1 automatic UV spectroradiometer was designed, constructed and


         successfully tested capable of  measuring UV radiation every.

.
         nanometer (ma) from 250 to 400  nra,  with a wavelength precision


         of,0.1 run (1A).  Broad- and narrow-band radiometers to  monitor the


         output of artificial UV sources for laboratory, growth  chamber, and


         greenhouse' experiments were also developed.


     2.  Substantially all major crops,  including many horticultural species


         and varieties, and some native  species have been screened for


         sensitivity to increased.levels of  UV-B. ...


     3.  Injury threshold levels were found  to vary widely among species


         ancl within cultivars and varieties  of the same species.


     4.  It was almost universally observed  that field grown plants were


         more tolerant to enhanced UV-B  levels than we're the identical


         selections grown in greenhouses or  controlled environment chambers.


         A seasonal difference in sensitivity to enhaiiced levels of UV-B


         was also observed, with plants  showing greater tolerance during


         the summer months.  Substantial evidence for the existence of a


         high-light intensity photorcpair or photoprotection mechanism in


         higher plants was obtained.

-------
     5.  Some economic plant species, such as cantaloupe, soybeans, and cotton




         have been shown to be sensitive to present levels of UV-Ji reaching




         the earth's surface.




     6.  Highly pigmentcd insects, especially those with a high melanin




         content, were found to be highly resistant to UV-B.  Honeybees,




         for example, were unaffected by very high levels of UV-B.  Some




         leaf disease organisms display similar responses.




     7.  We have established that "cancer eye" may have been induced in




         Hereford cattle by high levels of UV-B.




     These accomplishments provide a significant increase in knowledge




of the potential responses of terrestrial plants and animals to increased




UV-B irradiance.  Because of continued uncertainties involved in this




research, the results do not permit the development of conclusive




statements concerning the possible harmful effects to be expected from




increased UV-B reaching the earth's surface as a result of the projected




reduction in stratospheric ozone as a result of release of the chloro-




fluoromethanes in. the biosphere.




     The uncertainties are addressed below:




     Artil'icJat Enhancement Sj^rc
-------
                                      Figure 1
 10BO00
           JLS_1O..5SC!{ _	

           JES.40 .ZJ3CHi5J.IIl.JX
           J81i.4MLG.Mo-.LM	.	..Z
  100.000-
   18,009-
 k  l.B0a
1.J.1
C.J
eg
    0,109-
    0.010-
   0.001-
                              WAVELENGTH nm IRL AHRI FR USDA
                                       3 a

-------
     The first problem seen is that the region of; major interest 280-320 nra




is one of rapidly decreasing irradiance; decreasing over four orders of




magnitude.  Unfiltered lamps do not parallel this decrease in intensity,




displaying higher intensities as the. wavelength decreases.  As can be .seen,




this discrepancy can be partially overcome by use of suitable filters; 5 mil




cellulose acetate for example (+ 6 hrs. 5 m CA).




     The second, and perhaps as serious a difference is the greatly decreased




irradiance from artificial sources from approximately 310 nin and above.  The




possible effect of this reduced irradiance level in the UV-A region can only




be conjectured at our present state of knowledge.




     A third uncertainty, which we believe to be at least partially resolved




is the. fact that energy emitted by a fluorescent lamp and falling on a




horizontal surface parallel to the long axis of the lamp is unequal; being




highest directly under the center of the lamp and falling off rapidly in




all direction;; from the center.   By using improved instrumentation and




through development of equations defining this energy variable, careful




energy-defined biological research has been accomplished.




     However,  continued use of these sources for UV enhancement experiments




will require improvements in UV filters and in the sensitivity, accuracy




and utility of UV radiometer and spectroradiometer equipment.  Development




of new sources more nearly simulating*the sun will prove expensive and




probably require the development of new or improved technology.




     Ae_ticm Spectra.  Since the artificial enhancement sources do not




simulate the sun and because biological organisms and systems do not respond




equally to all wavelengths of UV radiance, the interpretation of data derived




from UV enhancement experiments requires the use of a suitable weighting

-------
function derived from an action spectrum to describe the comparative




biological effectiveness of different wavelengths in the. UV region to which




the organism is subjected.  At the beginning oC the short-term BACliR




program there was no universally accepted action spectrum for higher plants .




although several had been proposed (Figure 2).   The Uwo action spectra (AS)




which have been evaluated for data interpretation in the current program are




graphically shown as 9AS (AE9) and 21AS (Aฃ21).




     Weighting functions derived from these two spectra have been used by




participating.principal investigators.  The 9AS weighting function has




generally been applied by most researchers.   However,  the University of




Florida and the Florida-Antilles Area report their results using the 21AS




weighting function.                    .




     The Agricultural Equipment Laboratory report contains a presentation




of results obtained by Beltsville Agricultural  Research Center investigators




which suggest that the 9AS weighting function is more valid than the 21AS




weighting function.  It should be emphasized, however, that data are still




lacking as to how accurate or how universally applicable this weighting




function will prove to be.  Use of the 21AS weighting function in place of




the 9AS tends to overestimate the effectiveness of the longer wavelengths




thereby underestimating the amount of biologically effective UV radiation




(BUV) received.  Thus, tesฃ organisms are subjected to higher total UV




irradiances with the 2.1AS weighting function than with the 9AS weighting




function for a supposed equal amount of biologically effective UV.




     To provide a. basis for interlaboratory comparison of data,  all principal




investigators have provided irradiances used in experimental set-ups in




absolute uirweighted mW;n~ " thus, when a verified action spectrum has been




established, all submitted data may be reinterpreted on the basis of the




accepted action spectrum.

-------
                                          Figure: 2
  .
CD
CO
t"

C.J
                                   IIAVELENGTH nm IRL AMRI FR  U'S
                                              5a

-------
     Contro 1 Sii_nghino.  AnoLhpr major problem in interpretation of UV-


irracliance enhancement studies is the variation in the sun's UV-irradiance


as a function of latitude, elevation, zenith angle, and the sun's variability


from clay to day as the result of stratospheric and atmospheric changes.  This


is illustrated in Figures 3 and 4 showing linear and log plots of UV


irradianccs obtained every nanometer from 290-320 nm using a single or double


raonochromator spectroradiometer.  Starting with the sun spectrum with the


highest overall spectral energy, the data were obtained at:  Snowmass,


Colorado, 9,777 ft. (2980 in) on August .10, 1977, 1400 hours; Beltsvillc,


Maryland, 186 ft. (56.7 n), June 30, 1977, 1337 hours; Gainesville, Florida,


180 ft. (54.9 in), April 28, 1977., 1432 hours, (University of Florida


report. Table 10); and Be.Usville, Maryland, March 21, 1977, 1400 hours.


Asterisks on Figure 3 represent the control sunshine used by investigators


at BARC derived from data obtained by the Smithsonian Radiation Research


Laboratory, Rockville, Maryland, and the Instrumentation Research Laboratory,


Beltsville, Maryland.  It basically represents the average sunshine for


the months of June and July .1976 in the Washington, D.C., area.


     As can be seen on Figure 4, because of stray light response of the


instrument used at Gainesville, Florida, irradiances are overestimated at


the shorter wavelengths (290-300 nm); this presents a serious problem when

                                                          '• •
weighting functions are required since greater weight is applied to shorter


x>7avelengths.                                    .

                                               9
     Table 1 presents the total unweighted mWrn   from 290-320 nm, the Aฃ9 •

            -2
weighted mWm " and the fraction of one average control sunshine used in


the Beltsville investigations.  The biologically effective UV-B measured


at Snowmass,  Colorado,, was 2.7 times that of the Washington, D.C., area.


A 20 to 40 percent enhancement of the measured biologically effective UV-B

-------
  0. 8-
633. B
                          Figure 3
      J1QRLQA Jil^ J JB._Z7_2^.H JSI
      JEITSVUIH SUN.. G 32J7  i, 37 P;
      mORAl2Q^OJ3_ZLJ BOOT
.!
                      ., WAVELENGTH nr., IRL AHRI FR USDA
                              6a

-------
    Figure A
WAVELENGTH  nm  IRL AMRI PR USDA
        6b

-------
                                                                    _o
   the. Colorado location would range from 9.95 to 11.61 weighted mWm  ,



whereas an identical enhancement at Washington, D.C., would range from


                         -?
3.67 to 4.28 weighted mWm ~.  The need for biological research to cover  a



wide range of biologically effective UV-B irradiances is obvious.



     To properly evaluate the results presented here United States-wide



will require accurate knowledge of the sun's spectral energy in all geographic



regions.  The development of a program for regular monitoring of the UV



spectral irradxan.cc at selected locations is a necessity which cannot be



ignored in future programs.



     Research results obtained by each principal investigator are summarized



in the following pages.

-------
                                Table 1
Location


Snowmass, CO
Beltsville, MD (high)
Gainesville, FL
Beltsville, HD (low)
Control sunshine
Total
ir radiance
290-320 nm
o
mWra
4098
2499
1742
1274
2847
Biologically
effective UV Fraction of
irradiance AE9 control sunshine
290-320 nm
—2
v:eighted raVrm
8.29 2.7
3.94 1.3
2,, 68 0.7
1.60 0.5
3.06 1.0
(Beltsville)

-------
                           RESEARCH PROGRESS                            .  -



    lojinienI: of Ins 1:rument ation for MeasurIng UV--V> J
-------
scan, reverses the wavelength drive and returns the monochromator to the




starting wavelength.  At tlie completion of the scan, the data can be stored




on a magnetic tape for future analyses or transferred to an automatic plotter.




A miniature low-pressure mercury-arc lamp is used to provide a precise check




of wavelength accuracy.  The calculator computes the position of the 233.7-nin




and the 296.. 7-nm mercury lines to a precision of + 0.01 nm.




     In order to provide investigators in the program with a simple instrument




for monitoring the output of artificial UV sources in laboratory, green-




house, and growth chamber studies, broad-band and narrow-band UV radiometers




were also developed by the Instrumentation Research Laboratory.   These




instruments feature a teflon bubble cosine receptor, a so.lar--b.lind phototube,




a battery-powered photometer circuit and a small rugged housing.  In order




to provide a basis for interlaboratory comparisons of UV data, the sensitivity




of the broad-band radiometer was adjusted to give the same full scale




reading under a common UV source CFS40 fluorescent sunlamps filtered with
                                                                                        i
                                                                                        !


5 mil cellulose acetate).  Correction factors were developed for use under              j




other lamp-filter combinations.        .                                             •     j


                                                                                        i

     The specifications for these instruments were made available to industry,




and commercial models have now been developed and obtained by cooperating •




locations.                        v




Spectra.'l. Characteristics of Fluorescent Lamps arid Testing of Weighting •




  Functions




     The Agricultural Equipment Laboratory at Beltsville, Maryland,  developed




mathematical equations describing the distribution of normalized UV irradiance




levels in any desired combination of lamps,  either parallel or end-to-end,




at any defined distance of the lamps from the illuminated surface.   A computer
                                  10

-------
program was written which permitted scientists to accurately design




biological experiments and interpret the results.




     Lamp fixtures were fitted with specially designed reflectors to provide




greater uniformity and rcproducibility in UV radiation experiments.  Multiple




fixture assemblies were designed and built.




     An average or standard spectral distribution of UV energy of the sun




for .the Middle Atlantic area was derived from data collected by the




Smithsonian Radiation Laboratory, Rockville, Maryland, in June-July 1976,




and by the Instrumentation Laboratory, Beltsville, Maryland.  This is now




being used by Beltsville scientists as a base, for design of UV enhancement




experiments.




     In conjunction with the Florist and Nursery Crops Laboratory and the




Plant Stress Laboratory, experiments with higher plants were undertaken




to more clearly define the weight, ing function that must be used to interpret




results, since the spectral distribution of the fluorescent lamps does not




duplicate that of the sun.  Several weighting functions were investigated.




The equation providing the best fit was derived and is being used by the.




majority of investigators.  We are continuing to obtain data testing the




applicability of this weighting function.




Biological E ffects of UV—B Radiation/von Plant Growth and Function




     Greenhouse, growth chamber, laboratory, and -field studies were conducted




by the Plant Stress Laboratory at Beltsville, Maryland, on a wide range' of




vegetable and agronomic crops to determine the relative sensitivity or




resistance to increased L1V-B radiation.  Data were collected on various




physiological responses to increased UV-B radiation including:  photosynthesis,




respiration,  ion uptake, translocation of rad.ioisotopcs, stomatal activity,




changes in chlorophyll and anthoc.yanin content, leaf movement, germination,
                                  1.1

-------
seedling growth and reproductive development.  Studies were also conducted




On UV-B interactions with plant disease organisms.




     Broad-band UV-B studies were conducted in the greenhouse and growth




chamber on over 20 species and cultivars of vegetable and agronomic crops.




Plants were exposed to a gradient of IJV-B radiation representing a 50 to




500 percent increase in biologically effective UV radiation.  Plants studied




included cotton, peanut, wheat, rice, alfalfa, cucumber, pea, beet, tomato,




rutabaga, okra, bean, radish, and turnips  Most plants were exposed to UV-B




for 4-5 weeks from time of planting the seed, but a few (wheat:, rice, alfalfa,




cotton) were grown to maturity under elevated UV--B.  Visual injury was observed




in over half of the species and cultivars s.tudied.  In most cases only slight




or moderate UV damage was noted even when the. plants were exposed to an




increased level of biologically effective UV radiation as high as 300-AOO




percent.




     The most dramatic evidence of UV-B injury was chlorosis in pea and




cucumber, necrosis in pea leaves and pods, and reduction in leaf size in




pea and cucumber.           ^




     Dose-response studies conducted on cucumber varieties demonstrated




significant differences in UV-B sensitivity; Poinsett cucumber was extremely




sensitive and Ashly cucumber was only slightly sensitive.   Evidence, was




obtained for UV-B induction of chlorosis of the-leaves, inhibition of leaf




enlargement, and reduction in biomass.   These effects were most pronounced




under conditions for low photosynthetically active radiation, and high UV-B




exposure.




     High levels of UV-B irradiation in the greenhouse (100-400 percent




increase in biologically effective UV)  reduced the total number of kernels




in Pacific Triple Uwarf wheat by 20 percent, but had no appreciable effect




on the average yield.
                                  12

-------
     Translocation of radioactive zinc from the cotyledons to other plant




parts of the young cotton was not inQuencod by a lOO-AOO percent increase




in biologically effective UV; however, the transport of radioactive calcium




was depressed 12-30 percent over this range of UV irradiation.




     Based on linear regression analysis of plant data .obtained in the




greenhouse of one of the more sensitive plants (Poinsett cucumber) exposed




to-increased UV irradiation  (from 50-30CH- percent increase in biologically




effective UV), it was estimated that a maximum decrease in stratospheric




ozone content of 20 percent would cause a 10 percent reduction in dry matter




accumulation and a 15 percent decrease in leaf area.  It is not possible




at the present time to determine whether these estimates 'can be applied to




oUier species of higher plants.




     Measurements were made on net photosynthesis rates, plant biomass




production, stomatal diffusive resistance, and transpiration rates in




selected plants of snap bean, clover, cotton,  cucumber, and wheat irradiated




in the greenhouse and growth chamber.  In general UV-B effects on net COp




exchange rates and foliar gas exchange were correlated with the amount of




visible injury induced.




     Chromatography and subsequent UV and visible spectroscopy of acidic




methanol extracts of Cole us bljjmejL_ 'leaves taken from UV-B irradiated plants




demonstrated a degradation in UV absorbing compounds.  Similar results were




obtained with reflectance measurements.   Increasing the UV-B. irradiance




resulted in increased degradation of anthocyanin pigment, reduction in the




rate of leaf expansion, inhibition of apical growth, and abnormal development




of the leaves.
                                  .13

-------
     Field studies were conducted on UV effects at Be.ltsville on a  range




of agronomic and vegetable crops using a gradient of UV radiation




developed by the Agricultural Equipment Laboratory,  Crop plants studied




included Contender bush bean, Early Prolific, straightneck yellow squash,




Amsoy-71 soybean, sugar beet, Golden Cross Bantam corn, R-720 sorghum, and




Waltham 29 broccoli,  A fall crop of winter grains was also grown that




included Potomac, Redcoat, and-Abe whea.t, Pennard and Monroe barley, and




Abruzzi rye.,




     Increasing the biologically effective UV radiation by 100 percent had




no visible or consistent effect on crop performance under field conditions.




P.lant Disease T.nterac tion _w.iLth UV-B Radiat_icm




     The Plant Stress Laboratory has studied the effects of UV-B radiation




on plant diseases.  The results of increased levels of UV-B irradian.ce on




spore germination indicate that although plant leaf pathogenic fungal




species vary considerably in sensitivity to UV-B, relatively high irradiance




levels are required to reduce germination percentage.  Pigmented spores such




as Cladosporium, Steinphyllium, and Alternar.ia were found to be more, resistant




to increased UV-B irradiance than hyaline spores (Mycosphaerella t




ColletotrichuTTi).              -




     Disease .severity of Co 11 eto tr i c hum lagenarium on cucumber v;as decreased




with increasing UV-B irradimic.es.  A linear decrease in the percentage of




leaf area diseased with increased irradiances was found.




     Increased levels of UV-B irradiance did not affect disease severity




ฐ^ Cladosporium cucumerinum.  The disease tended to reduce plant growth




equally regardless of UV-B irradiance levels.




    . There was no noticeable UV-B effects on either Che Ste-mphyllium




botryosuni pathogen or the host,  alfalfa.
                                  14

-------
     In summary, recognizing that our results represent only a small


sampling of leaf disease organisms and of plant disease-interaction


experiments, they appear to support the following:  (1) considerably higher


levels of UV-B irradiances than those expected from the projected ozone


depiction will be required to adversely affect germination and growth of


pathogenic fungi, and (2) where fungal germination and growth are affected,


disease severity in the host plant can be expected to be reduced as UV-B


irradiances increase.


Response of Florist arid -Nursery Crops to Incre ased UV-B Rad.1 at ion


     Greenhouse and growth chamber studies were conducted by the Florist


and Nursery Crops Laboratory at Beltsville, Maryland,  on a wide range of


florist and nursery crops to determine their relative sensitivity or


resistance to increased UV-B radiation.  Selected plants were also chosen


for reflectance and fluorescence measurements and for microscopic examination


in the laboratory.   After 2-8 weeks of exposure,  visible injury was observed


in eight of the 58 species irradiated, and then only when applied in excess


of projected levels of UV-B radiation expected to result from CFM-cata.lyzcd


reduction of stratospheric ozone.                 .


     The most typical response to high levels of UV-B irradiation (100


percent or greater increase in biologically effective UV) included break-


down, of chlorophyll and anthocyanin and a glazing and browning of the tissue,


generally attributed to the presence of oxidized, polymerized,  phenolic
                                                                                        )

compounds.  Other effects observed in some of the test plants included                  [
                                                                                        !

abnormal leaf growth, characterized by reduced size,  twisting and distortion,           j

                                                                                        [
and reduced plant height.  Plant bioinass was generally unaffected when mature           i

                                                                                        I
plants were irradiated;  bioinass of young seedlings,  however,  was frequently             !


depressed under high UV-B.                                    .      •                    ;
                                  15

-------
     There was considerable variation in sensitivity to UV-B exposure,




depending upon species and cultivar, stage of development:, time of. year, and




level of exposure.  In general, herbaceous plants were more sensitive to




increased UV-B than were woody plants.  Fat si3 japonica was the only woody




species of the ten tested that showed inhibitory effects of high UV-B.




     Plants irradiated during the summer months in the greenhouse showed




little or no UV injury, even under the highest levels of UV-B used.  This




was in sharp contrast to the spring and winter months when they showed




considerable injury under the same level of UV irradiance.




     Poinscttia, Coleus, and Browallia were among the most sensitive examined.




Other species sensitive to increased UV-B irradiation included aster,




hollyhock, vinca, and impatiens.




     In order to develop a capability for understanding the basic cellular




and ultrastructural mechanisms of UV-B effects, a UV microspectrophotome.ter




was obtained and. assembled in the Florist and Nursery Crops Laboratory.




This instrument will enable researchers to irradiate single cells or




cellular constituents as small as 0.5  micrometer with narrow band UV radiation,




to make rapid scans of absorbance and reflectance in the region of 250-




1000 ntn, and to make precise measurements of UV fluorescing materials.




A programmable calculator was also obtained to control the instrument and to




provide on-line data acquisition, processing, storage, and display.




Influence pf Solar UV-B Radiatj.cm on Crop Productivity




     Greenhouse, growth chamber, and field studies were conducted by the




Fruit Crops Department, University of Florida,  Gainesville, Florida,




on a wide range of vegetable and agronomic crops to determine their




relative sensitivity or resistance to increased UV-B radiation.
                                  16

-------
     Field studies were conducted at Gainesville under specially constructed


UV-B gradients obtained by mounting the fixtures at an angle over raised


plant beds.  Crops grown to marketable size and maturity included corn,


potatoes, tomatoes, field peas, peanuts, rice, squash, mustard, and radish.


Visual effects were observed in corn and rice under high UV-B irradiances


(100 percent or greater increase in biologically effective UV).  Both crops


appeared dwarfed and the grain head of the-rice plants were slower to mature


than the unirradiated controls.


     Growth chamber studies were conducted by University of Florida researchers


at the Phytotron at Duke University in Durham, North Carolina.  Over 100


species and varieties' of agronomic, horticultural and forest plants were


grown from seed for 4-12 weeks under increased UV-B radiation.  Under high


levels of biologically effective UV-B radiation (100 percent or greater


increase) plants exhibited a number of abnormal responses.   These included:


Marginal and inte.rveinal chlorosis; cupping and epinasty of the leaves;


changes in pigmentation; increased branching; reduced vineness; and reduction


in height, leaf area.> and biomass.


     In general., plants within the same family responded similarly to increased


UV-B radiation.  By using controlled environment studies it was possible to


identify varieties of soybeans that are sensitive to present levels of
                                   <,

UV-B radiation at Gainesville, Florida.


     Preliminary studies conducted in the Phytotron with Jori wheat and


liar dee soybean, at four levels of UV-B radiation and four levels of visible


radiation showed that the extent of UV-B radiation damage was greatly


influenced by the amount of visible radiation present.  Other research


accomplishments of the Univcristy of Florida scientists included developing
                                   17

-------
an action spectrum for pigment induction in the avocado leaf having a




maximum effectiveness in the UV-B region at 293 nm.  These investigators




also found that increasing the level of biologically effective UV radiation




by up to 100 percent had no significant effect on structural changes or •




chemical composition of surface waxes of tomato and pepper plants.  Agronomic




plants subjected to high levels of UV-B radiation also produced increased




amounts of ethylene and accumulated larger quantities of abscisic acid than




control plants.




Response of Vegetable Crops to High UV-B Radiation at Ilipji Elevations




     UV-B enhancement and exclusion studies were conducted by the Department




of Horticulture, Colorado State University, at a 3000 m site elevation in the




Rocky Mountains.




     Supplementing natural solar radiation with additional UV-B radiation had




no significant effect on the growth and biomass of pea, radish, potato, and




wheat grown at this elevation.  Shielding wheat plants from natural solar




UV-B, however, resulted in an increase in size of the plants.




     Other accomplishments included the development of transmission spectra




for a chlorinated-fluorinated resin film "Aclar" found to be useful in




aquatic studies as a UV transparent film;  development of an assay for detecting




loss of electrolytes from UV-irradfated plant tissues; characterization




of the influence of low temperatures on decline1 in lamp output of UV fluorescent




sun lamps; and design of a solar UV-B collector and irradiator.




Response of Arid and S_emi--arid Plants to Increased UV-B Radiation




     SEA scientists at Las Cruces,  New Mexico, investigated selected native




and economically important species indigenous to the arid southwest United




States.   Plants were exposed to increased  UV-B irradiation in the greenhouse.
                                  18

-------
Dose response studies were conducted on alkali sacaton  (Spgrobolus avroidcs




Torr.)> mesa dropseed (J3. flexuosus) and Chile pepper  (Capsicum frutosceiis) .




Alkali sacaton and Chile pepper plants exposed to high UV-B showed a marked




reduction in leaf growth with increasing UV-B.  Mesa dropseed plants, however,




showed no differences in leaf growth between UV-15-irradiated and control




plants.  Dock plants (Rumex patientia L.) exposed to high levels of UV-B




showed a reduction in protein synthesis.




Impact of Solar Radiation on Crops and Crop Canopies__




     Physiological and ultrastructural studies were conducted by SEA researchers




at Gainesville, Florida, on selected vegetable, agronomic, and citrus crops




exposed to increased UV-B radiation in the field and the greenhouse.




     Citrus plants irradiated for 4 weeks under supplemental UV-B radiation




in the field showed no significant reduction in average daily photosynthctic




rate as compared with unirradiated control plants even under a 200 percent




increase in biologically effective radiation.  Similar results were obtained




in stomatal diffusion resistance of eight soybean varieties.




     Broad-band UV-B enhancement studies were conducted in the greenhouse on




soybeans (Bragg and Altona),  peas (Lit;tie Marvel), tomatoes (Rutgers) and




sweet corn (Golden Cross Bantam).  Plants were grown for 4-6 weeks under




three levels of UV-B irradiation ranging from approximately a 100-200




percent increase in biologically effective UV.  Da'ta were taken on biomass,




C02 uptake rate, chlorophyll content, Hill reaction, RuDP carboxy.1 ase,




PEP carboxylases soluble proteins, absorption spectra of pigment extracts,




and ultrastructural changes in selected cultivars.
                                  19

-------
     Ill general, plants exposed to high UV-B irradiation in tlie greenhouse


showed physiological changes.  For example, soybean plants showed          .              •'•;


a decrease in chlorophyll content, RuDP carboxylnse. activity, soluble protein


content, COo uptake, and fresh and dry weight as compared with control                   •,
                                                                                         ?•"

plants receiving only UV-A  (320-400 nm) irradiation alone or unirradiated                !•.
                                                                                        .i.

control plants.  In contrast, plants given supplemental UV-B irradiation                 r
                                                                                         '('

in the. field showed little or no effect.                                                 r

                                                                                         l
     Differences in species and cultivar response to increased UV-B irradiation          j'
                                                                                         i

were also observed.  Differences in chemical and structural makeup of the                j'

                                                                                         l;
epidermis and palisade parenchjina cells were thought to play a role in •                  j


the response of different plants to enhanced UV-B radiation.       "                  •    ;


Response of V?pp_dy Plants to Ipcreased UV-B Radiation^                                     j
   '                   —                     —              .                             i;

     Various physiological disorders of agronomic and horticultural crops                ,


and woody species have been ascribed to high levels of solar irradiation,            •    i

                                                                                         l'.
especially at high elevations.  In order to determine the role of UV-B


radiation in solar injury of certain woody plants at high elevations, Forest


Service scientists conducted UV-B enhancement and exclusion studies at the


Rocky Mountain Forest and Range Experiment Station in Fort Collins,


Colorado.  Englemann spruce was chosen, as a sensitive species and Lodgepolc


p.ine was chosen as resistant specias.                                         •  "


     Seedlings were irradiated under artificial UV lamps for a total of


AGO hours over a 67-day period or were grown under various filters to exclude


natural UV-B radiation.  No evidence of UV injury was observed in any of the


treatments during the first year of the study.   Since Engelmann spruce


seedlings transplanted to the natural environment do not show symptoms of


solar radiation injury until after the first winter, seedlings will be


observed for symptoms during the second growing season.
                                 20

-------
Rejjj>on_s c o f N :L t r o g en - f :i. x ing 0 r v; a n isms to Increased UV-B Radiation



     Nitrogen fixation of Anabe.na floss-aquae and other blue-green algae,



free-living and in symbiosis with the water fern Azolla, .is important in



rice culture and in worldwide soil fertility.  Laboratory studies were,



therefore, conducted at the SEA Northern Regional Research Center in Peoria,



Illinois, on- the influence of increased UV--B radiation on the nitrogen fixing



abilities of Anabena alone and in association with A/.olla.



     Results indicated that while viability and photosynthesis of Anabena



cells were unaffected by UV-B irradiation, nitrogen fixation  (as measured by



nitrogenase activity) was markedly reduced by high levels of UV-B irradiation.

                                                                 *^ •
     When cultures of blue-green algae were exposed to 10 watts/in  of UV-B



for 3-5 hours, the algal nitrogenase was inhibited to about one-half of the



activity of the control cultures.  In spite of this observed reduction in



nitrogenase activity there was no reduction in the treated cells'



reproductive capacity as demonstrated by plate count studies.



     When blue-green algae and its symbiont Axo_lla_ were exposed for 4-6


                    *)                           1 /
days with 10 watts/m  of UV-B, photosynthesis (C  CU fixation) was not



affected, and nitrogenase activity was reduced to 30-40 percent of that



observed in the control.



Response of Farm Animals to Increased UV-B Radiation



     Studies conducted by SEA researchers at the National Animal Disease



Center in Ames, Iowa., investigated the carcinogenic, effect of high levels of



UV-B irradiation on the eyes of four Hereford cattle.  Exposing .eyes of



these cattle to high levels of UV-B irradiation for 2 hours per day induced



ocular changes that were consistent with chronic irritation.  After 7



months of exposure, one animal developed ocular changes that were considered



ncoplastic after biopsy and pathological examination.  Cancer eye, a squaiiious
                                  21

-------
 cell carcinoma,  normally takes 5-6 years  Co  develop  in  cattle  under  natural

 conditions.

       Inspection of;  slaughterhouse condemnation .records was  made  to  determine

 the extent and incidence, of cancer eye  in cattle,  using USDA Meat  and.  Poultry

 Inspection data.  Based on the total  cattle  slaughtered since  1950,  the

 increase in cancer eye was about two-fold.   Since  other diseases  of  cattle

 have also increased  during this time  period,  it  is difficult to interpret

 these data.

 Response ^of Insects  to Increased UV-B Radiation

      Studies were conducted in the Chemical  and  Biophysical  Control

 Laboratory at Beltsvillc,  Maryland, on  the influence of increased  UV-B

 radiation on the physiology and behavior  of  selected beneficial and

 harmful insects..  These studies demonstrated  that  lightly  pigmented  insects,

 such as the pink bollworm,  codling moth,  and  the face fly, were much more

 sensitive to exposure to UV-B irradiation than heavily  pigmented  ones  such

 as  the house fly.

      Brief exposure  of pink bollworr.t. eggs to  UV-B  radiation  levels  (10-50

 percent above natural levels at Beltsvillc),  for 1 to 3 hours  greatly

 reduced the life span, of larvae hatching  from these  eggs.  High UV-B levels
..                       ••         v
 (100 percent increase in biologically effective  radiation) also had  a

 highly lethal effect on face fly pupae  irradiated  for 1 hour per day for

 3 days.

      Adult honeybee  workers,  however, were able  to tolerate  a  10-50  percent

 increase in UV-B radiation for 6 hours  per clay for 6 days without apparent

 injury.
                                  22

-------
     High UV-B irradiation increased pigment;  formation  in  the  .larvae  of


butterflies and moths, and the pupae of  face  flies,  but  had  little  or no


effect on the pigment content of  tobacco budworm  larvae, house  fly  pupae,


or honeybees.


     Physiological studies on respiration  indicated  an  increase  in  oxygen


uptake in codling moth larvae irradiated 6  hours  per  day for 2  days,  but no


effect on honeybee workers.


     Tobacco budworms allowed to  feed on bean leaves  exposed to  UV-B  irradiation


did not show increased mortality.  The eggs of pink  bollworms,  however,


irradiated on cotton leaves showed a redxiction in  number that  hatched.


Stab i_lity _of_ Ag_r icult ural Chem ic.a Is Under  In c r_e_asฃd_ JlYzlLJLl? r'   li
     The Organic Chemical Synthesis Laboratory at  Beltsville, Maryland,


constructed and successfully put into operation a  "merry-go-round"  type


photolysis apparatus for investigating the  stability  of pesticides  and other


agricultural clie.mi.cals under increased UV-B irradiation.   Such  studies are


being conducted to determine the efficacy of various  agricultural chemicals


under a high UV-B environment.


     Photoclegradation of test compounds was obtained  by exposing the  samples


to 313 nm radiation in the UV-B region and  quantum yields  measured.


Preliminary studies with aqueous solutions  of pesticides confirmed  the
                          ..         %

dependence, of quantum yield (the number of  pesticide  molecules  consumed


per quantum of UV-B radiation absorbed) upon concentration.

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                        FINAL REPORT
   EFFECTS OF UV-B RADIATION ON SELECTED LEAF PATHOGENIC
              FUNGI AND ON DISEASE SEVERITY
                       H. R. Cams
                       J* H. Graham
                       S. J. Ravitz
                   Plant Stress Laboratory
                  Plant Physiology Institute
           Beltsville Agricultural Research Center
                  Beltsvilleป Maryland 20705
                       EPA-IAG-D6-0168
                       Project Officer:

                       R. .J. McCracken
Agricultural Research,  Science and Education Administration
                 U.S.  Department of Agriculture
                   Washington, D.C. 20250
                         Prepared for
                Environmental Protection Agency
                         EAGER Program
                      Washington, D.C. 20460

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                              ACKNOWLEDGMENT






     We acknowledge the dedicated involvement of Ms.  Sheryl Olsen who




provided valuable aid in conducting experiments, data collection, and




analyses throughout these investigations.

-------
                               ABSTRACT

     The results of increased levels of UV-B irradiance on spore

germination indicate that although plant leaf pathogenic fungal species

vary considerably in sensitivity to UV-BS relatively high irradiance levels

are required to reduce germination percentage.  Pigmented spores such as

Cladosporium, Stemphylium, and Alternaria were found to be more resistant

to increased UV-B irradiance than hyaline spores (Mycosphaerella,

Colletotrichum).

     Disease severity of Col1etotrichum on cucumber was decreased with

increasing UV-B irradiance.  A linear decrease in the percentage of leaf

area diseased with increased irradiance was found.

     Level^of UV-B irradiance did not affect severity of disease incited

by Cladosporium.

     There were no noticeable UV-B effects on either the Stemphylium

pathogen or the host,  alfalfa.  Alternaria, the tomato pathogen, reacted

similarly to Cladosporium on cucumber,  disease severity being unaffected by

UV-B irradiance levels.

     Recognizing that  our results represent only a  small sampling of leaf

disease organisms and  plant disease-interaction experiments,  they appear
                                \
to support the following:  considerably higher levels of UV-B irradiance

than those expected from the projected  ozone, depletion will be required to

adversely affect germination and growth of leaf pathogenic fungi;  and,  where

fungal germination and growth are affected, disease severity in the host

plant can be expected  to be reduced as  UV-B irradiance increases.

-------
                            INTRODUCTION




     The impact of increased radiation in the 280-320 nra region  (hereafter




referred to as UV-3) on overall plant growth, development, and critical




metabolic processes involved is a major concern in the short-term Biological




and Climatic Effects Research Program.  Of equal 'concern is the possibility




that crop yield and quality may be affected indirectly by influencing plant




susceptibility to various plant pathogens and/or by affecting the pathogens




directly.




     Numerous studies have been made on the influence of visible light on




sporulation of fungi; a limited number of studies have been conducted on




the effects on plant infection and disease severity.  A few reports have




dealt with the effects of ultraviolet radiation; however, most researchers




used germicidal lamps (major energy in the 254 nm range) (UV-C).  UV-C




from germicidal lamps enhanced sporulation of some fungi and inhibited or




retarded it in others.  UV-C effects on pathogen spore germination, mycelial




growth and subsequent infection and disease severity have also been studied




and generally an adverse effect on the pathogen has been reported.   In




addition, limited information is available on the effects of other UV




unspecified or poorly defined wavelengths on fungal behavior.   Numerous




references are cited in reviews by Marsh et al.  (1959) and by Leach (1971).




     Our research was undertaken to provide preliminary information on the




effects of UV-B radiation on spore germination,  mycelial growth, infectivity




and disease severity of three fungal leaf pathogens of cucumber (Cucumis




sativus L.)s  one of tomato (Lycopersicon esculenturn Mill.),  and two of




alfalfa (Medicago sativa L.).   The pathogens selected were:   Colletptrichum




lagenarium (Pass.) Ell.  & Halst.,  causing cucumber anthracnose;

-------
 Cladosporium  cucumerinum Ell.  & Arth., causing cucumber scab;




 Mycosphaerella  melonis  (Pass.) Chiu & J. C. Walker, causing cucumber black




 rot;  Stemphylium botryosum Wallr., causing an alfalfa leafspot; Uromyces




 striatus  Schroet. var medicaginis  (Pass.) Arth., causing alfalfa rust;




 and Alternaria  solani  (Ell. & Mart.) L. R. Jones & Groot, causing tomato




 early blight.




                        MATERIAL AND METHODS




 UV-B Radiation  - Measurements, Instrumentation, and Methodology




     UV-B enhancement facilities were developed cooperatively with the




 Agricultural  Equipment Laboratory  (AEL), Beltsville Agricultural Research




 Center.  UV-B enhancement was provided by the required assembly of




 Westinghouse—  FS40 or FS20 fluorescent sunlamps, either filtered with 6-




 hour-aged 5 mil cellulose acetate  (CA) (plus UV-B) or 5 mil Mylar (minus




 UV-B).




     Spore germination and mycelial growth experiments were carried out in




 temperature-controlled incubators equipped with one FS20 lamp each as the




 UV-B source.  Three incubators were equipped with one FS20 lamp as the only




 radiation source and one incubator was additionally equipped with two




 Westinghouse 14-watt cool white fluorescent lamps to provide visible energy.




     Greenhouse and growth chamber experiments dealing with pathogen




 infactivity and disease severity were carried out in a fiberglass green-




 house or in plant growth chambers equipped with FS40 lamp assemblies




 provided by AEL.
_!/ Mention of a trademark, proprietary product, or vendor does not




   constitute a guarantee or warranty of the product by the U.S.  Department




   of Agriculture and does not imply its approval to the exclusion of other




   products or vendors that may also be suitable.

-------
     UV-B  irradiance  levels were  determined  for each plant or  fungal




 location in  each  experiment with  either an Optronics Laboratories,  Inc.




 Model  725  UV-B  Radiometer  or  an Instrumentation Research Laboratory (IRL)




 UV-B Radiometer described  in  the  IRL  final report.  Radiometer readings




 were verified by  spectral  irradiance  determinations (250-369 nm) with an




 automated  spectroradiometer as described in  the IRL report at  selected




 locations  in the  experimental irradiation areas.



                                                   ~2
     Weighted irradiance levels are reported as mWra  BUV, the biologically




 effective  UV derived  from  the AZ9 weighting  function, and unweighted


                  __ f\

 irradiance as mWm  obtained  by summing the measured or calculated  values



                                                —2                    _9
 at each nanometer from 280-320 nm.  Dividing mWm  BUV by 3.06  (the  mWm  BUV




 of control sunshine)  provides the fraction of BUV received by each  plant or




 fungal location relative to that of one control sunshine.




     Since all  UV irradiation for experiments reported here was filtered




 through cellulose acetate, BUV was limited to the UV-B region  (280-320 nm).




     For details  concerning average control sunshine,  spectral character-




 istics of  UV fluorescent lamps and filters, and the weighting function,  see




 the EAGER  final reports of the Agricultural Equipment Laboratory, and




 the Instrumentation Research  Laboratory, Beltsville Agricultural




 Research Center.




                          Spore ''Production




     Sporulating  cultures of  Cladosporium c me urnerinurn and Mycosphaerella




melonis were grown on potato  dextrose agar at room temperature (22-24ฐC).




Colletotrichum  lagenarium was maintained under the same conditions on




V-8 juice  agar.    For  Stemphylium botryosum,  sporulation was induced by




placing V-8 juice agar cultures in a 22ฐC incubator fitted with four

-------
Westinghouse 14-watt cool white  fluorescent lamps on a  12-12 hour




light-dark cycle.  Alternaria solan! spores were obtained by growing




mycelia on lima bean extract agar.  After  7 days of culture, the mycelia were




scraped with a scalpel in a sterile transfer chamber and the Petri dish lid was




removed.  Spores were produced the following day,.  Uredospores of Uromyces




striatus var. medicaginis, an obligate parasite, were obtained from infected




alfalfa plants.




                         Spore Germination




     From a distilled water suspension of  fungal spores, a drop was pipetted




onto 2 percent water agar in polyethylene  plastic Petri dishes and allowed to




.dry.  As the uredospores of Uromyces striatus do not readily suspend  in




water, spores were dispersed onto the agar surface by dusting with a  sterile




camel's hair brush.  The lids of the dishes were removed and replaced by




filter squares of 5 mil CA, pre-solarized  for 6 hours, or 5 mil Mylar as




required.• Dishes were then transferred to incubators at predetermined




positions under the FS20 lamp such that they were subjected to weighted




irradiance levels of 6.25 mWni~2BUV (644 mWm~2), 6.69 mWm~2BUV




(691 mWnT2),  8.06 mWm~2BiJV (8.33 mWm~2) and 10.52 mWm~2BUV (1087 mWm"2).




     Samples were irradiated for 6 hours, then left in the dark for 18 hours




after which microscopic counts of ,spore germination (200-500 spores per




dish) x^ere made.   Each UV-B irradiance was duplicated within each of  three



                                                                     —2 —1
incubators.   A fourth incubator was additionally supplied with 25 yEm  s




of photosynthetically active radiation (PAR) provided by cool-white




fluorescent  lamps.   These lamps x^ere allowed to remain on during the 6-hour




UV-B irradiation and for an additional 6 hours thereafter.




                          Myce1ia1 Gr owt h




     Fungal  cultures were exposed to UV-B in the same incubators used for




spore germination.   The 5 mil CA filter was placed as a collar around

-------
 Che FS20 lamp, lids were removed and the Petri dish bottoms were enclosed




 by UV-B transparent polyethylene bags to keep the agar from drying out




 over the course of the experiments.




     Samples were inoculated by placing a 7 nun diameter core of mycelia




 upside down in the center of the agar plates which were then placed at the




 predetermined positions in the chambers.  Samples were irradiated daily for




 6 hours until growth reached the perimeter of the Petri dish or sufficient




 data points had been accumulated.  Growth was determined by daily measuring




 the diameter of the colonies.




                          Disease Development




 Cucumbers




     For epidemiological experiments with cucumbers, the UV-B sensitive




 cultivar, Poinsett, was germinated in a synthetic soil mix of peat and




vermiculite (Jiffy Mix) in 12.5 cm pots in a fiberglass greenhouse, five




seedlings per pot.  Temperatures ranged from 24-27ฐC during the day and




19-21ฐC at night.  Plants were subjected from emergence to UV-B radiation




supplied by eight FS40 lamps, filtered by pre-solarized 5.mil CA.  Filters




were changed every fourth day.   Plants were irradiated with'UV-B between




the hours of 1000 and 1600 dailv for the duration of the. experiment.   Figure




1 shows a typical experimental design used in the plastic greenhouse.  '




     As the cotyledons became fully expanded (5-7 days after seeding), the




seedlings were selected for uniformity and thinned to one per pot.   Plants




were inoculated when the first  leaf was fully expanded (11-14 days).




     Colletotrichum lagenarium.  Figures 2 and 3 diagram the experimental




design and provide the weighted and unweighted UV-B irradiances, respec-




tively, for each pot location.

-------
Figure 1.  Typical experimental set-up used in UV-B enhancement




          "studies in the greenhouse containing a four-fixture,




           txtfo lamps per fixture, array of FS40 fluorescent sunlamps




           (filtered with 5 mil CA).  'Poinsett cucumber plants infected




           with Colletotrichura are shown.   The typical chlorotic lesion




           response induced by UV-B irradiation is evident.  Disease




           symptoms are visible at the bottom of the photograph on




           plants receiving the lowest levels of UV-B irradiance.

-------








•


          •••

      -

            .






-------
        4.6   6.1   7.7    9.2   10.1   10.1   10.1   9.5    8.3  7.0
                                                                5.8   4.3    3.1  2.1    1.5   1.2   1.0
CO
4'9   6'4   8.0    9.5   10.4   11.0   11.0 .10.1    9.2  7.7   6.1   4.3    3.4  2.1    1.5   1.2   1.0


5.2   6.7   8.6   10.4   11.3   11.9   11.6  11.0    9.8  8.0   6.4   4.6    3.4  2.1    1.9   1.2   1.0


5.5   7.0   9.2   11.0   11.9   12.6   12.2  11.3   10.1  8.3   6.4   4.6    3.4  2.5    1.8   1.2   1.0


5.2   7.0   9.2   10.7   11.6   12.2   11.9  11.3   10.1  8.3   6.4   4.6    3.4  2.5    1.8   1.2   1.0
                                **


5.2   6.7   8.6    9.8   11.0   11.6   11.6  10.7    9.5  8.0   6.1   4.6    3.4  2.1    1.5   1.2   1.0


4.6   6,1   8.0    9.2   10.1   10.4   10.4   9.8    8.6  7.0   5.8   4.3    3.1  2.1    1.5   1.2   1.0


4.3   5.8  I 7.0    8.3    9.5    9.8    9.8   9.2    8.0  6.7 I  5.2   3.7    2.8  1.8    1.5   1.2   1.0
         Figure  2.  Colletotrichum greenhouse experimental arrangement.  Each number represents the biologically
                   effective UV-B irradiance in mWnf^jjUV at each plant canopy.  Plants were 0.15 m apart.
                   Brackets indicate positions of fixtures, with the outer fixtures being 0,8 m, and  the  inner
                   fixtures 1.0 m from the top of the pot.

-------
 474   631
791   948  1043  1043  1043   982   854   724   599   441   316   221   158   125     98
 506 •  665   823   982  1072  1138  1138  1043   948   791    631 '   441   349    221    158   .125    98
 537   696   BbB  1072  1168  1233  1199  1138  1010   823    665    474    349    221    190   125    98
 563   727   948  1138  123'J  1296  1263  1168  1043   854    665    474    349    253    190   125    98
                            4*


 537   727   948  1105  1199  1263  1233  1168  1043   854    665    474    349    253    190   125    98


 537   696   858  1010  1138  1199  1199  1105   982   823    631    474    349    221    158   125    98
 474   631   823   948  1043  1072  1072  1010   858    724    599    441    316    221    158   125    98
  441    599
727   854   982  1010  1010   948   823   696   537   380   284   190   158    125     98
Figure 3.   Colie totriehum  greenhouse  experimental arrangement.  Each number represents  the  total
           unweighted  UV-B irradiance in mWm~2 at each plant  canopy.  Plants were 0.15  m apart.
           Brackets indicate  positions of  fixtures, with  the  outer  fixtures being 0.8 m, and  the
           inner fixtures  1.0 m from  the top of  the pot.

-------
     An inoculum, consisting of 30,000 spores/rni suspended in distilled




water, was prepared from 8-day old Colleto tr ichum cultures.  Using an




electric sprayer, the leaves were covered with fine, droplets of inoculum.




Control plants were sprayed with distilled water.  To attain the high




relative humidity necessary for infection, the sprayed plants were enclosed




within UV-B transmittable polyethylene bags for a 48-hour inoculation period.




     Photographs, fresh weight, dry weight, area of first leaf, and percent




of first leaf diseased were recorded and used to determine UV-B-disease




interaction.




     Cladosporium cycumerinum.  The weighted and unweighted UV-B irradiances




for each pot location are shown in Figures 4 and 5, respectively.  The




inoculum, containing 70,000 spores/ml, was applied as described for




Colletotrichum and the inoculated plants were sealed in polyethylene bags




for 48 hours.  Plants xvere harvested 8 days after inoculation.   Fresh weight,




dry weight, and area of first leaf were used to determine UV-B-disease




interaction as disease symptoms did not permit precise scoring.




     Mycosphaerella melonis.  The weighted and unweighted UV-B irradiances,




respectively,, for each pot location are shown in Figures 6 and 7.  The




inoculum, containing 60,000 sporesVml, was applied as for Colleto trichum.




Similar data were also taken; however, as with* Cladosporium,  disease




symptoms did not permit precise scoring.




Alfalfa




     For the evaluation of the effect of  UV-B on leaf rust of alfalfa, the




cultivar Arc was grown in a greenhouse for 5 weeks arid then transferred




to two plant growth chambers.  Temperature in the chambers was 25-20ฐC



                                                                         9 —1
day-night with a relative humidity of 90  percent.  An average of 200 pEm  s
                                  10

-------

4.9 6.7
5.2 7.7
6.1 8.3
6.7' 8.6
6.7 9.2
6.7 8.6
6.1 8.0
5.8 7.0
Figure 4.

9.2 10.
	 9_._5 11.
u ••ซ•
10.7 12.
11.3 13.
11.6 13.
11.0 12.
7 11.9 12.9 12.9 11.9 10,4 8,9 7,0 5.2 3.7 2.8 1.8 1.2 0.9
9 13.2 14.4 14.4 13.5 11.'6 9.5 7.7 5.8 4.3 2.8. 1.8 1.2 0.9
9 14.4 15.0 15.3 14.4 12.6 10.4 8.0 6.1 4.6 3.1 2.1 0.9
5 15.0 -15.9 15.9 15.0 13.8 10.7 8.3 6.1 4.6 3.4 2.1 0.9
5 15.0 15.9 15.6 14.7 12.9 10.4 8.3 6.1 4.3 3.1 2.1 1.2 1.2
9 14.4 15.0 15.0 13.5 11.9 9.8 7.7 5.5 3.7 2.8 1.8 1.2 0.9

10.1 11.
9.5 11.

9 12.9 13.5 13.5 12.6 11.0 9,2 7.0 5,2 3.7 2.8 1.8 1.2 0.9
3 12.2 12.9 12.6

Cladosporium greenhousc0experiment arrangement. Each number represents the biologically effective
UV-B irradiance in mWm  BUV at  each plant  canopy.   Plants  were  0.15 m  apart.   Brackets  indicate
position of fixtures,  with the  outer fixture being 0.8  m,  and the  inner  fixture  1.0  m from the
top of the pot.

-------
505
537
631
696
696
696
631
599
Figure
696
792
854
885
943
885
823
727^
5.
948
981

1105
1168
1199
1138

1043
981
1105
1233

1328
1389
1389
1328
1233
1168
Cladosnorium
1233
1358
1484
1547
1547
1484
1328
1263
1328
1484
1547
1641
1641*"
1547
' 1389
1328
greenhouse.
1328
1484
1579
1641
1611
1547
1389
1235
1233 1072 915
1389 1199 981

1484 1235 1072
1547 1421 1105
1515 1328 1072
1389 1233 1010

1235 1138 948

experiment arrangement.
727 537 380 284
792 599 441 284
823 631 474 316
854 631 474 349
854 631 441 316
792 568 380 284
727 537 380 284
Each number represents
190 125 95
190 125 95
234 95
234 95
234 125 125
190 125 95
190 125 95
the total unweightec
UV-B irradiance in mWm   at  each plant  canopy.  Plants were 0.15 m apart.  Brackets indicate
position of fixtures,  with the outer  fixture  being  0.8 m, and the inner  fixture  1.0 m  from
the top of the pot.

-------
4.9 6.4
5.2 7.0
5.8 7.7

6.4 8.7
6.4 8.7
8.7 10.
9.2 11.
i
9.8 11.

10.7 12.
10.4 12.
1
0
6

9
9
11.
12.
13.

14.
14.
3
2
2
.**
4 '
4
11.
12,
14.

15.
15.
9
9
1

0
0
11.
12.
14.

15.
15.
9
9
1

0
0
11.
12.
13.

14.
14.
6
2
5

4
4
10.
11.
11.

12.
12.
4 7.7
0 8.0

9 8.7

6 10.1
6 10.4
6.1 4,
6,4 4,
7.0 6.

8.3 5.
8.3 5.
6
9
1

8
8
3.7
3.7
4.0

4.3
4.3
2.8
2.8
3.1

3.1
3.1
1.
1.
2.

2.
2.
8 1.2
8 1.5
1 1.5

1 1.5
1 1.5
.9
.9
1.2

1.2
.9
J6.1    8.0    10.1    12.6    14.1    14.7    14.7    13.8    11.9    9.8    8.0    5,8     4.0    2.8    1.8    1.2
 5.8    7.7     9.5    11.6    12.9    13.5    13.5    12.6    11.3    9.2    7.3    5.2     3,7    2.8    1,8    1.2
 5.2    6.7     8.1    10.4    11.6    11.9    11.9    11.3     9.8    8.3    6.7    4.6     3.4    2.1    1.8    1.2    .9
 Figure  6.   Mycosphaerella  greenhouse  experiment  arrangement.   Each number  represents  the  biologically
            effective UV-B  irradiance  in mWm~2BUV at  each  plant canopy.   Plants v;ere 0.15  m apart.   Brackets
            indicate position  of  fixtures, with  the outer  fixture  being  0.8 m, and  the inner fixture 1.0 m
            from the top  of the pot.

-------
506   661   900   1043  1166   1227   1227   1197  1072   796  628    A66   381   291    186   124    93
537   722   949   1134  1259  1331  1331  1259  1134   827  661    506   381   291.   186   154    93
598   796   910   1197  1520  1455  1455  1394  1227   900  722    628   414   320    217   154   124


661   900  1104   1331  1485  1547  1547  1485  1301  1043  858    .598   444   320    217   154   124

                            ซ•
661   900  1072   1331  1485  1547  1547  1485  1301  1072  858    598   444   320    217   154    93


628   827  1043   1301  1455  1518  1518  1425  1227   910  827    598   414   291    186   124    93
598   796   981   1197  1331  1394  1394  1301  1166   949  753    537   381   291    186   124    93
537   692
835   1072  1197  1227  1227  1166   910   858  691    466   352   217    186   124    93
Figure 7.  Mycosphaerella greenhouse experiment arrangement.  Each number represents the total
           unweighted UV-B irradiance in mWm~  at each plant canopy.  Plants were 0.15 m apart.
           Brackets indicate position of fixtures, with the outer fixture being 0.8 m, and  the
           inner fixture 1.0 m from the top of the pot.

-------
visible radiation was provided by sixteen 165-watt cool white fluorescent .Tamps



and twelve 50-watt incandescent bulbs with a photoperiod of 16-hour day


8-hour night.  The plants were allowed to acclimate to chamber conditions


for 5 days before being irradiated with UV-B.  UV-B radiation was provided


by two FS40 Westinghouse sunlamps (with no reflector) filtered by 5 mil CA



that had been pre-solarized for 6 hours.  Filters were changed every fourth


day.  Figures 8 and 9 diagram the plant arrangement within the chambers


and show the weighted and the unweighted UV-B irradiances, respectively, at


the canopy height of each plant.  Plants were clipped during the experiment


to maintain a distance of 0.37 m from the fixture.


     Uromyces striatus.  The plants were inoculated with spores of the rust


fungus after 7 days of UV-B irradiation.  Inoculum was prepared by scraping


the spores from 10-day-old _!5. botryosum agar cultures, suspending them in


distilled water and filtering the mixture through cheesecloth to remove


mycelial fragments.  The resulting spore suspension contained 10,000


spores/ml and was applied to the alfalfa leaves in fine droplets by means


of a chromatography sprayer.  Control plants were sprayed with distilled


water only.  All plants were then covered for 24 hours with UV-B transmit-


table polyethylene bags to achieve maximum humidity necessary for good


infection.  Five days after the end  of the inoculation period,  all leaflets
                                  v

in the upper 4 centimeters of the plant were scored for type of lesion as


fellows:  1 = pinhead size brown flecks; 2 = lesion approximately 1 mm in


diameter with a brown margin and tan center;  3 = lesion approximately two


to three times larger than #2 type and often with obvious yellow halo out-


side of the brown margin;  and 4 = a  large blighted area most often found


on leaflet margin.
                                   15

-------

7.3 8.6 8.6 8.6 8.3 ' 7.0
2,1 3.4 5.8
9.1 10,4 10.7 11.0 10.1 . 8.6
2.1 3.4 ! 6.1
8.6 10.1 10.7 10.4 10.1 8.6
2.1 3.4 5.8
• •
6.7 7.7 8.3 8.0 7.7 7.0
. 0.9
•
! 0.9
*
ป
. 0.9
•
•
•
. 0.9
Figure 8.
Growth chamber experimental arrangement.  Each number represents the biologically
effective UV-B irradiance in rr.Wm  BUV at each plant canopy (0.37 m from the
fixture).  Plants were 0.15 m apart.  Brackets indicate the position of the
fixture.  The dotted line represents a 5 mil Mylar barrier.

-------
 221
 221
 221
349    599
349
611
349    599
                         759       885      885      885      854     727
                         948      1072     1105     1138     1043     885
                         885     1043     1105     1072     1043    885
                         696      792      854      823     792    727
                                                                         95
                                                                         95
                                                                         95
                                                                         95
Figure 9.- Growth chamber experimental  arrangement.   Each number represents the total
           unweighted UV-B irradiance  in mWm   at  each plant  canopy (0.37 m from the
           fixture).   Plants were 0.15  m apart.   Brackets indicate  the position of
           the fixture.   The dotted line represents  a 5 mil Mylar barrier.

-------
 Tomato




      The cultivar  Chef  was germinated  and grown in the same plant growth




 chambers used for  the alfalfa  experiments except that the FS40 lamps  were




 repositioned to  adjust  UV-B irradiance levels and temperatures were




 maintained  at 26ฐC .day  -  20ฐC  night.   Figures 10 and 11 diagram the plant




 arrangement within the  chamber and  list  the weighted and unweighted




.irradiances, respectively.




      Alternaria  solani.   Twenty-two days  after seeding when the third leaf




 was  well expanded,  buds were pinched  out;  and four days later the plants




 were inoculated  as follows:  The  spore suspension was prepared by placing




 two  spore mats and 100  ml distilled water in a blender for 15 seconds and. then
                     h



.filtering the resulting suspension  through cheesecloth to remove mycelia




 and  agar fragments.  The  suspension was applied in a fine mist to the second




 and  third.leaves by means of a chromatography sprayer.   Relative humidity in




 the  chamber was  maintained  at  90-100 percent for 24  hours by  a chamber




 humidity regulator .and  the  placement of a polyethylene canopy over the plants.




 The  second  and third leaves were  harvested for dry weights 48 hours after




 inoculation when blighting  became severe.




      For analyses  of plant-disease  response as a function of  UV-B,  in the




.above experiments, .data from .individual plants xsrere  combined  into irradiance



                            -?     v
 groups of increasing 1.5  mWm   BUV and  subjected to .analyses of variance.




 If P•= 0.05 or less, the  data  were  further analyzed  using linear regression.




                           RESULTS  AND DISCUSSION




 Spore Germination




      The results of increased  levels of UV-B irradiance on spore germina-




 tion .are shown in  Table 1.  They  indicate  that  even  in  the more  sensitive




 species,  high UV-B irradiance  levels are  required  to  reduce germination




 percentage;  generally, more than  double the  UV-B  of  one  control  sunshine
                                      18

-------
1.8
1.5
1.5
1.2
1.5
1.5
2.5
2.8
2.8
2,8
2,8
2,5
3.7
4.6
4.9
4.9
4.6
3.7
4.6
5.8
6.7
7.0
6.4
5.2
5.5
7.0
8.3
8.3
7.7
6.1
6.1
7.7
8.9
9.2
8.3
6.7
6.7
8.3
9.5
9.5
8.9
7.0
7.0
8.6
9.8
9.8
8.9
7.3
7.0
8.6
10.1
10.1
9.2
7.0
6.7
8.3
9.5
9.5
8.6
6.7
5.5
7.3
8.3
8.3
7.3
5.8
4.6
5.5
6.4
6.1
5.5
4.3
3.1
3.4
3,7
3.7
3.7
3.1
1.8
1.8
1.8
1.8
1.8
1.8
Figure 10,   Alternaria growth chamber experimental arrangement.   Each number
            represents the  biologically effective UV-B irradiance in mWra~^BUV
            at  each plant canopy.

-------
                  190   253....38.0   474   568   611   696    727     727   696   568   474    316   190


                  159   286   474   597   727   792   854    885     885   854   759   568    349   190


                  159   286   506   696   854   915   981   1010    1043   981   854   665    380   190
O
                  125   286
506   727   854   956   981   1010   1043   981   854   611
380   190
                  159   286   474   665   792   854   915    915    956   885   759   568   380   190
                  159   253   380   536   611   696   727    759    727   696   597   441   316   190
                  Figure 11.  Alternaria growth chamber experimental arrangement.   Each number represents
                              the total unweighted UV-B irradiance in mUm   at each plant canopy.

-------
was required before a reduction in spore germination was noted.  In the

more resistant species, more than three times the level of control

sunshine UV-B was needed to inhibit spore germination.

     For Colletotrichum, the most sensitive species, spore germination was

reduced by less than two times control sunshine as indicated by comparison

of the Mylar-filtered controls and the lowest irradiance level used.

     Resistance to UV-B appeared to be correlated with spore pigmentation.

Spores of the resistant species Cladosporium, Stemphyliura, Uromyces, and

Alternaria are all darkly pigmented, whereas, spores of Mycosphaerella and

Colletotrichum are hyaline.  Our results suggest that pigmentation provided

protection from damage by UV-B.

     In the incubator supplied with PAR in addition to UV-B, germination

values were consistently, but only slightly, higher than those observed in

the incubators irradiated by UV-B only, with the most notable increase in the

sensitive Colletotrichum spores.  Furthermore, we observed that germ tube length
 ซ>
appeared to be considerably increased in nearly all tests in the presence

of PAR.  This observation is consistent with other BACER research which provides

evidence for the existence of a photorepair or photoprotection mechanism.

Linear regression analyses indicate that within the range 'of irradiance

levels tested,  there is a significant correlation between reduction in

germination and increased UV-B irradiance in the susceptible species.

Mycelial Growth

     A measure of the mycelial growth rate under identical UV-B irradiances

and environment used for the spore germination experiments is presented in

Table 2.   Growth rate is expressed as increase in colony diameter with time.

In contrast to  the spore germination,  increase in colony diameter, (as

expressed in terms of percentage of the Mylar control)  at the end of the
                                  21

-------
Table 1.  Influence of UV-R irradiation on spore germination of six pathogens.
          Spores were irradiated in Petri dis-hes in an incubator at 22ฐC under
          an FS20 fluorescent sunl'amp filtered with either 5 mil cellulose
          acetate or 5 mil Mylar.


Mylar-5 mil
Disease organism <

Colletotrichum lagenarium
Test A
B
Mycosphaerella melonis
Test A
E
C
D
E
Alternaria solani
Test A
B
Stemphylium botryosum
Test A
B
Cladosporium cucumerinum
' '': -' Test A '" "' 	 '•- ••""••'"•
B
Uromyces striatus
Test A
B
0.25
Mean
17.82
49.29
52.46
50.21
41.30
33.06
63.08
98.17
98.88
98.03
93.46
93.69
94.73
86.85
90.24
mWm
2BUV



Cellulose acetate-5 mil
6.26
percent
12.70
10.31
57.75
45.48
47.85
29.10
60.04
98.58
99.01
97.44
93.48
90.53 '
90.93
86.70
89.62
6.69
8.06 10
.52
germination
17.79
8.20
57.34
42.34
46.34
28.49
57.41
98.49
99.38
97.35
93.48
89.02
t89.65
85.54
89.90
5.13
2.80
47.85
30.95
37.96
24.59
47.28
98.72
99.14
95.49
93.04
86.37' '
87.80
86.25
89.03
2.03
1.22
35.44
22.94
26.62
19.62
33.90
98.75
99 . 01
94.09
93.29
81.33
83.78
86.10
89.88

r2 '
0.691
0.851
0.945
0.934
0.924
0.885
0.935
0.942 '
0.909
                                   22

-------
Table 2.  Influence of UV-B irradiation on mycelial growth of five pathogens.
          Mycelia were irradiated in Petri dishes in an incubator at 22 C
          under an FS20 fluorescent sunlamp filtered with either 5 mil
          cellulose acetate or 5 mil Mylar.
-2
rnWm BUV
Mylar- 5 mil Cellulose acetate-5
Disease organism
No.

days
< 0.25

irradiated
Colletotrichum lagenarium



Percent of Mylar Control
Mycosphaerella melonis



Percent of Mylar Control
Alternaria solani





Percent of Mylar Control
Stemphylium botryosum




Percent of Mylar Control
CladosDorium cucumerinum




Percent of Mylar Control

4
6
8


2
3
4


2
3
4
5
6


3
5
7
11 V


3
5
7
11


22.8
34.0
40.5


45.0
64.0
82.3


29.8
40.0
50.3
60.5
71.2


28.7
41.5
55.7
. 78.0
*

18.2
32.9
47.5
76.0

6.25

Colony

17.5
27.0
35.3
87

44.0
62.0
79.7
97

29.3
39.0
49.7
60.7
70.7
99

14.8
23.2
41.0
72.5
93

12.7
25.0
37.2
59.5
78
6.69

diameter

15.5
26.0
35.0
86

44.5
62.7
80.8
98

29.3
38.7
49.3
59.5
70.2
99

14.0
21.7
39.3
. .68.3
88

14.0
24.8
36.0
58.5
77
8.06

- mm

14.0
26.3
35.8
88

43.2
60.7
78.3
95

28.8
38.8
48. ,8
59.0
69.7
98

13.5
22.3
37.7
.68.3
88

13.8
23.7
34.2
57.7
76
mil
10.52



12.8
24.0
29.3
72

42.7
60.2
76.2
93

28.7
37.5
48.5
59.3
69.7
98

13.3
18.3
33.3
65.0.
83

13.2
23.3
34.3
55.5
73
                                  23

-------
         growth period did not differ greatly from colonies receiving no UV-B.

         As with spore germination, Colletotrichum appeared to be most sensitive to

         relatively high UV-B irradiance levels used.  Cladospgrium and Stemphylium

         were intermediate in response, while Alternaria and Mycosphaerella showed

         little .if any reduction at the highest UV-B Irradiance level used.  However,

         with all species, mycelial density was visibly reduced when compared to the

         Mylar controls.  Attempts to obtain dry weights of colonies revealed

 .        differences between CA-filtered and Mylar-filtered colonies, but the method

         used was insufficiently sensitive to distinguish between CA-treatments.  We

 *        conclude that, as with spore germination, relatively high UV-B irradiance

 ?        is required before, the growth of these fungi is impaired.

 i        Cucumber

 {             Growth and disease responses of cucumber to UV-B radiation are shown as

 *       'follows:  Colletotrichum,  Figures 12 through 17; Mycosphaerella, Figures 18

 '•        through .20; and Clad o s porium,  Figures 21 through 23.   Growth responses of the

 f        uninoculated control plants, as measured by fresh weight, dry weight, and
 I
 :        area of first leaf,  responded similarly in all cucumber disease experiments,

 i        showing increased repression with each increase in UV-B irradiation level

 r.        applied.
 \
 I             Disease severity of Colletotrichum and Myco sphaerella on cucumber

 j        decreased with increasing UV-B irradiances.  This is  shown graphically for

 ''        Colletotrichum (Figure 12) for which percentage of diseased leaf area is plotted

 |        against UV-B irradiance levels.  Pictorial representation of the response is

 if        presented in Figures 16 and 17.  Figure 16 depicts the disease response in -the

 \        absence of UV-B enhancement (Mylar control)-'  In Figure 17, disease
v   .                                   -•      •    ' • .
(        response is compared to uninoculated/controls subjected to high and low
•i
'.        levels of UV-B irradiances.  The differences in disease response are obvious.
                                              24

-------
         Leaf  Area. -Diseased, %
       .30
                           5
                       •Weighted mWm
                                            -2
 Figure ..12,  ..The effect of .increased  UV-B .irradiance on the percent
'-'-. ••'- -'  —•   '.diseased 'area of cucumber leaves  infected with Colletotrichum
            '.lagenarium.  .r  _= 0.81,  ^standard  error := _2._2.
                                25

-------
           Fresh  Weight,
          19r
                                              Control
                          5              10
                         Weighted
                              *
Figure 13.  The effect of increased UV-B irradiance on fresh weight of
           Colletotrichum lagenarium  infected (r2 = 0.77,  standard
           'error = 0.63) and rtoninfected  (r2 = 0.03, standard  error =
           0.A3) cucumber plants.
                                  26

-------
       Dry  Weighting
     75ฎ
     5OO
                                             Control
                                   Inoculated
                        5      .       1O
                      Weighted   mWrn"2
.Figure 14.   The effect of UV^B  irradiance on dry weight of
            Colletotrichum Jsagenarium infected (r^  = 0.32,
            standard error = A0.6) and noninfected  (r^ = 0.79,
            standard error = 64.43) cucuciber plants.
                              27

-------
       First Leaf Area,
                                              Control
                                              o
                       Weighted mWm
                                          -2
Figure 15.  The effect  of UV-B irracliance on area of first  leaf of
                             V.O
           Colletotrichum lagenarium infected  (rz = 0.01,  standard
           error = 2.36) and noninfected (r^ = 0.80, standard error
           = 5.74) cucumber plants.
                               28

-------

Figure 16;  Disease response to inoculation of cucumber with Coiletotrichum spores in the




            absence of UV-B (Mylar filtered);  left to right, uninoculated and inoculated




            plants.

-------
   .



   •

XA
         •
               u
          „



                               , •

                                 '
                                    •


                                 .
5
                 30
                                   ;

-------
-Figure :17. .'Disease > response ; to 'inoculation' 'of'cucumber'plants' with  Colletotric'hurn spores




            r sub j ec te'd ; to '-high-arid'low ; level'UV-B'ir radiance.  'Left'to  right:   high UV-B




            juninoeulate'd; I high1' UV-B 'inoculated;  'low'UV-B'uni'riocul'ated;  and 'low UV-B inoculated.

-------
r
              ...

                                                                                                                                                                                                                                     •

                                                  M	   -ป.  -
                                                                                             •
                                                                                    --
                                            -    .

                                               ~
                                                                        .  •
•
-••                  •                     -
                   -    -       .      -
                                                                                                                                                 .
                                                                                                                           -
                                                                                                                                                              •
                                                                                                                                    	,'  :,  ...
v . '-
,
,
•**.*ป*>

-------

.Figure 18.  The  effect of increased UV-B irradiance on fresh weight
            of Mycosphaerella melonis,  infected  (r- =• 0,43.,      .
            errorr= 0.19) and noninfected  (r'2 =  0.91* standard error
            - 0.11) cucumber plants.
       Fresh  Weight,  g
                                        Control
                        Weighted mWm'
                                   33

-------
        Dry Weightjin|

     25%-
     15ฉ
                         5
Fieu-r-e 19.  The. ef-f-ec-t  of- increased- UV-3- irradiance on dry weight of
            Mycosphaerella melonis infected  (r2  =  0.24, standard
            er5oi"'€ 2_0';i)"and noninfected  (r2. =r. 0.89,  standard
            er-r-qr- =?,• 10.95. cuc_umber. plants.._.
                                    34

-------
      Leaf  Area, cm'
      60r
      40
                                          C ontrol
                                   Inoculated
                       5             1O
                     Weighted mWm~2
15
Figure  20.  The effect of  increased UV-B irradiance on area of first
           leaf of Mycosphaerella melonis infected (r  = 0.91, standard
           error = 2.61)  and noninfected  (r  = 0.95,  standard error =
           2.31) cucumber plants.
                               35

-------
       Fresh  Weight, g
         20 r
         16


         12


          8
          O
                                                        
-------
       ^;*~^A*^^                 A^^A
          Dry Weight, g
       2.ฉf
       1.5-
                                              0   Control
                       Weighted  mWm
Figure 22.   The effect of increased  UV-B  irradiance on dry weight of
            Cladosporium cucumerinum infected (r^ = 0.89,  standard
            error = 0.07) andvnoninfected  (r^ = 0.94, standard
            error = 0.07) cucumber plants.
                                37

-------
          Leaf  Area,
                                                  Control
                          5              T&
                       Weighted mWm~2
Figure 23.   The effect of increased UV-B  irradiance on area of  first
            leaf of Cladosporium cucumerinum infected (r  = 0.92,
            standard error = 4.0) and  noninfected (r^ = 0.92, standard
            error = 4.06) cucumber plants'.
                                38

-------
     The decrease  in disease  infectivity and severity are reflected in


area of first leaf and  fresh  and dry weight of both Colletotrichum and


Mycosphaerella.  With both pathogens, the measured growth responses of


inoculated and noninoculated  plants approach unity in the region of 15

   _o
mWra  BUV irradiance.  Colletotrichum and Mycosphaerella both have hyaline


spores.  These disease-UV-B interactions are consistent with the spore


germination and mycelial growth data presented earlier.  They suggest that


disease response to these organisms is due to the direct effect of UV-B


irradiance on the pathogen, although increased resistance to the pathogens


by the irradiated cucumber plants could also be a factor.


     Cladosporium, possessing a pigmented spore, showed no such pathogen-


UV-B interaction.  Observed disease symptomology and measurement of first


leaf area, fresh and dry weight, shox^ed a uniformly reduced growth of


inoculated plants regardless of UV-B irradiance levels (Figures 21 through


23); again, this observation is consistent with the effects noted on spore


germination and mycelial growth.


Alfalfa


     At the UV-B irradiances used, there were no noticeable UV-B effects


on either the Stemphylium pathogen or the host plant,  alfalfa.   Stemphylium


spores are also pigmented.  Table 3 shows the results of these experiments


undertaken in plant growth chambers and indicates that similar lesion types


occurred at all UV-B irradiance levels.   The differences between experiments


were due to harvesting the first experiment 3 days later than the second.


The results indicate that the lesions progressed equally over time under all


UV-B levels.   Again,  this response is consistent with that of other pathogens


having pigmented spores.  Using Uromyces in an experiment with alfalfa grown


in a fiberglass greenhouse,  similar results were obtained and supported our

-------
''"'fa'ble'-3.  nThe"'reff e'ctrroฃ ''-UV-B^Dn' 'disease''development-"of ifche- alfalfa1:pathogen>:':'Stemphyp.ium..bQtryosum
v Wallr
Ml :Biolo'gically
effective
'UV-B
T;''mUm~2BUV
;-2.i4
"•3'.67
'•-5l'!20
6.73
8.26
9.76
." : torv A'r cf ' tultivar •


:;'No.
•1'leaifle'e's/'scoye
'''•"48
''';48
/!;'48
108
240
96
:ofi alfalfa.

Experiment
''Growth
' 'chamber. A
:•' 2
'• 2
/ 2
1
2
2


1
Growth
chamber'.
l 1
: 1
., 2
2
2
1

i 'Lesion score

• Growth
B . : chamber
i 1
2
; 1
1
1
1


Experiment. 2
, Growth
A . chamber. B
i 1
; 2
-, 1
1
1
1

-------
belief that the progression of rust on alfalfa is not likely to be affected




by enhanced UV-B, except perhaps at very high irradiance levels.




Tomato




     Alternaria, a pigmented spore, reacted similarly to Cladosporium on




cucumber, disease severity being unaffected by UV-B irradiance levels




(Table 4).  The differences in dry weight between UV-B irradiances appear




to be more closely correlated with PAR than with UV-B radiation.




                                SUMMARY




     Recognizing that our results represent only a small sampling of leaf




disease organisms and plant disease interactions, they appear to  support




the following: ' 1) considerably higher levels of UV-B irradiances than those




expected from projected decrease in ozone that might be caused by chloro-




fluoromethanes will be required to adversely affect germination and growth




of leaf pathogenic fungi, and 2) where fungal germination and growth are




affected, disease severity in the host plant can be expected to be reduced




as UV-B irradiance increases.
                                  41

-------
Table 4.  The effect of UV-B on disease development of the tomato pathogen,
          Alternaria solani (Ell. & Mart.) L. R. Jones and Groot.

UV-B
weighted
inWnT2BUV
2.5
4.0
5.5
7.0
8.5
10.0




Inoculated
148.5
168.1
190.6
172.6
198.1
131.8
Mean 168.3
r2 < 0.01

Dry weight - mg
yninoculated
177,3
1?2,0
. 211,4
202.9
195,6
162,5
!?0f3
QtP5

-------
                                REFERENCES




Leech, Charles M.  A practical guide, to the effects of visible and




     ultraviolet light on fungi.  Methods in Microbiology.  C. Booth, Ed.,




     Chap. XXIII.  pp 609-664.  Academic Presss London and New York, 1971.






Marsh, Paul B., E. E. Taylor, and L. M. Bassler.  A guide to the literature




     on certain effects of light on fungi:  Reproduction, morphology,




     pigmentation, and phototropic phenomena.  Plant Disease Reporter.




     Supplement 261.  U.S. Department of Agriculture,  pp 251-312, 1959.
                                    43

-------
                                              FILL-
                        FINAL REPORT
       THE EFFECT OF ULTRAVIOLET  (UV-B) RADIATION ON
       ENGLEMANN SPRUCE AND LODGEPOLE PINE SEEDLINGS
                       M. R. Kaufraann

      Rocky Mountain Forest and Range Experiment Station
                     U.S. Forest Service
                 Fort Collins, Colorado 80521
                      EPA-IAG-D6-0168
                      Project Officer:

                      R. J. McCracken
Agricultural Research, Science and Education Administration
              U.S. Department of Agriculture
                Washington, D.C. 20250
                      Prepared for
              Environmental Protection Agency
                      EAGER Program
                   Washington, D.C. 20460

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                                   ABSTRACT
     Engelmann spruce and lodgepole pine are conifers found at high elevations
 (2,700 to 3,300 m) in the central Rocky Mountains.  UV-B enhancement and ex-
 clusion studies were performed at about 2,980 ra on transplanted seedlings.
 Enhancement studies were performed using  standardized light banks consisting
 of  two FS-40 sunlamps in each of two fixtures 60 cm apart and 110 cm above the
 seedlings.  Cellulose acetate and Mylar filters were used along with, an un-
 treated control to provide appropriate UV-B treatments, with seedlings arrayed
 to vary the amount of supplementary radiation.  Treatments extended for 67
 days, with supplementary radiation for a cumulative period of 400. hours.
 Exclusion studies were performed to compare the effects of UV-B in natural
 sunlight.  Seedlings were placed under filters of cellulose acetate or Mylar,
 under lath shade, or in the open to provide different UV-B treatments.

     Careful visual observations of the seedlings by several scientists in-
 dicated no symptoms (color, necrosis, growth form, etc.) for any of the
 treatments, either during the study period or in the subsequent 2-1/2 months
 before the seedlings were covered by snow.  Analyses of growth QLength. of
 terminal leader, number and length of lateral branches) indicated no -major
 effect of treatments, either in the enhancement or in the exclusion study.

     Importantly, however, in the natural environment transplanted seedlings
 of Engelmann spruce do not show symptoms of solar radiation damage until the
 summer following exposure.  Consequently, seedlings are being kept for con-
 tinued observation to determine whether differences among treatments may
 appear during the second growing season, perhaps as a result of effects on
 food reserves and ability to x^ithstand the harsh winter environment of high.
 elevation areas.              *

     This research was done by the U.S.  Forest Service Rocky Mountain Forest
 and Range Experiment Station, Fort Collins,  CO,  under a cooperative agreement
with the Agricultural Research Service,  Beltsville, Mt>. and under an agreement
with ARS and the Environmental Protection Agency.   This report covers the
period of Oct.  1,  1976 to Sept.  30,  1977.   Except for continuing experimental
observations of plants,  this work was completed on Dec. 15, 1977.

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                                        CONTENTS
:          Abstract	,	,  .  ,  .  .      i
          Figures  	 ,,,.ซ,...,    ill
;          Tables	 , ,  ,  ,	,  ,  .   iv

                Introduction 	 ,.,.,,..,,..,,,.,      1
                Conclusions  ,	  i	,      2
                Recommendations  	  .,.....,..,,.,,      3
i                Text	      4
                     General Objectives  	      4
I                     Experimental Procedures  	      4
[                     Results 	      6
'                     Discussion	     14
I                References	     15
i:          Appendix	     16
                                            ii

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                                    FIGURES

Number                                                                 Page

  1    Distribution of seedlings among 7 radiation levels
         beneath the lamp fixtures .....  	  ....   5

  2    Effect of supplemental radiation levels  on vegetative
         growth of Engelmann spruce  	  12

  3    Effect of supplemental radiation levels  on vegetative
         growth'of lodgepole pine  	  13
                                     111

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f
[                                         TABLES

     Number                                                                 Page

|       1    UV-B Radiation Levels at Different Positions Beneath.
I              Lamps, Measured with IRL Spec D Spectroradioroeter 	   7
ป
,l       2    Effect of Natural Sunlight, Shade, and UV-B on Terminal
i              Bud Failure and Seedling Mortality in Engelmann
*•              Spruce and Lodgepole Pine (.Exclusion Study)	   8
            Effect of UV-B Radiation Levels on Terminal Bud
              Failure in Engelmann Spruce and Lodgepole Pine
              (Enhancement Study)	   9,

            Effect of UV-B Radiation Levels on Mortality in
              Engelmann Spruce and Lodgepole Pine (Enhancement
              Study)   	10

            Effect of Natural Sunlight, Shade, and UV-B on Vegetative
              Growth  of Engelmann  Spruce and Lodgepole Pine
              (Exclusion Study)	,	11

            Effect of UV-B Radiation Levels on Vegetative Growth of
              Engelmann Spruce (Enhancement Study)  ,..,,,.,,,.,  16

            Effect of UV-B Radiation Levels on Vegetative Growth, of
              Lodgepole Pine (Enhancement Study)   ,...,....,.,,  17
                                            iv

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                                 INTRODUCTION

     The possibility that the intensity of ultraviolet light (UV-B,  280-320 nm)'
received at the earth's surface will increase is of considerable interest
in the high-elevation forest zone of the central Rocky Mountains,   Ronco
(1970 a and b) concluded that mortality of open-grown Engelmann spruce seed-
lings was related to intense solar radiation, whereas seedlings of lodgepole
pine were not adversely affected by full sunlight.  The possibility  that
solar radiation damage of spruce is caused in part by high levels  of UV-B
stimulated interest in evaluating the effects of increased levels  of UV-B
on growth and development of these species.

     The research reported here was designed to determine the physiological
effects of enhanced UV-B radiation on Engelmann spruce and lodgepole pine and
to establish UV-B tolerance levels for these species in high-elevation forest
ecosystems.

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                                  CONCLUSIONS

     Observations were made of the effects of UV-B on vegetative growth,
foliage color, and morphological development of new foliage of Engelmann
spruce and lodgepole pine seedlings during and after a treatment period
extending 67 days (400 total hours of supplemental UV-B radiation).

     No significant effects of UV-B enhancement or exclusion were observed
during the treatment period or in the subsequent 2-1/2 months before the
seedlings were covered by snow.  It must be emphasized, however, that in the
natural environment transplanted seedlings of Engelmann spruce do not show
symptoms of solar radiation damage until after the first winter.

     Consequently, it is premature to reach conclusions about the impact on
high elevation conifers of UV-B enhancement or exclusion after a single
growing season.

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                                RECOMMENDATIONS
     No effects of UV-B enhancement or exclusion were observed during a
single growing season.

     From an experimental standpoint, it is recommended that observations
on the treated seedlings continue into the second growing season and that
future studies on tree species be long enough to accommodate the perennial,
long-term nature of growth and development.

     In the context of environmental impact of UV-B,  no assessment or
recommendation can be made at this time regarding tolerance levels for
Engelmann spruce and lodgepole pine.

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                                     TEXT
                              GENERAL OBJECTIVES
     Because of  thinner atmosphere, solar radiation is attenuated less at
high elevations  than at low elevations.  In most portions of the solar
spectrum,  including UV-B, intensities are higher at high elevations than
near sea level.  Thus the study of growth and development of high, elevation
conifers was designed to include exclusion of natural UV-B as well as en-
hancement  of UV-B, as might occur through ozone depletion in the stratosphere,

     The field research was divided into two parts.  The. exclusion study was
conducted  to determine if the presence or absence of natural UV-B or a general
reduction  across  the entire solar spectrum had an effect on physiology and
vegetative growth of conifer seedlings.  Simultaneously, an enhancement study
was conducted to determine seedling response to various levels of enhanced
UV-B which might result from ozone depletion,

                            EXPERIMENTAL PROCEDURES

     Experiments were conducted near the Elk Camp Restaurant in the Snowmass
Ski Area,  Snowmass Village, Co. at an elevation of 2,980 m.  Experiments were
performed  using  3-year old Engelmann spruce (Picea engelmannii Parry)  and
2-year old lodgepole pine (Pinus contorta Dougl.) seedlings.  The seedlings
were grown at the U.S. Forest Service Mt. Sopris Nursery located at Carbondale,
CO, about  20 km northwest from the study site and at an elevation of about
2,800 m.   Seedlings were potted June 13-15 and exposed to selected treatments.
                               \
     In the exclusion study, groups of 25 seedlings of each species were
exposed to one of four treatments:  (1)  natural sunlight control; (2)  lath
shade providing 50 percent interception of noon-day sun; (3) natural UV-B,
using a 5 mil cellulose acetate filter; and (4)  excluded UV-B,  using a 5 mil
Mylar filter.  Treatments were replicated, and 200 seedlings of each species
were used  in the study.

     The enhancement study utilized natural sunlight plus supplemental  radiation
from FS-40 lamps.  Seedlings were exposed to three treatments:  (1)  natural
sunlight control; (2)  plus UV-B, consisting of natural sunlight augmented by
FS-40 lamps filtered with  5 mil cellulose acetate;  and (3)  natural UV-B,
consisting of natural sunlight and  FS-40 lamps filtered with 5  mil Mylar.
Both cellulose acetate and Mylar remove wavelengths below UV-B; cellulose
acetate passes UV-B'and Mylar removes  UV-B.   Lamp pairs were located 60  cm
apart and 110 cm above the seedlings.   Lamps were operated for  6 hr. per  day
for 67  days of treatment.   Seedlings were distributed  beneath the lamps  to
provide seven levels (A through G of UV-B  intensity (Fig.  1))..  The  positions

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 were selected  to  provide  supplemental  UV-B  radiation  in  the  following  approxi-
 mate relationship:  A:B:C:D:E:F:G =  0.2:0.4:0.6:0.8:1.0;1.3:1.6.  Nighttime
 measurements with an  IRL  Spec D spectroradiometer  at  each, seedling  indicated
 these relative values to  be  substantially correct  for the seven  radiation
 levels (Table  1).  Thirty-six seedlings  of  each  species  were used in each of
 the  three  treatments,  and all treatments were  replicated.  Six seedlings were
 subjected  to each of  the  five lower supplemental UV-B levels (A  through E) and
 three to each  of  the  two  highest levels  (F  and G).  A total  of 216  seedlings
 of each species were  used in the enhancement study.

      Throughout the treatment period,  observations were  made of  the breaking
 of dormancy of terminal and  lateral buds.   Particular attention  was paid to
 visual observation  of color, deformity,  or  dwarfing of new foliage.  On
 Aug.  22-24, 1977, after 67 days of  treatment,  measurements were  made of mor-
 tality, length of terminal leader (.if  present) ,  and number and lengths of all
 lateral branches.   No records were  made  of  color or of needle length.,  since
 variation  within  treatments  obviously  greatly  exceeded variation among
 treatments.

    .  On Aug. 30,  1977, seedlings were  moved to the Fraser  Experimental Forest,
 CO.  (elevation 2,740 m).  Visual observations  were continued until  mid-November
 when  snow  covered the seedlings.  Additional observations  of mortality and
 color  and  condition of 1977  and 1978 growing season foliage will be made
 during 1978.

                                    RESULTS

     During the 67-day treatment period, no treatment differences of any kind  .
 were  observed  in  rate of  dormancy break  or  in  appearance  of  foliage from the
 new flush  or from the previous growing season.   Failure of the terminal bud
 of Engelmann spruce to break dormancy  ranged from 30  to 52 percent  in the
 exclusion  study (Table 2) and from  25  to 83 percent in the enhancement study
 (Table 3).  In  lodgepole  pine, terminal bud failure was 0  to 8 percent in the
 exclusion  study and 0 to  33 percent in the enhancement study.  No significant
 effects were found among  treatments or supplemental radiation levels in either
 study.  The high bud failure in spruce is not  important, since a lateral bud
 on the terminal leader quickly assumes dominance.  Seedling mortality ranged
 up to 8 percent for spruce and to 17 percent for pine in the two studies
 (Tables 2 and 4), but again,  no treatment or radiation level effects were
 significant.

     Vegetative growth data for the exclusion study are summarized in Table
 5.  No significant treatment effects were observed in terminal leader
 length (measured on plants whose terminal bud broke dormancy), number of
branches,  and  total and mean branch lengths.  Vegetative growth data for
 the enhancement study are given in Figures 2 and 3 (means with, standard
 deviations and statistical evaluations appear in Tables 6 and 7 in the.
Appendix).   Significant treatment effects were observed on total branch
 length in  Engelmann spruce (Fig.  2C) and on  number of  branches in lodgepole
 pine (Fig.  3B).  Supplemental radiation effects were significant only for
mean branch length of Engelmann spruce (.Fig. 2D)  ; however this effect was
also observed in the untreated  control plants and is probably due to random
experimental error.   Treatment  and radiation level effects in the enhancement
                                       6

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TABLE 1.  UV~B RADIATION LEVELS AT DIFFERENT POSITIONS BENEATH LAMPS,
MEASURED WITH IRL SPEC D SPECTRORADIOMETER.  UNWEIGHTED POWER CONVERTED
TO WEIGHTED POWER EQUIVALENTS WITH THE AI9 WEIGHTING FUNCTION.  VALUES
MEASURED AT NIGHT WITH NO NATURAL UV, AT SEEDLING HEIGHT BENEATH LAMPS
WITH 5 MIL CELLULOSE ACETATE FILTERS.

Location
beneath
lamps
A
B
C
D
E
F
G
Unweighted
power
(mW.m )
47..
9.4
17Q
2Q4
273
.385
478
Weighted
power*
(mW.m )
0.44
0.88
1.59
1.89
2.52
3.57
4.34

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TABLE 2.  EFFECT OF NATURAL SUNLIGHT, SHADE, AND UV-B ON TERMINAL
       BUD FAILURE AND SEEDLING MORTALITY IN ENGELMANN SPRUCE AND
	LODGEPOLE PINE (EXCLUSION STUDY)	

                      Terminal bud failure (%)*Mortality (%)
	Treatment	Spruce	Pine	Spruce    Pine

Natural sunlight        32                  10            0        8
  (control)

Lath shade              52                  10            0        8

Natural UV~B            40                   4            24
  (cell, acetate)

Excluded UV-B           30                   020
  (Mylar)

    Significance        N.S.**               N.S,         N.S.      N.S,
*  Includes trees which died.

** N.S. = not significant at P = 0.05.

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t
I


|        TABLE 3.  'EFFECT OF UV-B RADIATION LEVELS ON TERMINAL BUD FAILURE
I           IN ENGELMANN SPRUCE AND LODGEPOLE TINE (ENHANCEMENT STUDY)

I                                        Terminal bud failure (%)*
                                        Supplemental radiation level
         	Treatment	A	B	C	D	E	F	G_

         Engelmann spruce

         Natural sunlight       42**   33     67     33     50     67     67
           (control)

         Plus UV-B (lamp        42     58     58     33     58     33     50
           with cell, acetate)

         Natural UV-B (lamp     25     50     42     67     83     50     50
           with Mylar)

        ** Radiation level and treatment effects not significant (P = 0.05)
         Lodgepole pine

         Natural sunlight        8**    0      0      8      8     17     17
           (control)

         Plus UV-B (lamp
           with cell, acetate)    0      0      8      0     17     17     33

         Natural UV-B (lamp      0888800
           with Mylar)

         *  Includes trees which died

         ** Radiation level and treatment effects not significant (P ••= 0.05)

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TABLE  4.' EFFECT OF UV-B RADIATION LEVELS ON MORTALITY  IN  ENGELMANN
	SPRUCE AND LODGEPOLE PINE  (ENHANCEMENT  STUDY)	

                                       Mortality ~(%T"
                                Supplemental  Radiation Level
     Treatment	A	B      C	D	E	F_

Engelmann spruce

Natural sunlight        0*     0      0       8      8       0
  (control)
Plus UV-B (lamp
with cell, acetate)
Natural UV-B (lamp
with Mylar)
* Radiation level and
Lodgepole pine
Natural sunlight
(control)
Plus UV-B (lamp
080
080
treatment effects

0* 0 0
000
0
0
8
0
not significant

8
0

0
17
0 0
0 0
(P = 0.05)

0 17
17 17
  with cell, acetate)

Natural UV-B (lamp      0888800
  with Mylar)

*  Radiation level and treatment effects not significant (P = 0.05)
                               10

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 TABLE  5.   EFFECT  OF  NATURAL  SUNLIGHT,. SHADE, AND UV-B  ON VEGETATIVE
   GROWTH  OF  ENGELMANN  SPRUCE AND  LODGEPOLE  PINE (.EXCLUSION STUDY) .
              VALUES  ARE MEANS AND STANDARD  DEVIATIONS
    Treatment
Terminal
 leader
 length
   (cm)
 Number
   of
branches
 Total
branch
length
 (cm)
 Mean
branch
length
 (cm)
Engelmann spruce

Natural sunlight
   (control)

Lath shade

Natural UV-B   -
   (cell, acetate)

Excluded UV-B
   (Mylar)

    Significance
4.7+3.0    19.8+10.9   67.4+36.0  3.5+0.8


5.1+2.0    17.4+11.2   60.5+39.1  3.5+0.7

4.6+2.4    17.3+10.3   59.2+33.4  3.3+0.7


5.0 + 2.4    17.1 +  8.0   57.4 + 25.7  3.5 + 0.5
   N.S.*
   N.S.
  N.S.
 N.S.
Lodgepole pine

Natural sunlight
  (control)

Lath shade

Natural UV-B
  (cell, acetate)

Excluded UV-B
  (Mylar)

    Significance
6.4+2.1     7.0+  3.3   15.1+  8.0  2.2+0.8


6.2+2.4     7.8+3.8   15.2+  8.0  2.1+1.0

6.4+2.5     9.3+  4.1   18.5+  8.0  2.1+0.8

      t
6.0 + 2.4     9.5 +  4.9   16.2 +  8.2  1.8 + 0.7
   N.S.*
   N.S.
 N.S.
N.S.
* N.S. = not significant at P - 0.05
                                   11

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    study are judged to be minor and inconsequential for the 67-day treatment
 •   period.
 I
 I                                     DISCUSSION

 i        Most effects of UV-B radiation on higher plants have been observed
 I   on herbaceous species.  Studies on woody species have been very limited,
 |   although Biggs and Murphy (1977) reported damage of several conifers
 Jl   exposed to high UV-B levels shortly after germination.  The effects of UV-B
 ,.;   on tomato were cumulative (Caldwell et alf, 1974),   Hart et al. (.1974) observed
 j   increased branching of chrysanthemums during exposure to UV-B.

 |        From these and other studies, it was anticipated that UV-B treatment
I   effects might appear on new foliage produced during a treatment period exceed-
    ing 2 months.  Based on other research, particular  attention was paid to               i
    stunting of needles and branches,  color of foliage, and frequency of branching
    (e.g. dormancy break of buds and subsequent growth).

         The nearly complete lack of response of Engelmann spruce and lodgepole pine      i'
    to any of the treatments or radiation levels suggests two possibilities.   First,
    neither species may be sensitive to the UV-B levels used during this study.            !
    Secondly, because of the perennial nature of these  species, a treatment and           i-
    observation period confined to a single growing season may be too short for           r
    effects to appear.                                                                     |

         Ronco's (1970 a and b) observations that solar radiation damage of
    Engelmann spruce does not appear until the second growing season suggests
    that the second possibility is more realistic.   Clearly,  it is  premature  to
    conclude that UV-B has no effect on these two species,

         It seems advisable to continue visual observations of the  seedlings  into
    the second growing season, and for this reason the  plants were  moved to the
    Fraser Experimental Forest where studies can be continued more  conveniently.
    It has not yet been decided,  however,  if the plants should be subjected to
    the UV-B treatments again during the second growing season.   It can be argued
    that continual treatment during successive growing  seasons is most  realistic,
    yet to do so would  confound the second year's observations of carry-over
    first-year effects.   High variability  and limited numbers of  seedlings  per
    treatment prevent dividing the plants  into two  groups,  one for  observation
    and the second for  retreatment.
                                         14

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 i
 A
 i
 j.
 }
 |                                    REFERENCES
 i
 .'   Biggs, R. H. and S. K. Murphy.   1971.   Impact of solar UV-B radiation on
         crop productivity.  Third Quarterly Report} EPA-BACER, 7 pp.

    Caldwell, M. M., W. B. Sisson,  F.  M.  Fox, and J. R.  Brandle.   1974.
 j        Plant growth response to elevated UV irradiation under field  and
 3        greenhouse conditions.   Impacts  of Climatic Change on the Biosphere.
 1        CIAP Mono. 5, pp. 4-253 to 4-259.

j   Hart, R. H., G. E. Carlson,  H.  H.  Klueter, and H,  R,  Cams,  1974,
 1        Responses to economically  valuable species to ultraviolet radiation,
j        Impacts of Climatic Change on the Biosphere,  CIAP Mono,  5,  pp.
         4-263 to 4-273.

    Ronco, F.  1970a.  Chlorosis of planted Engelmann  spruce seedlings unrelated
         to nitrogen content.   Can. J.  Bot.   48:851-853.

    Ronco, F.  I970b.  Influence of high  light intensity  on survival of planted
|         Engelmann spruce.  For. Sci.  16:331-339.
                                         15

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APPENDIX

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TABLE 6.  EFFECT OF UV-B RADIATION LEVELS ON VEGETATIVE GROWTH OF ENGELMANN SPRUCE (ENHANCEMENT STUDY)
                              VALUES ARE MEANS AND STANDARD DEVIATIONS
Radiation Level
1 Treatment
Terminal Leader Length (cm)
Natural sun (control)
Plus UV-B (cell, acetate)
Natural UV-B (Mylar)
A

3.9 +
2.8 +
4.1 +
No significant differences (P
Number of Branches
Natural sun (control)
Plus UV-B (cell, acetate)
Natural UV-B (Mylar)
24.2 +
19.3 +
16.1 +
No significant differences (P
Total Branch Length (cm)
Natural sun (control)
Plus UV-B (cell, acetate)
Natural UV-B (Mylar)

71.9 +
57.7 +
50.5 +


2.1
1.7
2.6
= 0.05)
8.8
8.8
8.9
= 0.05)

30.0
23.6
28.4
B

3.8 +
3.1 +
3.3 +

22.6 +
14.7 +
21.3 +


72.4 +
43.9 +
60.6 +


2.4
2.6
1.8

9.1
8.9
7.9


33.9
25.3
24.0
. Natural sun significantly different from plus UV-B
Mean Branch Length (cm)
Natural sun (control)
Plus UV-B (cell, acetate)
Natural UV-B (Mylar)
Significant radiation

3.0 +
3.1 +
3.3 +
effect

0.6
0.9
1.0

3.3 +
3.0 +
2.8 +
(P = 0.05) ; no

0.7
0.6
0.3
C

2.0 + 1.5
3.9 + 2.9
4.9 + 2.1

17.0 + 9.3
19.0 + 6.6
14.9 + 9.8


49.6 + 27.1
58.2 + 18.4
47.1 + 32.6
and natural

3.0 + 0.6
3.2 + 0.6
3.1 + 0.5
D

2.4 + 1
4.2 + 2
3.0 + 2

18.3 + 5
18.0 + 8
16.2 + 8


59.5 + 21
55.5 + 29
49.7 + 27
UV-B (P = 0

3.2 + 0
3.1 + 0
3.1 + 0


.8
.2
.2

.7
.1
.5


.7
.0
.2
.05)

.8
.5
.8
E

3.0 + 2.0
2.1 + 2.1
5.3+ 0.7

18.4 + 8.9
12.6+ 5.8
20.3+ 6.8


63.8 + 29.3
41.5 + 22.4
64.7 + 17.0
F

6.2 + 1.9
4.1 + 1.2
2.4 + 2.3

24.5 + 13.4
15.5 + 7.7
10.8 + 5.0


84.1 + 41.4
45.5 + 25.6
37.1 + 18.4
; no significant radiation

3.5 + 0.5
3.2 + 0.7
3.3 + 0.4

3.6 + 0.6
2.9 + 0.6
3.4 + 0.5
G

4.3 +
6.3 +
6.2 +

17.3 +
13.7 +
19.3 +


60.4 +
44.3 +
68.0 +
effect

3.7 +
3.8 +
3.6 +


0.6
1.0
2.0

12.3
12.5
11.1


39.5
35.5
41.4


0.9
1.0
0.7
significant treatment differences.


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TABLE 7.  EFFECT OF UV-B RADIATION LEVELS ON VEGETATIVE GROWTH OF LODGEPOLE PINE (ENHANCEMENT STUDY)                                                     {
	VALUES ARE MEANS AND STANDARD DEVIATIONS	•_•	;	                  i

                                                                        Radiation Level                                                                   [

	Treatment	A	B	C	D	E              F              G                             |


Terminal Leader Length (cm)                                                                                                                              |


Natural sun (control)        6.6 +  2.1     5.7 + 2.9      5.0 + 2.3      5.1 + 1.6      6.3 +  1.7     4.7 +  1.3     5.5 +   2.0                        j


Plus UV-B (cell, acetate)    5.7 +  2.8     5.5 + 3.0      4.9 + 1.7      6.1 + 2.0      5.6 +  2.9     4.8 +  2.7     5.4 +   3.0                        !


Natural UV-B (Mylar)         5.1+  2.5     4.5+1.7      5.3+2.1      5.4+2.4      5.0+  2.2     5.8+  3.7     5.1+   2.0                        f


     No significant differences (P = 0.05)                                                                                                               t
                                                			                   l


Number of Branches                                                                                                                                       I
Natural sun (control)        6.9+  3.8   ^6.2+3.2      7.3+3.7      9.4+4.7      8.7+  3.9     9.8+  5.6     9.2+   3.6

Plus UV-B (cell, acetate)    8.7 +  3.1     9.2 + 3.3      7.5 + 4.3      8.8 + 3.0      7.5 +  3.4     8.6 +  3.1     8.0 +   4.7

Natural UV-B (Mylar)         6.5+  3.3     7.2 + 2.7      6.2 + 2.9      5.5 + 2.3      8.4+  4.8     6.2+  2.6     6.2+   2.8

     Natural UV-B significantly different from natural sun and plus UV-B (P = 0.05); no significant radiation effect



Total Branch Length  (cm)

Natural sun (control)       16.0+  6.8    13.3+6.9     15.3+8.9     15.5+6.3     15.9+  5.8    16.7+14.7     17.1+   6.8

Plus UV-B (cell, acetate)   15.6 +  6.5    16.4 + 7.3     13.5 + 6.1     16.3 + 6.7     16.5 +  8.5    14.2 +  8.2     13.9 +  11.0

Natural UV-B (Mylar)        14.0+10.0    12.3+4.6     13.0+6.6     12.4+7.0     15.6+11.0    13.2+  7.1     10,7+   3.7

     No significant  differences (P = 0.05)



Mean Branch Length  (cm)

Natural sun (control)        2.5+  1.0     2.2 + 0.5      2.0 + 0.5      1.8 + 0.5      2.1+  0.8     1-5+  0.5     1.9 +   0.5

Plus UV-B (cell, acetate)    1.8+  0.6     2.0+1.3      2.5+2.2      1.9+0.6      2.3+  0.8     1-7+  0.9     1-9 ฑ   1-6

Natural UV-B (Mylar)         2.3+  1.1     1.8 + 0.7      2.1 + 0.8      2.2 + 0.8      1.8+  0.5     2.1+  0.8     2.0+   1.0
                                                                                                                                     i
     No significant  differences ( P = 0.05)                                                                                         1^

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