ON PAGE

-Is REPORT SEC%..RIT'1 CL.ASS ME.JM2IU 297 r7AICTIVE MARK~INGS

Unclassified ii111iiinH

2o. SECURITY CLASSIFICATIC liii1 IN I II liii III II I1111 IIBUTION/AVAI 1.ARILTY OF REPORTived for public release;

ý.,zibution unlimited2b. OECLASSIFICATION/OOWNGRAOINQc UPILE'

4,PERFORMING ORGANIZATION REPOR?'* Y~s S MONITORING ORGANIZATION REPORT NUMBER(S)

AFOSR.TR. ~ <'1

6&. NAME OF PERFORMI1NG ORGANIZATION ~b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATIONtII'nPplica bl).

Alabama ALM University . AFOSR/NL

6c. ADDRESS (City State and ZIP Cadet 7b. ADDRESS lCiy. Staue and ZIP Code,

'110 Duncan Avenue, Suite B115

P.O. BOX 14004, Huntsville, AL Bolling AFB DC 20332-000.l35815

&a. NAME OF FUNOING/SPONSOAING 8b. OFFICE SYMBOL 9. PROCUREMENT' INSTRUMENT IDENTIFICATION NUMBERORGANIZATION Air Force OIf applicabl) AOR 462-1i06

ýOffice of Scientific ReseaCh N PS: 46091C06Sc. ADDRESS lCity. Stage and ZIP Code) 10. SOURCE OF FUNDING NOS. _____________

PROGRAM PROJECT TASK WORK .. NiT

110 Duncan Ave. Suite B1.15 ELEMENT NO . NO. NO. NO

Boiling A F B, DC 20332-000111. TITLE tinclude Se r ClauAficat In 61103D 3484 RS*Effects ofeWAlocente'd hy drocarbon

aquattc orgl ni~sms ____________ __________I12. PERSONAL AUTHORISC Nahasin G. Tadros

~13s. TYPE OF REPORT 13b. TIME COVERED -i.J&17g3 1 14. DATE OF REPORT (Yr.. Mo.. Day) 15. PAGE COUNT

Annual Tecuical FROM 8/L1/92 T08/l/93 1993, August 30 I 45

116. SUPPLEMENTARY NOTATION

17 COSATI CODES 18l. SUBJECT TERMS (Continue on rUevese If neceucery and identify by block nui

FIELD IGROUP I sue. GR.

* BSRAT CatLa Algae-Halogenlated hydrocarbons

19. ABTATlotneon reverse if necessary and identitfy by block numbert

This report summarizes progress for the first year of the subcontract, AFOSR F49620-

91-C-0063 entitled "Effects of Halogenated Hydrocarbons on aquatic organisms'.

This research dealt with several experiments evaluating the reponse of different algal=

species towards selected haloganated hydrocarbons.Two groups of algal species were

assayed. The response of the algal species towards the chemical was evaluated under ~ __

various growth medium composition. With respect to changes in the growth medium

composition it was clear in this work that depletion of nutrients nitrogen or phosphate in ()__

case of green algal species or silicate in the case of dia-toms lowers the percentage of

survival of the organism. Green algal species were more or less tolerant to changing

growth media composition than diatoms. In conclusion, when bioassaying the

halogenated hydrocarbons, various algal species as we~l as growth medium

composition should be considered.2'o. DiSTRiBUTION/AVAILABILITY OF ABSTRACT 21. ABSTRACT SECURITY CLASSIFICATION

JNCLASSIFIED(UNLIMITEO (5SAME AS APT. 0 OTIC USERS 0 Unclassified

22s. NAME OF RESPONSIBLE INDIVIDUAL 22b. TELEPHONE NUMBER 22c. OFFICE SYMBOL

tlncludEr A4#m Code)

Dr Walter J. Kozumbo 1(202) 161-5021 AFOSR/NL

QO FORM 1473, 83 APR EDITION OF I .IAN 73 IS OBSOLETE. _______________

19 .SECURITY CLASSIFICATION OF TH-S PAGE

93 101 2 24'

I

Effects of Halogenated Hydrocarbons on Aquatic Organisms

First Annual Technical Report

to

Air Force Office of Scientific Research

Contract AFOSR: F49620-91-C-0063

August 30, 1993

Mahasin G. Tadros

Biology DepartmentAlabama A & M UniversityNormal, Alabama 35762

SUMMARY

This report summarizes progress for the first year of the subcontract, AFOSR F49620-

91-C-0063 entitled "Effects of Halogenated Hydrocarbons on aquatic organisms".

This research dealt with several experiments evaluating the reponse of different algal

species towards selected haloganated hydrocarbons.Two groups of algal species were

assayed. The response of the algal species towards the chemical was evaluated under

various growth medium composition. With respect to changes in the growth medium

composition it was clear in this work that depletion of nutrients nitrogen or phosphate in

case of green algal species or silicate in the case of diatoms lowers the percentage of

survival of the organism. Green algal species were more or less tolerant to changing

growth media composition than diatoms. In conclusion, when bioassaying the

halogenated hydrocarbons, various algal species as well as growth medium

composition should be considered.

2 ------- -

S. . .. .

9J

TABLE OF CONTENTS

SUMMARY ........------------------------------------------------ 2

INTRODUCTION --------------...---- ..............---------.-.------- 4

OBJECTIVES ----------------------.-- .............-------------- 5

MATERIAL AND METHODS ----------------------------------------------------- 5

RESULTS & DISCUSSION -------------------------------------------------.--- 8

CONCLUSIONS -.--------------- ..................--------------------- 12

FUTURE PLANS -.-.--- ..............------------ ----......--------- 13

REFERENCES .....------------------------------------------------- 14

FIGURES ---------- ---------------------------------------------- 15

PERSONNEL ....--------------------------------------------------- 45

3

INTRODUCTION

This report summarizes progress for the second year of the subcontract, AFOSRF49620-91-C-0063 entitled 'Effects of Halogenated Hydrocarbons on aquatic

organisms.Chlorinated hydrocarbons are natural components of oil deposits and commonlyfind their way into surface waters as a result of discharges from refineries, wasteoil, disposal, and accidental spills. Municipal wastewater discharges have alsobeen recognized as sources of aliphatic and aromatic hydrocarbons (Barrick,1982). Chlorinated hydrocarbons may enter the environment as a result of theiruse as solvents, heat transfer fluids, flame retardants or chemical intermediatesor as waste products of the elector-industry (Jan and Malnersic, 1980). Amongthe most common solvents used form halogenated hydrocarbons are:trichloroethylene, tetrachloroethylene and dichloroethlene. These compoundsare among the dominant contaminants detected in gound water (Barber et. al.,1988: Love and Eilers, 1982). Organic solvents can make their way into theenvironment as industrial wastes. Because of their carcinogenic potential,contamination of soil and water by solvents is cause for serious concern.

Relatively few reports have been published on the comparative toxicity ofsolvents towards tests organisms, and these dealt primarily with fish and aquaticinvertebrates (Alexander et. al., 1878: Bouman et. al., 1981: LeBlanc andSuprenant, 1983). However, only few data of toxicity effects of solvents on algaehave published (pearson and McConnell, 1975; Lay et. al., 1984; Stratton,1987).

Algae have been considered to be good indicators of bioactivity of industrialwastes (walsh et. al., 1984). Algae are ubiquitious in aquatic ecosystems, wherethey incorporate solar energy into biomass, produce oxygen that is dissolved inwater and used by aquatic organisms, function in cycling and mineralization ofchemical elements, and serve as food for herbivorous and omnivorous animals.When they die, they sink as food for herbivoruous and amnivorous animals.When they die, they sink to the sediment where their chemical constituents aretransformed, solubilized, and recycled into the water. These functions aredependent upon phytoplankton population dynamics which, in turn, depend

4

upon seasonal variability in temperature, intensity of solar radiation, nutrientconcentrations in the water, and grazing by animals. Natural and anthropogenicalterations of water, and grazing by animals. Natural and anthropogenicalterations of water quality can upset the balance of these controlling factors-andbring about changes in species composition of the algal community, rates ofproduction, biomass, and water chemistry. If water quality is altered by toxicantsor growth stimulants from industrial, agricultural or municipal sources, normalalgal function may be upset, causing gross changes in structure and function ofthe receiving aquatic ecosystem.

OBJECTIVE:

During the second year of the project the following studies were performed inorder to:.Compare the response of fresh water and saltwater (estuarine) single algalspecies, to different concentrations of the halogenated hydrocarbons, underdifferent growth parameters: nutrients and salinities

EXPERIMENTAL DESIGN:

The response of algal species to chemicals was determined at 20 0 C and 30 0 C,under two light irraditions : 80 and 120 uEm- s-1

MATERIALS AND METHODS:

Algal species:Assays were conducted with freshwater and saltwater algal species:Fresh water Green :

Gleocystis sp., Tetraselmis sp., Chlorella sp.,Nannochloris 3 sp., Selenastrum capricomutum sp.,

Nannochloris sp., Scendesmus basilensis sp., and Chloroccus sp

Salt water (estuarine): Diatom :Cyclotella, Nitzschia pusilla, Navicula saprophila, Nitzschia dissipata,

Thalassiosira weissflogii, Skeletonema, Amphiprora hyalina, Thalassiosira

5

pseudonana, Cyclindrotheca, Cyclindrotheca, Chaetoceros muelleri, and

Minutocellus sp.

All algal species were obtained frorm the University of Texas algal

collection (UTEX). The algal species were checked for bacterial contamination

before use.

Culture Medium:

OF/2" Guillard and Ryther (1962)

Macroelements: (concentration mM/L medium)NaN0 3 0.88 NaH2PO4 0.036 Na 2SiO 3 0.107

Trace Metals (concentration uM/L medium):Zinc 0.08 Copper 0.04

Manganese 0.90 Iron 11.70

Cobalt 0.05 EDTA 11.50Molybdenum 0.03

Vitamins: (concentration ug/L medium)

Cyanocobalmin 0.05

Biotin 0.05

Thiamine, HCL 100.00

The culture medium was used for all species.For marine species, the mediumwas enriched with commerical artifical sea salt mix (Instant ocean, aquarium

system, Inc. East Lake, OH.) to 20 parts per thousand (ppt) salinity.

Distilled water was used for preparation of media. The pH of media was adjusted

to 8.0 with sodium hydroxide.

Inoculum:Inoculations were prepared with cultures in log growth phase, obtained by

frequent replinishment of medium. Cultures were acclimated to the growth

conditions of the treatment for 72 h prior to the exposure by maintaining thegrowth rates constant. The initial inoculum was standarized to 7 x10 4 cells/ml. in

all treatments.

Culturina

All cultures were performed in triplicate in sterile optically matched tubes.

Cultures were incubated on shakers in incubators at one temperature (300 C)

6

under one light irradiation (80 uE m-2 s-1), in light-dark cycle (16hr.light: 8hr.dark).Chemicals for tesin:The following volatile halocarbons were tested:

Carbon Tetrachloride, Chloroform, Trichloroethylene and Tetrachloroethylene.

Test compounds were ordered form. J.T. Baker Chemical Co.

Concentrations and Treatments:All test organisms were assayed in water-solubale fractionconcentrations of 0.05, 0.1, 0.2, 0.3. The 100 % solution was preparedby adding part of chemicals to 100 parts dilution water (volume tovolume) and stirring in a covered glass bottles with Teflon-coating-lined screw caps for 2 hours. After allowing the solution to settle forlh, the water-soluble fraction was siphoned into another containerfor distribution to the test containers. The assay was carried out intubes containing 25ml medium. All assays were conducted intriplicate test tubes.

All algal cultures were treated with different concentartions of the halocarbon.The concentration of halocarbons was not measured, because the gas liquid

chromatograph was not yet operated.

Growth MonitorinM:Cultures were incubated for 96 h. The population density was determined by cellcounting using a hemacytometer. Ten microscopic fields were counted andaveraged.Responses of species were estimated by:

A. Population density measured by cell counting using Hemacytometer.From population density the growth rate (u) of each species was

calculated from the expression:u = log 10 N - log 10 No

t - toWhere:

N = population density at the end at the testNo= population density at the beginning of the testt - to = length of time of the test

B. Toxicity was calculated in percentages of the control

7

QUALITY CONTROL AND STATISTICS:Culturing media were sterilized by autoclaving before treatment withhydrocarbons. All glass used for experiments were also sterilized byautoclaving. The temperatures of autoclaves were monitored on a per-use basis.Spectrophotometers, pH meters, and analytical balances were calibrated on aregular basis. All glassware (pyrex) were cleanedusing 1% HCL followed byrinsing thoroughly with deionized water.The triplicate tests analyzed at each

parameter (e.g. Temperature, salinity...) each test was performed twice. All errorswere expressed as the standard error of the mean (SEM) Occupational safetyand Health Administration (OSHA) regulations regarding the safe handling ofchemicals and safety of personnel were followed.

RESULTS AND DISCUSSION:

The chemicals were tested, after being dissolved in acetone as we proposed inthe proposal.We find that acetone, alone stimulates the growth more than thecontrol.Therefore the chemicals were dissolved in water at very lowconcentration (see methods).

Response of species to solvents under different growth conditions.

I. Growth Media Composition:A. Green species:

1. Gleocystis sp. (Figure 1)The response of the alga was more or less the same to all chemicals in all mediaexcept trichloroethylene was some what affected by the deficiency fo P or N,when compared with its effect in complete medium. The survival rate of the algawas higher in complete media than media depleted in N or P.

2. Tetraselmis sp. (Figure 2)The survival rate of the alga was less in the media deficient in P or N than in thecomplete medium especially in tests treated with 0.1% solvents

8

3. Chlorella sp. (Figure 3)The survival rate of the alga was not affected by changing the composition of the

medium because the alga was sensitive to the solvents when tested in complete

medium

4. Nannochloris 3 (Figure 4)

The alga was sensitive to changes in the nutrients of the medium at

concentration 0.05% Increasing the concentration of the solvents lowered the

survival percentage of the alga

5. Nannochloris sp. (Figure 5)

The survival rate of the alga was sensitive to changes in the nutrients of the

medium at concentration 0.15%

6.Scenedesmus sp. (Figure 6)

The survival rate was more affected by the medium nitrogen deficiency than

phosphate deficiency

7. Selenastrum sp. (Figure 7)

The survival rate was reduced in media deficient in P and N. It was clear media

deficient in N affected the survival rate more than P.

8. Chlorococcus sp. (Figure 8)

The survival rate was not affected by changing the medium nutrient

concentrations. However at concentration 0.15% the depletion of N from the

growth medium affected the survival rate than the other media

Comparison of the chemicals in terms of medium conditions:

Comparison of the chemicals (Figures 9,10,11) in terms of the response of the

green species to Carbon tetrachloride, Chloroform, Trichloroethylene and

Tetrachloroethylene under growth conditions

The survival rate of all species was reduced in all treatments especially in media

deficient in nitrogen

9

In case of green algal species it should be concluded:N deficient media was 3nective in reducing survival rate

B. Diatoms

1. Cyclotella sp. (Figure 12)The survival rate of the algal was not affeted by P and N deficiency

2. Nitzschla pusilla sp. (Figure 13)The growth rate was affected in media deficient in N specially treated withchloroform and carbon tetrachloride

3. Navicula saprophila sp. (Figure 14)The species was not affected by depleting the media from Si or N

4. Nitzschla dissipata sp. (Figure 15)The survival rate of the alga was affected by Si and N defficiency in the growthmedium

5. Thalassisira weisflogii sp. (Figure 16)The survival rate of the alga was lowered in media deficient in Si and N

6. Skeletonema costatum sp. (Figure 17)The survival rate of the alga was affected by Si and N deficiency

7. Amphiprora hyalina sp. (Figure 18)The survival rate of the alga was not affected by changing the nutrient of themedium.

8.Thalassiosira pseudonana sp. (Figure 19)The survival rate of the E ',a was reduced in media deficient in N

9. Cylindrotheca sp. (Figure 20)The survival rate of the alga was reduced in media deficient in Si and N

10. Chaetoceros sp. (Figure 21)

10

The survival rate of the alga was not much affected by changing the nutrients of

the medium

11. Minutocellus sp. (Figure 22)

The survival rate of the alga was not much affected by changing the nutrients of

the medium

Comparison of the chemicals in terms of growth conditions:

comparison of the chemicals (Figures 23, 24,25,26) in terms of the response of

the diatoms species to Carbon tetrachloride, Chloroform, Tetrachloroethylene

and Trichloroethylene under growth conditions.

Carbon Tetrachloride: (Figure 23)Nitzschia dissipata, Skeletonema and Minutocellus were more sensitive to Si

and N deficiency than the other species and as a result their survival rate was

lowered

Chloroform: (Figure 24)

When species treated with the chemical in growth media deficient in Si and N,

the survival rate of Nitzschia dissipata and Thalassiora weissflogii were lower

than the other species.

Tetrachloroethylene: (Figure 25)Thalassiosira weissflogii, skeletonema, Amphiprora, Thalassiosira pseudonana,

Cylindrotheca, and Chaetoceros: The mentioned diatom species were sensitive

to nutrient deficiency.

Trichloroethylene: (Figure 26)

Nitzschia dissipata and Thalassiosira weissflogii although they were tolerent of

the solvents in complete media, they were sensitive and their survival rate was

reduced in the media depleted from Si and N.

11

Il. Salinity:DIATOMS: (Figure 27, 28, 29, 30)

The salinity did not change the survival rate of the diatoms in response to salinityhowever, they responded at different salinities sometimes even higher thanlower salinity. The reason for that is that some of the diatoms grow at different

salinites some of them require 18g/L others require 36g/L and others require54g/L for growth.

Conclusion:

• with respect to changes in the growth composition it was clear in this work thatdepletion of nutrients nitrogen or phosphate in case of green algal species orsilicate in the case of diatoms lowers the percentage of survival of the organism.

. Green algal species were more or less tolerant to changing growth mediacomposition than diatoms.

12

FUTURE PLANS:

We will continue to investigate the effect of halogenated hydrocarbons on the

aquatic organisms in the following experiments:.The effect of the chemicals will be assayed in growth media complete and

deficient in one element (nitrogen as nitrate, phosphorus as phosphate or silican

as silicate)..The response of algal species to the chemicals will be determined in the

original medium after being enriched with various sea salt concentrations 15, 25or 35 ppt (parts per thousands).

.The above experiments will be performed with mixed species.

. The accumulation of the solvents will be determined by the algal species using

Gas liquid Chromatography.

13

REFRENCES

Alexander HC, McCarty WM, Bartlett EA (1978) Toxicity ofperchloroethylene, trichloroethylene, 1,1, 1-trichloroethane, andmethylene chloride to fathead minnows. Enviorn Contam Toxical 20:344-352.

Barber LB, Thurman EM, Schroeder MP (1988) Long-term fate oforganic micropollutatns in sewage-contaminated groundwater.Environ Sci Technol 22: 205-211.

Barrick RC, (1982) Flux of aliphatic and polycyclic aromatic hydro-carbons to central Puget Sound for Seattle (Westpoint) primarysewage effluent: Environemntal Science and Technology, V. 16, p.682-692.

Jan J, Malnersic S, (1980) chlorinated benzene residues in fish inSlovenia (Yugoslavia): Bullentin of Environmental Contamination andToxicology, v. 25, p. 824-827.

Lay JP, Schauerte W, Klein W, Korte F 19784 Influence oftetrachloroethylene on the biota of aquatic system. toxicity to phyto-and zooplankton species in compartments of a natural pond. ArchEnviron Contam Toxical. 13: 135-142.

LeBlance GA, Surprenant DC (1983) The acute and chronic toxicity ofacetone, dimethylformamide, and triethylene glycol to Daphniamagna (Strauss). Arch Environ Contain Toxicol 12: 305-310.

Love OT, Eilers RG, and Jr (1982) Treatment of drinking watercontaining trichloroethylene and related industrial solvents. J AmWater Works Assoc. 74: 413-425.

Pearson CR, McConnell G (1975) Chlorinated C and C hydrocarbons inthe marine environment. Proc R. Soc Lond 189: 305-332.

Stratton GW (1987) Toxic effects of organic solvents on the growth ofblue-green algae. Bull Environ Contam Toxicol. 44 (accepted).

Walsh GE, Merrill RG (1984) Algal bioassays of industrial and energyprocess effluents, p. 329-360. In: L.E. Shubert (ed) Algae as ecologicalindicators. Academic Press, New York, NY.

14

.8 GLEOCYSTIS0 250

z0 200 COMPLETE MEDIA * c,,Tw,

o 1 TISO Na ym

C3 100I-z

Ww 5

a. 0

0.05 0.1 0.15

GLEOCYST71-.h 2500I-z 200 P-DEFICIENCY0C.)

u.150U0Il ' 100

CL 500.05 0.1 0.15

S20 GLEOCYSTISo 250Wzo 200 N-DEFICIENCY

mL.o: 150

Lu

a 100zLU

0 o-Lu 0L~tu

0.05 0.1 0.15

Concentration

Figurel: Effect of chemicals on growth of green alga Gleocystis sp. in completeand deficient media as a percentage of the control. Standard deviation did notexceed 2%

15

TETRASEL MIS-j 250

O1 Dcmomftm. 2

z COMPLETE MEDIA

0 IOI yweloiuwh

L• 150 *] TebmAhm4~my•

0

0 100 .4CIP-z

I~l. 0-

0.05 0.1 0.15

TETRASELMIS

..J 250

I-- S200S20P-DEFICIENCYC.,

u. 1SO0'U03 100 -I,,-z'U so0

&uIL 0

0.05 0.1 0.15

TETRASELMIS-I 2500I-z 200 N-DEFICIENCY0C-.)

U. 1500

'U0 100

LUI.-

Z'.' 50L bkaB. 0

0.05 0.1 0.15

Concentration

Figure 2: Effect of chemicals on growth of green alga Tetraselmis sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

16

S250CHLORELLA0: 250 13 ' ID w.,

C.) 200 COMPLETE MEDIA

•)150 fl Tamch,•ys,,,w

( 00

L. 150

CL 0 0.05 0.1 0.15

CHLORELLA

150

0 250CLOEL

Z 200 P-DEFICIENCY0

.150

a~ 100

z

0.0S 0.1 0.15

250 CHLORELLA

ex0e 2%O

0 N-DEFICIENCY

LL150.

100

0.05 0.1 0.15

Concentration

Figure 3: Effect of chemicals on growth of green alga Chiorella sp. in completeand deficient media as a percent-,,: of the control. Standard deviation did notexceed 2%

17

NANNOCKLORIS-J 25020

z 200 * -COMPLETE MEDIA

L. 150 -

0

0 100 T

z0,)'•InuIIq Iso -

U

0.05 0.1 0.15

NANNOCHLORIS_j 2500

z 200 P-DEFICIENCY0

IL. 150

0

100

s -

0.06 0.1 0.15

NANNOCHLORNS.j 250

0

I-.Z 200 N-DEFICIENCY0

IL0 150

0100

zU0 5

C L 00.05 0.1 0.15

Concentration

Figure 4: Effect of chemicals on growth of green alga Nannochloris 3 sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

18

NANNOCHLORIS-. 250 oaZ 200 -0TA-abo COMPLETE MEDIA Tug

' 13

0OI10:0~~.•I-10 50

0.05 0.1 0.15

.-j NANNOCHLORIS0 250I--z0 200o P-DEFICIENCY

IU.0150

4C

P-100UI- 10

'U 50

00.05 0.1 0.15

NANNOCULORISo 250I-.z0 200 N-DEFICIENCY0U-o 150

oc 100I-z'U

IL S00.05 0.1 0.15

Concentration

Figure 5: Effect of chemicals on growth of green alga Nannochioris sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

19

SCENEDESHUS BASILENSMS-J 2SO

Z0 200

Z00 COMPLETE MEDIA *T~~m~~0

u. ISO0

C3 100

z su 0w o

WA" 0.05 0.1 0.15

.$ SCENEDESMUS BASILENSiS0 250

o -z0 200

- P-DEFICIENCY

1. 1 5 0

0Lu

, 100 -

zuJ 50

0.05 0.1 0.15

SCENEDESMUS BASILENSIS2250

S200 N-DEFICIENCY0U

0

100

Ia. 0.05 0.1 0.15

Concentration

Figure 6: Effect of chemicals on growth of green alga Scenedesmus sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

20

SELENASTRUM CAPRICORUTUN

-j 2500Q 0

I.- NoCOMPLETE MEDIA N TWM06"MM

5 Te•h~u~Wbf

0

L"

1 100

z'Li

- 150

uLI

00.05 0.1 0.15

SELENASTRUN CAPRICORUTUN

0I.-Z 200 P-DEFICIENCY0QuL.o 150'U

4 100.-z

'U

'LI

0

0.05 0101

SELENASTRUM CAPRICORUTUN

.j 2S0- z -- NDEFICIENCY

0

'U 1004

I- o -

W 50LU

a_ 0.05 0.1 0.15

Concentration

Figure 7: Effect of chemicals on growth of green alga Selenastrum sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

21

CHLOROCCUS-.J 250 Cmsem

0

01 COMPLETE MEDIA

0 0.05 0.1 0.15

-j CHLOROCCUS0 250

z P-DEFiCIENCY0 200

0

U.)0 ISO0 100 f

so -

LU

00.05 0.1 0.15

CHLOROCCUS-J 25O0

I-= 200 -N-DEFICIENCY

0

LU

150

Lu

0.05 0.1 0.15

CONCENTRATION

Figure 8: Effect of chemicals on growth of green alga Chlorococcus sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

22

CARBON TETRACHLORIDECOMPLETE MEDIA

250 ao . o.00- U - % I N C. 0.10 200 2

V- ~j-a 21 *co.n1s

IS

* 150

ono

CC

4I) CY50 caQ

0)

CARBON TETRACHLORIDE hiP-DEFICIENCY 00250 Co._o

200 -- I , cco 1!€'

m 150 0C.

U)C

u 100 a) 0

00)0

so

00uCD

iijilili .1_ 0 m

0 (D)CARBONTETRACHLORIDE 00N-DEFICIENCY U )

260 cc- C1

250

cus

OSO

co) 100

o sa

I0-

Concentration

23

CHLOROFORMCOMPLETE MEDIA

250 .. j 13 cow Om

I- 200kZ * Cm o.l

0

S100

" " 50 oJ Ia,-

IIo ('-

*- x

a)0

CHLOROFORM --

E -P-DEFICIENCY 0 C

250 00

200 It

o 150 -ISOm

00

100 0

0UU

00o (0 (D

- I-CHLOROFORM 0 a)N-DEFICIENCY 0.0

250 d:.C

100

"• so a

Concentration

24

TETRACHLOROETHYLENECOMPLETE MEDIA

250

Cc Mal

so

150)

00

UB

a.~

E00

TETRACI4LOROETHYLENE EVCP.DEFICIENCY 00

250

20

IS 150

o~0~

* 0

1000

0) cco*r Co

TETRACI4LOROETHYLENE C

N-DEFICIENCY a).2S a:-S2000 "2.

~100

0

Concentration

25

CYCLOTELLA.250 CW.m

Z 200 COMPLETE MEDIA

LL. 1500

rr

I 100

z

u504

C.)

*, 0 0.2 0.25 0.3

CYCLOTELLA

-j250

Z 200 -SI-DEFICIENCY

0u. 150

LUla 100

S50

C. 0.2 0.25 0.3

CYCLOTELLA0 250

13 C ~rooMI-- N.DEFICENCY E CW.e.W.Z 2000

150 0 TdaworV"b,

U. DT ni0 ISO0

ILl

100

I--

L .so 0

'U

0.2 0.25 0.3

Concentration

Figure 12: Effect of chemicals on growth of diatom, cyclotella sp. in completeand deficient media as a percentage of the control. Standard deviation did notexceed 2%

26

NITZSCHIA PUSILLA0 250 ,

z0 2O00U COMPLETE MEDIA ,

fl! T..,Ilmethy14wm

U.o 150

'U

, 100I--z

U 50

00.2 0.25 0.3

NITZSCHIA PUSILLA--J 2500I--Z 200 SI-DEFICIENCY

0 u. 150

UJ0 100

'U M

'U

0.2 0.25 0.3

NIZSCHIA PUSILLA250

,cc

z u N-DEFICIENCY0

U-

NILLHAPUlL

0U- 100 N1 50

'U0 SOILl

0. 0.2 0.25 0.3

Concentration

Figure 13: Effect of chemicals on growth of diatom, Nitzschia pusilla sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

27

NAVICULA SAPROPHILA-1 250

0~ a Ca.,.MmIý-Z COMPLETE MEDIA

0L 150 .T.tftuieUym

0

a 100

z

ILlL 0o0.2 0.25 0.3

NA VICULA SAPROPHILA-I 2500ccI.-Z 200 SI-DEFICIENCY0

UL

05 100

z0 5

C.LU

0.2 0.25 0.3

NAVICULA SAPRROPHILAO 250

CO 200 N-DEFICIENCY

U.o 150ILu

0Ix 100I.-zuLJ(€, 50so -

-Li OAkUU0.2 0.25 0.3

Concentration

Figure 14: Effect of chemicals on growth of diatom, Navicula saprophila sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

28

NITZSCHIA DISSIPATA

-0 250o 0 C€ f•i

z 200 COMPLETE MEDIAo U TdetemlwhIu.•

U. 1501.aaTia•m~hvWe0

zJ 50

a. 0

0.2 0.25 0.3

NITZSCHIA DISSIPATA.I

0 250I-zo 200 SI-DEFICIENCYU.)

o 150

Uii

0 100I.-zC.)

•. 00.2 0.25 0.3

NITZSCHIA DISSIPATA

.J 2500I-

z 200 N- DEFICIENCYrC.)

I. 1500

C. 100I--z

'UCL 0

0.2 0.25 0.3

Concentration

Figure 15: Effect of chemicals on growth of diatom, Nitzschia dissipata sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

29

THALASSIOSMRA WEISSFLOG5

- 250 13I-

U COMPLETE MEDIA U ThI_ * Taemhismwyem.,o '150L.I

,• 100I--

zLu

C.)LU

0.2 0.25 0.3

THALASSISIRA WEISSFL0011.. 2500I.-Z 20000U SIL-DEFICIENCYu_150

ILu

100

zLUoJ 50

L 0

0.2 0.25 0.3

"THALASSISIRA WEISSFLOGII0Wr 250I--zo 0N-DEFICIENCYU.0 150

LUa 100

-. SOk Ek a

0. 0.2 0.25 0.3Concentration

Figure 16: Effect of chemicals on growth of diatom, Thalassisira weisfiogii sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

30

SKELETONEJA COSTATUM-j 250o 0 CNisudsrcc•b.- 0 Cm4wmwd~mei0 200 COMPLETE MEDIA * TdaIeawhmU•) 1,50 ]Tm ,, ,,

0UA100

I-

C.,

W

UJ AI-z

20o SIL-DEFICIENCY

0

Ui

- 250[

p-

0 200 N-DEFICIENCY

Ui

oU 150 71W

Q. 0.2 0.25 0.3

Concentration

Figure 17: Effect of chemicals on growth of diatom, Skeleonema costatum sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

31

AMPHIPRORA HYALINA250

z 20 o ~COMPLETE MEDIA *LL.

o 150

LU04C 100z

so 5

ai 9L 00.2 0.25 0.3

AMPHIPRORA HYALINA250

0re-

z 200 SI-DEFICIENCY0

0

UJ

0 100

z

W• 5o

ILl

0.2 0.25 0.3

ADIPHIRORA HYALINA

2502

z 200 N WDEFICIENCY

15

'Ii0 100

U1 s

0.2 0.25 0.3

Concentration

Figure 18: Effect of chemicals on growth of diatom, Amphiprora hyalina sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

32

-j0 THALASSI$1RA PSEUDONANAW 250

z0 200 COMPLETE MEDIA c,,0U

2100 •

z

) 150 [ ~ m

CL 0.2 0.25 0.3

THALASSIOSIRAPSEUDONANA-j 2500- 200 SI-EFICIENCY

THLS.SR PIESONANZ) 200 SN-DEFICIENCY

0

0

z 50

0j0.a. 0.2 0.25 0.3

THALASSISIRA PSEUDONANA

I--Z 200 N-WDEFICIENCY

0

a 100

0 0.2 0.25 0.3

Concentration

Figurel 9: Effect of chemicals on growth of diatom, Thalassiosira pseudonanasp. in complete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

33

CYLINOROTHECA0 250 [ CMO,,.g

0 200 COMPLETE MEDIA

0 150

IA-

q 100zwC)• 50LU

010.2 0.25 0.3

CYLINDROTHECA0 250IxI.-zo 200 Si- DEFICIENCYC)IL.0 150

uJ

.q 100I--zLUC) 5o

LUCL o[A

0.2 0.25 0.3

CYLINDROTHECA2500

o 0 N-DEFICIENCY

LL0 150

0100

a.-

0 so

0.2 0.25 0.3

Concentration

Figure 20: Effect of chemicals on growth of diatom, Cylindrotheca sp. incomplete and deficient media as a percentage of the control. Standarddeviation did not exceed 2%

34

CHAETOCEROS MUELLERI250 CMuuetum

0 [ Cw4mdome

200 * T1SM1111" snCOMPLETE MEDIA Tai,,,ilI,,0

0

a. 1001

zw 50

L oa- 0

0.2 0.25 0.3

CHAETOCERO MUELLERI_j 250

3-200Z) SI-DEFICIENCY

lu 150

.

Aus 0.2 0.25 0.3

IL

CHAETOCERO$ NUELLERI-250

zma

I--z0 200SN-DEFICIENCY

U.

0' ISOI-o 150a 100

z

0.2 0.25 0.3

Concentration

Figure 21: Effect of chemicals on growth of diatom, Chaetoceros sp. in completeand deficient media as a percentage of the control. Standard deviation did notexceed 2%

35

MINUTOCELLUSz 20

0 200 COMPLETE MEDIA *C)

0 15 r Telmbmaftytuia15'U04 100_9.-z

0 0

0

0.2 0.25 0.3

MINUTOCELLUS. 250

cc

0

I--

Z 0

150LU0 S100

I,-

zLU

0.2 0.25 0.3

HINUTOCELLIJS0 250

ILL0 150LU0I-- 100zU.ILU

LU

0.2 0.25 0.3

Concentration

Figure 22: Effect of chemicals on growth of diatom, Minutocellus sp. in completeand deficient media as a percentage of the control. Standard deviation did notexceed 2%

36

CARBON TETRACHLORIDECOMPLETE MEDIA0: 2Sq 0, •! C=mO..

S/iii! I i --- I.

z -o

o 200

= m I i *i f 1 ._2

o. Us '' t _<

C.,., ILL M-DEowIE0.30

100 0WU 0

cr.)

Co

250 al

-L. -

2 50 - u , ):

0 S%go

uJ S

1 00 00

0 .. ..04).

00 r

0 '

CARBON-TETRACHLORIDE 0R

307

N-DEFICIENCY C

o) 2002 h. )

ID 2005za0C

0 150 iD

100

IL 0

CONCENTRATION

37

CHLOROFORMCOMPLETE MEDIA

2501

0 t) *c.omaa

AU0 10

500

AUU

0i 00

-a -

E00

CHLOROFORM OCVSI-DEFICIENCY oC

200

z LU

0ot0U0

a0100 0

4i 00

a. Cu-Ln.0

0

0.0

250(

0150 cc C 0)

2 000

00

Concentration

38

TETRACHLOROETHYLENE

20COMPLETE MEDIA

25u. cmoco

0 0.

'U'

0~00

SI-FCINY4

250 "0)

2000

0 c

ISo -ouiOCL

25 .2-0

Cr. so 0

IL CmU) 0

500 a)0)

(Di

TETRACHLOROETHYLENE C

N-DEFICIENCY c250 LO0)

3 200 S CF)a)

100

Cm

IL

Concentration

39

TRICHLOROETHYLENECOMPLETE MEDIA

260

a 13 I

0 50

b- 200 Co k u

CD 100•

C C

AD

oo

1500

o so

100100

CD

0

40:

TRICHLOROETHYLENE Q CSI.DEFICIENCY r

250

200 4E-

IS - 0

1000

50 Eu

00

000)0

TRICHLOROETHYLENE CCN-DEFICIENCY OW

250 (- ~0)0

co-

SMC)

0 0

400

CARBON TETRACHLORIDESALINITY:I6 g _/L250

o so Ca.0.

ARBO TERAHORDE V.l

- 100 " )

o X

s- s

0

CARBON TETRACHLORIOE .-

SALINITY: 36 giL ug >,2150

I0

0 a) &i-100 - E

0 00

~0)

CARBONTETRACHLORIDE-SALINITY: 54 gIL W

20-F "

41\

g &co OW

100 Y-LLw

Concentration

41

CHLOROFORM

250 SALINITY:ll 9/L

_ •q I ! •

0 2004 L m 0 * .3

0 1ISO33

U) 100 I t - I i

C

so 04

CHLOROFORMSALINITY: 36 VIL 0

a 0200 E .

-~Cu

co1 00 E C''D

o 0150 L,

I 1 0

CHLOROFORMlW250 SALINITY: 54 9& _ E.. E -

• -• 100 n

2-=

UUu

Concenratio

4(D

.) 200 0 to

IO

C q L

1a 0) c

C6 0)100S

0.LLC

Concenratio

42

TETRACNLOROETHYLEMESALINIY: 18 gil.

b- 0 Camef

200 -3-

9i C a- a .

40 100 4

so

TETRACHLOROETHYLNEESALINITY: 36 giL._____

250

0 200~ -

o~~~ 15 U)~~E

.2 100 -C O

00

00TETRACHLOROETHYLENE 0SALINIY: 54 gIL 0-

5 250 (

aa r

o 200~ 3~

B 100

a S100

C u

0

Concentration

43

TRICHLORETHYLENE260 SALINITY: II g/L

C 200 it ! G-

150 *Cog 0.N

sooCE a a)

&X

0:5 0

TRICHLORETHYLENE -SALINITY : 3i g/.

250 a00

200 -

150 ,

E"

100 100

o soo

Q50 0

a.

0 0.0)

TRICHLORETHYLENE 0SALINITY: I OIL C

S 250 0c

'nco 200 ~ 8

s CdCV)

so c

Concentration

44

PERSONNEL

The following personnel have been involved in this project:

Mahasin G.Tadros, Ph.D. Principal Investigator

Janelle Phillips, M.s.Havovi Patel, B.s.

Vinod K Pandiripally, B.s.

45