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QO FORM 1473, 83 APR EDITION OF I .IAN 73 IS OBSOLETE.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