Rila sea salts dissolved in distilled water was used in all studies at a salinity of 15 ppt unless
otherwise noted.

Microcosms were incubated in the dark at 25 C unless noted.

To sample the sediment in the microcosm, the entire contents were thoroughly mixed with a
mechanical stirrer. The desired amount of sediment was then removed with asterile wide tip pipet.

All dilution water blanks were made with artificial sea water diluted to the same salinity as the
water in the microcosms being sampled. Incubation was carried out for 10 days at 25 C. The base
medium was composed of 0.5% peptone and 0.1% yeast extract prepared with artificial sea water of the
desired salinity and modified by the addition of appropriate materials. Solidified media were
prepared by the addition of 2% agar (Difco). The conventional spread plate technique was employed
with incubation under air for aerobic counts and incubation under an atmosphere of 95% N,-5% CO,
for anaerobic counts. The spot plate technique employed for determining starch-, gelatin-, casein-,
lipid-, and chitin-utilizing populations was conducted as follows: Three 0.01 ml portions of each sam-
ple were spotted on plates of media. After incubation, the plates were developed, and the number of
positive reactions out of the three possible were recorded at each dilution. Numbers of organisms were
calculated using a standard three-tube MPN table.

The standard three-tube MPN procedure was employed for estimating the numbers of sulfate-
reducers, organisms capable of producing H,S from organic sulfur, nitrate-reducers and am-
monifiers.

Tests on dehydrogenase activity were carried out as follows: Two g portions of sediment were
weighed into glass centrifuge tubes. Fifty to 100 mg of CaCO, powder were added to each tube and,
with the exception of the blank, all tubes received 0.5 ml of a 1% solution of triphenyl tetrazolium
chloride (TTC) prepared in 15 ppt artificial sea water. Samples were mixed, placed in a vacuum desic-
cator, evacuated for 10-15 min, and incubated under vacuum at 30 C. At the end of 22 h the vacuum
was released and 10 ml of methanol added immediately to each tube. Samples were mixed and cen-
trifuged at low speed for 2-3 min. The supernatant then was filtered through a cotton plugged funnel
into a 50 ml volumetric flask and the methanol extraction procedure repeated using 10 ml amounts of
methanol until color could no longer be extracted from the sample. The filtrate was made to volume (50
ml) using methanol. The color intensity was measured at 485 nm on a B&L Spectronic 20 colorimeter
using the blank as a reference. Results were calculated from a standard curve and expressed as 73
tripheny] tetrazolium formazan (TPF) per g of sediment.

Studies on the oxygen consumption by organisms in sediments were conducted using conven-
tional Warburg manometric techniques with glucose as a substrate. Sediment samples were amended
with 1000 ppm carbofuran and incubated. This mixture was subsampled after 5 and 12 days incuba-
tion and 2.5 g wet weight was used in each Warburg flask. The salinity was 15 ppt and temperature
was 25 C.

For the glucose mineralization experiments, the procedure employed was a modification of
Harrison et al.}? The major change involved the amount of C-14 glucose added (0.1 pi per bottle) and
the incubation time (1 h).

Proteolytic activity studies were monitored in terms of the change in soluble protein concentra-
tion within a sediment slurry to which peptone had been added as the protein source.

Five g of sediment and ten ml of sea water (15 ppt) containing 10 mg of Bacto-peptone was placed
in 60 ml prescription bottles and incubated with shaking for 22h at 25 C. Theslurry was centrifuged at
high speed to separate the liquid from the cells and sediment. The supernatant was checked for pro-
tein content using the method of Lowry et al.!*

1


Table 33. MEAN BACTERIAL COUNTS (x 104) IN SEDIMENTS AMENDED WITH 100 PPM CARBOFURAN AND
HELD UNDER VARIOUS CONDITIONS OF SALINITY AND TEMPERATURE.

Incubation 15 ppt -10C 15 ppt - 21C 15 ppt - 30 S ppt - 21C 30 ppt - 21

period
after 8 s 5 § s
esticid 5 a 5 5 5 Ey
added. . 3 g £ & $ & 3 & 3 &
a o g ory o g
(days) e bet g 4 g 2 g 2 ¢ 2
8 é 8 8 & 8 8 8 iS) &
0 113" 167° 91 123° 96 147° 34 55 ql 105
4 442 638° 662 636 722 1075 645 589 532 530
9 743 880° 962 1009 933 1416° 1066 1227 807 660
16 802 1344° 892 1300° } 1155 14875 798 1359° 748 2053°
22 868 1463° | 1177 [5086]° | [3885] 1438° 710 2116" 966 = 1392°
30 680 1335° 980 1326" 608 1242° 745 1498° | 1080 © 1374°
44 972 1601" | (8300) 2013? 918 620° 3236 932° | [5638] [10275]
a

- Mean of three replicates with five plates counted on each replicate

{] - Poor agreement among replicates which differed by one log in count.
~ Treatment counts significantly different from control counts at 95% level as deter-
mined using t-test.

- Treatment counts significantly different from control counts at 99% level as deter-
mined using t-test.

Table 34, DEHYDROGENASE ACTIVITY IN SEDIMENTS TREATED WITH DIFFERENT PREPARATIONS AND CONCENTRATIONS
OF MIREX.

Addition

to sediment Incubation period after pesticide added

14 days 21 days 28 days
TOOOppa-=== =
Untreated 228 180 _ 190 a 127
Technical mirex 20 (0.1 182 (Ns) 174 (Ns) 155 (NS) 106 (.05)
Purified mirex 22 (NS) 174 (Ns) 196 (NS) 169 (NS) 139 (NS)
— — — ---100 ppm- - - ~ --

Untreated 23 164 137 22 172
Technical mirex 24 (NS) 165 (Ns) 211 (.001) 191 (NS) 181 (NS)
Purified mirex 25 (NS) 207 (.001) 244 (.001) 231 (NS) 213 (001)

Untreated 21 108 98 115 88
Technical mirex 23 (NS) 119 (NS) 105 (NS) 54 (001) 102 (.01)
Purified mirex 24 (.10) 158 (.01) 127 (.01) 139 (.05) 134 (.001)
manera n enna 1 ppm-—- wane nn nena nana
Untreated 31 152 158 150 104
Technical mirex 43 (.001) 181 (.01) 162 (NS) 151 (NS) 158 (,001)
Purified mirex 34 (Ns) 157 (Ns) 198 (.10) 148 (NS) 158 (.001)

@ Mean dehydrogenase activity (ug of TPF produced per gram of wet sediment) from six replicates.
> Significance level of difference between treated and untreated samples as determined by the t-test.
NS means not significant at the 0.1 level.

32


Table 27. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS FOUR WEEKS AFTER AMENDING WITH MIREX.

Amended with mirex

Control 0-1 ppm T0 ppm 1000 ppm 70,000 ppe

Microbial group #/nl “Tank Tank Tank Tank Tank Tank Tank” Tank Teak Teak
x 1 16 2 Uv 13 18 14 19 15 20

4.62 1.1 13.0 3.2 14 2.4 7.9 3.3 Le wy

Nitrate reducers roo 4.9) 2.2 1.8 11.0 9 0.5 33 21 4.9
Ec Ok 0.8

20 7 ua 7.9 0 2 1.34 0.30 LL

samontfers 5 4.9
HyS producers ro 4.9 24.0 17 1.9 3.3 3.3 2.2 4.9 2. 2.4
Ta 08S EOS OT, 0.8

Sulfate reducers rot 17 0.3 0.7 0.8 0.8 0.2 0.5 0.8 0. 0.5
$979.0 17-0 17.0 3.3 —-%6.0 3.3 ~—~33.0——T T.0

Lipid hydrolyzere 10) 4.9 79.0 «13.0 24.0 24,033.09 38.07 79.0
To 3.3 17.0 2.0 7.00.8 16 3.3

casein hydrolyzere wd 4.9 7.9 33.0 17.0779
33.0 24.0 130.0 240.0 33.0. 7.9 3,3 47-0 4.9 24.0

Gelatin hydrolyzers yo? 13.0 13.0 79.0 240.0 «33.0769 240 7933.0
Zl 4,9 3.3 0.8 3.3 0.8 1.7 0.6 1.32.4

Starch hydrolyzers 1054.9 2.2 3.3 3.3 4.9 1.3 1.3 28,0 2.4 17.0
a 210.7 1.10.7 20.5

Chitin hyérolyzers ao 0.4 0.2 2.3 14 0.2 0.8 1.2 0.5 0.8 17
19.8 25.4 47.0 62.0 15.0 8.38.7 9.5 7.1 ‘11.2

Aerobic plate count 106 18.4 23.0 57.0__59.0_216,7_—S6 976 7B BT 13H
20.3 101.0 130.0 158.0" 50.0 38.0 34.0 31-0 v 30.0

Anaerobic plate count 10 5 16.1 87.0 135.0 141.0 64.0 34.0 «32.0 24.0 36.0570

@ Replicate counts
Missing value

Table 28. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS IMMEDIATELY AFTER AMENDING WITH CARBOFURAN.

Amended with carbofuran

Control 0.1 ppa TO pea 1000 ppa 10,000 ppm

Microbial group #/ot Tank Tank Tank Tank” Tank Tank” Tank Tank Tank Tanke

x 25 26 27 28 29 30 31 32 33 34
Nitrate reducers 10° 3.304908
Anmoni fers 10° 29 La 1.3 ao 7.0 LT 7.0 Led 1711.0
HS producers 20> 1.3. 0.3 1.3 0.5 0.2 0.3 0.5 0.5 La 0.5
Sulfate reducers ao* L307 7.9 24 0.8 8
Lipid hydrolyzers 10° 49 49 49 2.4 49 4.9 24 49 3.3 1.3
Casein hvdrolvzers 10° 292k 8S 8D
Gelatin hydrolyzers 10° 0.8 7.9 24.0 2.2 33 OB
Starch hydrolvzers 10° Oe 49 3
Chitin hvdrolvzers 104 LL? 0.7 3.3 2.3 2.3 0.9 17.0 49 3.3 3.4
Agrobic Plate count 108 73 76 66 56 ST
Anaetobie plate count 10° 29 324 26 2 2B PST



Table 40. BIOACCUMULATION STUDIES OF MIREX BY CULTURE #260 (BENECKA).

Sample # Test system Total mirex Percent
in Liquid remaining
(ug) in liquid
1 200 ml Mineral salts
2 200 ml Mineral salts
3 200 ml Mineral salts + 1% glucose
4 200 ml Mineral salts + 1% glucose
3 200 al Mineral salts + 5% celle
6 200 ml Mineral salts + 5% cells
7 200 ml Mineral salts + mirex +
1% glucose 20.14 100
8 200 ml Mineral salts + mirex +
1% glucose 22.22 100
9 200 ml Mineral salts + 5% dead
cells + wirex + 1% glucose 0.94 4
10 200 ml Mineral salts + 5% dead
celle + tirex + 1% glucose 13h 6
sty 200 ml Mineral salts + 5% live
cells + mirex + 1% glucose 2.7L 2
22 200 ui Mineral salts + 5% live
cells + mirex + 1% glucose 271 12

DERIVATIVES OF MIREX AND CARBOFURAN

As reported previously, the inhibitory effect of mirex (technical) varied from batch to batch and
was erratic even when multiple tests were conducted using the same batch. Purified mirex samples
never exerted an inhibitory effect when tested using the disc assay technique. There was reason to
believe that contamination arises in aged mirex samples since older samples of mirex were toxicto the
greatest number of bacterial cultures.

Since hexachlorocyclopentadiene is the starting material for mirex synthesis, this compoundisa
potential contaminant in the mirex. However, disc assay tests failed to indicate any inhibition with
the 20 bacterial cultures tested when it was added to mirex at a concentration of 1:1000.

Chemical analyses of technical grade mirex demonstrated the presence of hexachlorobenzene
(HCB) in some of the mirex samples. However, this compound was not found to be toxic to any of the
20 bacterial cultures as determined by disc assay tests.

Since Alley et al.3 have identified 10-hydrogen; 5,10-dihydrogen; 8-hydrogen; and 2,8-dihydrogen
derivatives of mirex as photodegradation products, and Ivie, Dorough, and Alley reported kepone to
be a degradation product of mirex, these compounds along with reduced kepone were tested againsta
number of bacterial cultures using the disc assay technique. Neither the 8 hydrogen derivative of
mirex nor the 2,8-dihydrogen derivative of mirex inhibited any of the 10 cultures tested. The 10-
hydrogen derivative of mirex and the 5,10-dihydrogen derivative of mirex inhibited all 5 of the five
cultures tested, while the kepone and reduced kepone both inhibited 10 of the 20 cultures tested. It is in-
teresting to note that neither of the two products with substitutions on the 2 and 8 positions of the
mirex molecule were inhibitory, while the four products with substitutions on the 5 and/or 10
positions of the mirex molecule were highly toxic to the test microorganisms. Whether these com-
pounds were actual contaminants in the mirex employed in this investigation is unknown.

37


tacyclodecane, 4 were prepared from mirex by photochemical methods (Alley et al.° ‘). 1,2,3,4,6,7,8,9,-
10,10 decachloro-5 hydroxy-[5.3.0.02 -6.0%. 9.04: §] pentacyclodecane, 5 was prepared by the method of
Dilling®. (See Figure 2)

Medium

Unless otherwise noted, all media used in this study were obtained from Difco Laboratories and
prepared with Rila sea salts mixture to obtain 15 ppt salinity. The mineral salts medium contained 1.0
g KNO,; 0.5 g K,HPO,.3H,0; 0.2 ¢ MgSO ,e7H ,0; and 0.05 g FeCl e6H ,O per 1000 ml distilled water
(final pH adjusted to 7.0).

Cultures

Pure cultures of estuarine bacteria were obtained from the stock collection at Gulf Coast Research
Labortory, Ocean Springs, Mississippi. Methanomonas methanooxidans (Brown and Strawinski),
Mycobacterium, and Nocardia were obtained from stock collection at Mississippi State University.
Sediments used for the study of methane consumption and nitrification were obtained from Biloxi
Bay, Mississippi.

Figure 1, Carbofuran and derivatives

a
| ci

CHy

we cH,

°

ocn#3
Wh
OH

Carbofuran, Ry = Ry H
3-hydroxycarbofuran, Ry = H, Ry = OW

3-ketocarbofuran, Rj, Ry 0


14.

15.

16.

Pamatmat, M. M. and A. M. Bhagwat. Anaerobic Metabolisms in Lake Washington Sediments.
Limn. and Oc. 18:611-623, 1973.

Anonymous. U. S. Dept. H.E.W. Washington, D.C. Pesticide Analytical Manual 1:211-212, 1973.

Ivie, G. W., H. W. Dorough and E. G. Alley. Photodecomposition of Mirex on Silica Gel
Chromatoplates Exposed to Natural and Artificial Light. J. Agr. Food Chem. 22(6):933-935,
1974.

47


SECTION VII
DISCUSSION

The following paragraphs describe briefly the rationale whereby the conclusions were drawn.
from this study.

Mirex is known to be very insoluble in water; also, it is quickly adsorbed onto particulate matter
and ultimately settles to the bottom where it is not subject to photochemical degradation. It resists
decomposition and is virtually inert under normal environmental conditions. Therefore in nature, in
the aqueous environment, mirex will not remain long in the water column but may have an ap-
preciable lifetime in the sediments.

While carbofuran is not readily adsorbed, it has been shown that more than 75%of the carbofuran
in an aqueous environment disappears in a short time probably due to hydrolysis to the phenol.
Therefore, buildup of this pesticide is not likely.

The only microbiological activity appreciably effected by either mirex or carbofuran at a level
below 100 ppm was the inhibition of primary productivity. As a result of the low water solubility and
the high rate of depletion by adsorption in the case of mirex and the rapid disappearance of car-
bofuran by other mechanisms, neither are expected to have any significant effect on primary produc-
tivity in the environment since the majority of the phytoplankton are in the aqueous phase, rather
than in the sediments and most of the material would be attached to particulate matter prior to en-
trance into the water.

While 3-hydroxycarbofuran has been shown to be toxic in concentrations as low as 1 ppm, this
derivative was not produced from carbofuran in the fate studies. Although degradation products of
mirex have been shown to be toxic at concentrations of 1 ppm, they were not found in detectable quan-
tities in the fate studies. In another study *¢ of mirex under environmental conditions, the yields of
degradation products were extremely low. This low conversion, coupled with the probability that the
degradation products may be more biodegradable than the parent compound, leads to the expectation
that they will not cause problems in the environment.

Highly significant was the finding that purified mirex was not toxic to microorganisms while
some batches of technical mirex and UV irradiated moist mirex were toxic. It may be concluded that
other studies on the toxicity of mirex should be re-evaluated. This is especially true in view of the fact
that one of the breakdown compounds studied in this investigation has the same retention time as
mirex and because the concentrations of degradation products that would cause observable toxicity is
such that they would not be detected using normal gas chromatographic procedures. All of these com-
pounds are more polar than mirex and should have higher water solubility than mirex. These facts
may be important factors in toxicity studies in water.

45


Table 48. DEHYDROGENASE ACTIVITY IN SEDIMENTS TREATED WITH DERIVATIVES OF MIREX.

Addition Period after pesticide added
to sediment
Initial 7 days 14 days 21 days 28 days
Untreated 28 180 190 a7 v7
wing derivatives" 19 (.05) 168 (NS) 184 (NS) 193 (NS) 166 (.01)
of mirex

Untreated 23 164 137 212 a2

“wing derivatives" 24 (NS) 205 (.001) 222 (001) 260 (.02) 224 (.001)
of mirex

Untreated a 108 98 11s 88

“wing derivatives” 19 (Ns) 150 (.001) 129 (.001) 108 (Ns) 144 (.001)
of mirex

Untreated a1 12 158 150 104
“wing derivatives" 32 (NS) 147 (Ns) 205 (.01) 204 (.01) 176 (.002)
of mirex

@ Mean dehydrogenase activity (ug of TPF produced per gram of wet sediment) from six replicates.
NS means not significant at the 0.1 level.

43,


Adsorption Studies with Carbofuran

Two independent studies were made on carbofuran adsorption to clay. In the first, mont-
morillonite clay was employed and the analyses conducted by ultraviolet adsorption at 276 nm. The
apparent adsorption was about 10-15% after 35 days (Table 3). In the second study a solution of 189

Table 3, ADSORPTION STUDIES OF CARBOFURAN ON MONTMORILLONITE CLAY

Time

Absorbances

days 0 ug/ml 0.06 ug/ml 0.13 ug/ml 0.6 ve/ml
clay clay clay clay
1 1 - 0.84 a
2 0.93, - 0.87 -
3 ~ 0.85 0.84 0.87
7 i 0.93 0,84 0.87
4 ccs 0,92 0.86 0.91
au ut 0.96 0.89 ccs
28 + 0.98 0.82 0.83
35 + 0.95 0.83 0.83

ppm carbofuran in 0.05 M sodium acetate-acetic acid buffer at pH 5 was treated with acidified

kaolinite clay such that the clay concentration was 1200 mg/1 (Table 4).

Table 4, CARBOFURAN ADSORPTION ON KAOLINITE CLAY

Time

% Carbofuran remaining
in solution

36 min
81 min

180 min

240 min

9h - 46 min
21h

28h - 26 min

54h

100

Apparently about 25% of the carbofuran is very rapidly adsorbed under this set of conditions, and

then the system is at equilibrium and no further adsorption is observed.

Fate Studies with Mirex

The analytical method employed in this study had a coefficient of variation of about 25% Table5
lists the data obtained on disappearance of mirex from an estuarine sediment - water system. To this
system 0.1 ppm mirex was introduced. A blank control consistently had undetectable levels of mirex.

15


Table 35. DEHYDROGENASE ACTIVITY IN SEDIMENTS TREATED WITH DIFFERENT PREPARATIONS AND CONCENTRATION OF CARBOFURAN.

Addition to Incubation period after pesticide added
sediment

Initial 6 days 13 days 20 days

---Amended with 100 ppm--~

Untreated 138 89 57 62
Technical carbofuran 86 (.05) b 23 (.001) 16 (.001) 19 (001)
Purtfied carbofuran 105 (ws) 22 (,001) 25 (.001) 16 (,001)
Water washed carbofuran 124 (NS) 40 (,001) 24 (,001) 29 (,001)

-~—-~-----Amended with 10 ppm-—--~

Untreated 49 36 3” 34
Technical carbofuran 68 (.01) 38 (NS) 45 (.01) 42 (.10)
Purified carbofuran 47 (Ns) 36 (NS) 41 (NS) 21 (,001)
Water Washed carbofuran 71 (001) 37 (NS) 40 (Ns) 59 (,001)

Amended with 1 ppm—

Untreated 115 73 37 24

Technical carbofuran 121 (ws) 62 (,05) 43 (.001) 32 (,001)
Purified carbofuran 140 (.001) 77 (ws) 53 (Ws) 32 (.01)
Water washed carbofuran 148 (.001) 46 (.001) 41 (,001) 35 (,001)

@ Mean dehydrogenase activity (ug of TPF produced per gram of wet sediments) from six replicates.
Significance level of difference between treated and untreated samples as determined by the t-test. NS means
not significant at the 0.1 level,

The question arose as to why the dehydrogenase activity in carbofuran amended sediments
decreased, while the bacterial numbers increased. To answer this question, seven pure cultures of
bacteria of the types which predominated in sediments were isolated and inoculated into autoclave-
sterilized sediment which had been enriched with 1% glucose. As a positive control, enriched sterile
sediment was inoculated with fresh bay sediment. In all inoculated sediments, the bacteria increased
in number, but only in the sediments inoculated with the fresh sediment did the dehydrogenase activi-
ty increase. This indicated that these aerobic heterotrophic bacteria did not significantly contribute
to the dehydrogenase activity within the sediments, and their increase in numbers would not be ex-
pected to increase dehydrogenase activity.

Proteolytic Activity-

Protein utilization studies were conducted to determine if the pesticides affected the assimilatory
power of the sediment microflora. As shown in Table 36 neither of the pesticides at any of the concen-
trations tested appeared to greatly change the amount of protein utilized.

Glucose Mineralization-

Radioisotopic studies were employed to determine the effect of various concentrations of mirex
and carbofuran on the carbon dioxide released during the mineralization of glucose. Results of these
studies are shown in Table 37.

The addition of carbofuran in concentrations of 1000 and 100 ppm significantly depressed the
rate at which glucose was converted into carbon dioxide. After four weeks incubation with carbofuran

33


IL.
UL.
Iv.

vu.
VII.

CONTENTS

Page
Conclusions 1
Recommendations............. 600 c cece eee ener e ene n een e eens 2
Introduction . 2.0.0.0... 66. e rete et ne ent en tn ee eee 3
Materials and Methods..........-.. 60s sce eeee ence eee nett eee e teens 4
B01 14
SUMMALY 2.6 EEE ene ene eee nent e ee 44
Discussion 1.0.00... 000 c cee eee e ne eee ene ene 45
References ..... 6.6.6 c cece eee eee teeter ete ee 46

iii


Table 23. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD 50 PPM MIREX OR CARBOFU-
RAN (AS RESIDUE) IN THE PRESENCE OF 0.08% NUTRIENT BROTH.

Sample No. 1 2 3 4
Contents
in vial Cells in Rila cells in Rila Celle in Rile cells in Rila
contents in Acetone residue Acetone residue ‘Acetone residue Acetone residue
capsliery -—-- Mirex residue Carbofuran residue
*
© Rile Bile Rila
Nutrient broth Nutrient broth Nutrient broth
34 48 131 97
34 Aye. 60 141 Ave. 97
® 3535 73 143140 106 104
38 80 166 116
29 95 225 134
(b) 32 Ave. 107 Ave. 247° Aves 136 Ave.
No. cot 3836 19-10 251244 167153
io. colonies 43 121 255 175
on plage
at 107
dilution 26 93 249 118
(a) 34 Aves 105 Aves 283 Ave. 156 Ave.
43°36 us UL 307 289 156149
43 132 316 161
Overall ave. 36 95 226 135

(a) Replicates obtained from experiment in which time sequence began with sample no. 1 followed sequen-
tially by samples no. 2,3, and 4,

(b) Replicates obtained from experiment in which time sequence began with sample no. 4 followed sequen-
tially by samples no. 3, 2, and 1.

Table 24. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS IMMEDIATELY AFTER AMENDING WITH MIREX.

Amended with mirex

Control 0.1 pe 10 ppm 000 ppa 10,000 pps
Microbial group #/m1 Tank Tank Tank Tank Tank Tank Tank Tank
x uu 16 12 vv 3 18 evi 19
4.38 9.3 43 9.3 2.4 2.9 2.1 43
Nitrate reducers 105 2.48 2 94 60. 9.3. Dah 2ab
43 2:4 120 9:3. +93 24 2.4 2h
‘Amponi fers 105 1.59.3 4 LS. 4 4 44.3
4 2.3 2.3 15.0 43.0 43 9.3 15.0 4.3
H2S producers. lo! 4.3 3.9 3 13.0 9.3 43 4.3 4.0
24.0 43.0 4.3 15.0 24.0 15.0 43 15.0
Sulfate reducers 039.3443 A 24,0 9.3 15.0 9.33
2.4 is 1.2 43 1.5 0.93 4.3 2.4
Lipid hydrolyzers 105 42.4 4 4 93 gS
9.3 4.3 2.4 7.5 9.3 1.2 43 2.4
Casein hydrolyzers 105 3 2 a3 21.0 24.0 1 2a 203
4.3 24.0 43 15.0 2.4 43 4.3 43
Gelatin hydrolyzers 105 4 240 24.0 93a
2.4 2.4 0.43 9.3 2.4 2.4 9.3 3.9
Starch hydrolyzers woo 7 24 24.0 600 4d 4a
9.4 2.3 4.3 4.3 43 9.3 4.3 43
Chitin hydrolyzers 104 443 L120. 9:3 2.9 on aa
6 1.2 1.6 1.6 1.6 1.4 1.0 1.4 1.6
Aerobic plate count. 1o' L112 LL Li LA L.0. 2.9 16

b b b b b >’ b b

Anaerobic plate count

@ Replicate counts
b Missing value


It was found that carbofuran reacted with the reagents used in the protein determination produc-
ing an overestimation of the protein concentration. Blanks without added peptone were used to com-
pensate for this error.

Bioaccumulation-

These studies were conducted with culture #280 (Benecka) using mineral salts medium (15 ppt)
with static incubation at 30 C. Inoculum consisted of cells from a 24 h, 30 C nutrient broth-grown
culture which had been centrifuged, washed three times in saline and resuspended in the same solu-
tion. The dead cell controls were prepared by heating a portion of the cell suspension for 15 min at 80
C. For the studies utilizing mirex, a saturated solution was obtained by stirring 2 g of mirex in 31 of
mineral salts medium for 3 days. This solution then was filtered through a 0.45, membrane filter and
the clear filtrate was considered to be saturated with mirex (approximately 10-° g/ml). For studies
utilizing carborfuran, a 150 ppm concentration was obtained in the test flask by dissolving 40 mg of
carbofuran in 1 ml of ethanol and adding 0.75 ml into 100 ml of mineral salts medium.

The tests were conducted as follows: A 5% inoculum of both living and dead cell suspensions was
separately placed in flasks containing mineral salts medium with 1% glucose and the test compounds.
The test flask along with several control flasks was incubated for 48 h at 30 C under staticconditions.
Cells then were removed from the respective media by centrifugation followed by filtration through
0.45, im membrane filter. The clear filtrate was analyzed for the test compound to determine how much
of the material either adsorbed to the cells or was bioaccumulated. Bioaccumulation is defined here as
the difference between the amount of test material adsorbed to the dead cells and the amount of
material found in the live cells. Dry weights were determined initially and after 48 h on the test flask
containing live cells to determine the increase in cell mass.

Isolation of Mirex- and Carbofuran- Utilizing Microorganisms-

Throughout the course of this contract a considerable effort was put forth to isolate mirex- and
carbofuran-utilizing microorganisms. In a number of cases encouraging results were obtained but in
no instance was it proven that a particular culture could use either of these compounds.

In regard to carbofuran, it was not anticipated that problems would arise in the isolation of an
organism capable of utilizing this compound. Most of the work was directed toward obtaining a
culture that was capable of utilizing carbofuran either as a sole carbon and/or nitrogen source. In
most of the work minimal media were employed and it is entirely possible that richer media con-
taining vitamins, accessory growth factors, etc. might have yielded different results. While efforts to
isolate a carbofuran-utilizing microorganism were unsuccessful, this fact is not interpreted to mean
that such organisms do not exist, but different methodology, different inocula, etc. should be
employed in future work.

Similarly, no mirex-utilizing microorganisms were obtained even though a considerable amount
of effort was expended in the search; and, at times results seemed quite encouraging. Cultures
suspected of being mirex-utilizers (demonstrating growth in the presence of mirex and hexane and the
concurrent lack of growth in hexane alone) failed to give any concrete evidence of mirex utilization.
No conclusive verification could be obtained using manometric techniques and in general, results

12


The rate constants were calculated for both pseudo-first and second order kinetics. The second
order rate expression gives a slightly better fit, but it was discovered that the pH was dropping slight-
ly during the latter part of the reaction. When the experiment was repeated using 0.05 Mbuffer an ex-
cellent fit for first order kinetics was obtained up through 70% reaction. The rate constants in 0 and 15
ppt solution were identical within experimental error. The rate constant was greater in 0.05 Mthan in
0.005 M buffer as would be expected if the pH was dropping in the 0.005 M buffer. The first order rate
constant was 0.011 sec !. This is about the same as that observed by Metcalf et al.§ at pH 9 and 37 C.
Our earlier experiments showed that Metcalf’s measurements were not based upon disappearance of
carbofuran or appearance of phenol, but apparently were based on an artifact, and our experiments
indicate that the rate constants they obtained were not related to the rate of hydrolysis of carbofuran.

Disc Assay Studies

The disc assay technique was employed asa rapid screening method to test the inhibitory effect of
the mirex on a variety of pure cultures of bacteria isolated from an estuary. The first series of tests
with the mirex produced zones of inhibition on 18,2,1 and 7 of the 20 cultures tested using four separate
batches of mirex. The results were extremely erratic with three of the batches of mirexin that they did
not consistently and/or repeatedly produce zones of inhibition.

Ten new batches of mirex were obtained from Allied Chemical Company and tested by means of
“disc assay” against 20 bacterial cultures. No zones of inhibition were noted even when the experi-
ment was replicated 5 times. Because of the results cited above, and the fact that gas chromatographic
analyses indicated varying degrees of purity for the different batches, laboratory purified mirex was
employed in subsequent tests unless otherwise noted.

Carbofuran failed to inhibit any of the 20 bacterial cultures when tested using the disc assay
technique.

In another study using technical mirex and technical carbofuran, no inhibition was observed
when tested against 596 isolates from estuarine and non-estuarine origin.

Effect of Mirex and Carbofuran on Growth Curves of Selected Cultures

Tests to determine the effect of mirex and carbofuran on pure cultures in liquid media (15 ppt
salinity) were performed using 8 cultures and concentrations of the test compounds of 0 ppm, 0.1 ppm
and 10 ppm. Essentially no reduction in counts occurred after shake incubation for 72 h (Tables 10 and
11). It should be pointed out that these tests were conducted using technical grade mirex rather than
purified mirex.

In another series of tests optical density was employed to monitor growth. Mirex and carbofuran
at concentrations of 10 and 1000 ppm failed to alter the growth response of any of the cultures tested:
two species each of Bacillus (#222 and #275), Achromobacter (#223 and #274), and Vibrio (#221 and
#295), and one species each of Benecka (#242), Pseudomonas (#297), and Acinetobacter (#173).

Results of Qualitative Studies on Effect of Mirex and Carbofuran

Qualitative tests using mirex and carbofuran in concentrations of 0.1, 10.0, and 100 ppm failed to
give any evidence of inhibition of growth, glucose fermentation, or H,S production by any of the
cultures tested (7 glucose fermentors and 4 H,S producers). Visual observations on solid media in-
dicated no inhibition of growth or hydrolyzing ability of 16 gelatin-, 7 casein-, 1 lipid-, 6 starch- and 4
chitin-hydrolyzers.

18


SECTION V
RESULTS

MIREX AND CARBOFURAN

Adsorption Studies with Mirex

Water was saturated with mirex and the amount of mirex in the aqueous phase determined as a
function of time by extraction with hydrocarbon solvent and analysis made by electron capture gas
chromatography. The saturated solutions were made by stirring mirex in distilled water for 25h
followed by removal of the excess mirex with a millipore filter (0.45,)). Table 1 summarizes the results
for montmorillonite clay and Table 2 for kaolinite clay.

Table 1, ADSORPTION OF MIREX ON MONTMORILLONITE CLAY

Concentration mirex yg/al

Time

days | Ove/mt 0.9 ve/al «0.45 ug/ml «0,09 we/ml —= 0,045 g/m
clay clay clay clay clay

4 0,003 0.004 0,002 0,003 0,002
7 0.004 0.002 0,001 0.003 0,001
14 0,001 0.002 0.003 0.001 0.006
30 0.002 0.00007 < 0,00003 < 0.00003 < 0.00003

No detectable mirex residues were found in the samples containing only clay. Adsorption to the
glass vessels was not a major problem, as shown by the lack of disappearance of mirex in the systems
without added clay. The precision of the data is not high; it is typical of this type of low-level residue
analysis. For the zero clay samples which should have had constant mirex levels, the percent stan-
dard deviation is 52. It is clear that adsorption or slow chemical reaction does occur on the clay sur-
face, ultimately removing all the pesticide from the aqueous phase.

Table 2. ADSORPTION OF MIREX ON KAOLINITE CLAY

Concentration mirex ug/ml
Time
days | 0 ug/ml 0.9 ye/ml = 0.45 pg/ml 0.09 g/ml 0,045. g/ml
clay clay clay clay clay

4 0.003 0.002 0.002 0,003 0.002

7 0,004 0.0007 0.0008 0.0008 0.0008
4 0.001 0.0008 0.0007 0.0005 0.0004
30 0.002 0.00008 0.0001 < 0.00003 < 0.00003

14


10,000 ppm
Tank
20

Tank
15

1000 ppm
Tank Tank
19

14

Tank
18

Amended with mirex
0.1 ppm 10 ppm

Tank Tank

uy 13

Tank
12

Control
Tank
16

Tank

i

27

MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS ONE WEEK AFTER AMENDING WITH MIREX.

Table 25.

4/al
x

Microbial group

2
SAISIV AY IIIs yy 4s syq aa eyo go ie jodmuyononmumed sm a]n aloo)
Sal galn sad] safas|erlacfa ds clad F g EelggoSaga2 aaa ob G2 daa S
adadaeleaaanamaasjananed Fa SB Jy Jedeadedee, jodiaerdasle
Jal aa]sa]sdacfan|snfad|safas|rn EI So gals dog atoce gp elagse ds le
°
2
oMnanaa|sayals anols alain moo} 8
g3[saficilde]e glicja dla s|s dla slag) g Sal SITIES le o/b o [AVS
. s 4 g $2] sofcicl< ole gs al *|oioF ar eae
g ETS aS ag
g
a ze =
eqeqaaessr soso sods ayes & xe mmloc x ems aly dala
Bs anldaldalsslials digala dls slaa By 8S je woqzqemedes|, casas adas)
a he aie [Balu ls Mo Ai clo miles t]e3 oo
2 || am je"84 age g sg
a g | 2
saoyossaoqsasclasjasasioe e | 3 ro-49 a]e ol
Als s]s clea salaals loos alg e]e a o Bz jee 2H HS SI
. * 4 i z saat duoc)
a.
g | Be
aoladoalealmaoaaaaulmaladeo Be lo dmaueaed
S¥lxalsaldalaaly seals ]iofa sla 4 a 2 jgdequazge
4 dd Eaadadis|sol
x 3 ela
2 3 8
ededaalS<laalncloaaqaSaliwloo| 8 4
sslggled|odagsa]agles]ocjgalqal 2 jz Jegegerelesh, .|8 ce ob o(S3ee
sgas arrays sans g ESlede sols sep els Sela s|
s s an = e ja "Ss0q7 | | on Fajen val
| = &
2o[adqaalyalnalnaloo<4]<alocloo 5 in
Saagds|[Ksjagaalsaldiclialaula | & 5 Seo GS ae Segre se sys sas aes)
gales gyryas ARs 2 IS JE cde ae ded sis digdsciéda gad
a sy g 6 GRS ASar ass Faso
8 3 Be
ae yalonlsaoulnalmolns|<aeulo ol 3 eg Joa rda ol ain} lo in col
oA[Ralgalas|snfs alow] dale alot afyn o Bel ded goals ol? #44 ae * sda oie
gj 3 |Ee4 3 aq ages
ae aleulneleo 5 2
2+ ATs spo s/s spas sale Be SI 8 § [x eYoranienia ee cla nla sie ala ale:
Ha] ig)ssleia] scfeia sa]aisfein|aafeg g EEGs te i CE eC
Zz Eclgqe ie 3 2924
5 3S
“e} 2) SB] S} S] BB] “S] G} “S| g ag | al al st al af a} al af alo
=] 3) SB] 3} “Sl “S} “S} *S) ©) “s} “S| a, S| S| SS] a} a} al ey a] ele
8 2x 3] 8] 8) 9) “S| 8} 8) 8] 8] gg
y FA 2
2 Pe 2 «| wg
ad foe el # aos 8 ad foo a gl 2
ee} FH gg 3) 8 gq fs at a a gl
| ad oe # 3 8 3 z 3g l Jd oh sa Sl 8
q | s | 3 gage 8 3 ef 8 o
H e| 2 8 ad a ae sf 8 ; 8} 4 | 3) 4 444 4 3
3 sag ge eg a og g EB] 3 jd he 8 es a
3} | 8 3H 4 3 3 Jf PRGA eS S
se} ge) 5 eH 8 : s/ 4 as 2 8 og B 3d
"al a) Hos 2 2) a alg 2 r] 3] ya BE 3
3g) gg | J 4 a a oS 2 Z|] a 3 g | fs} d al 3
4 275 a oy gf vada ag & é s/s a a so Sd al a ae
Pia gd aaa agg a 2 S) 8 gs og 3 ee Sos Fy 8
2 4§aaad a ada 4 : gla Pa aa dg se a a

>’

Replicate counts
Missing value


Quantitative Physiological Studies -

Tests on the consumption of methane by estuarine sediments were conducted in Sdhngen units as
modified by Hutton and ZoBell®. The inoculum was prepared by making aslurry of 1000 g of sediment
and 3000 ml of 15 ppt salinity mineral salts medium (prepared with Rila sea salts). Aliquots of 50 ml of
the slurry containing 0.0, 0.1, 1.0, 10.0, or 100.0 ppm of the test compounds were placed in the “reac-
tors”. The air atmosphere of the “reactors” was replaced with a gas mixture consisting of 65% CH,,
30% 0,, and 5% CO,. Incubation was carried out at 30 C under static conditions. Gas consumption was
measured daily in terms of millimeter increase in height of the liquid in the “reactors”.

Experiments employing Sohngen units for the studies utilizing M. methanooxidans were con-
ducted in accord with the procedures described by Brown et at!°. Appropriate concentrations (same as
in sediments above) of filter-sterilized test compounds in acetone were added to empty “reactor”
bottles, and the acetone was allowed to evaporate prior to the addition of the mineral salts medium.
After gassing, the “reactors” were inoculated with 3 ml of a culture of M. methanooxidans, and the
units were incubated at 30 C under static conditions.

Medium for the studies on ammonification was peptone broth containing Rila sea salts mixture to
yield 15 ppt salinity. The culture vessels were 500 ml Erlenmeyer flasks each containing 300 ml of
medium. Filter-sterilized test compounds were added to the flasks so that the final medium contained
100 ppm. The inoculum consisted of 1 ml of a 24 h broth culture of the test organism, and incubation
was carried out at 30 C for 5 days under static conditions, in duplicate. Optical density and ammonia-
N were measured daily. Protein was determined on the filtered (0.45 mn membrane filter) medium ini-
tially and at the termination of the experiment.

Experiments on nitrate reduction were conducted in duplicate in 500 ml Erlenmeyer flasks each
containing 300 ml of Bacto-nitrate broth. The concentrations of test compounds in these tests were 0,
1,10, and 100 ppm. Each flask was inoculated with 1 ml of a 24h broth culture of Achromobacter #274
and incubated at 30 C under static conditions. Optical density, NO3-N, and NO,-N were determined
initially and thereafter at 6 to 12 h intervals up to 48 h.

Nitrification studies were performed in 500 ml Erlenmeyer flasks each containing 300 ml of
ammonium-calcium carbonate medium ata salinity of 15 ppt. Test compound concentrations were 0,
1, 10, and 100 ppm. The inoculum was 5 ml of a mixture of one part estuarine sediment to three parts
mineral salts medium ata salinity of 15 ppt. All tests were conducted in duplicate, and incubation was
carried out under shake conditions at ambient temperature. Substrate depletion (NH) and product
formations (NO,, NO,) were determined initially and every 5 days thereafter up to 20 days.

Primary productivity studies were conducted utilizing estuarine pond water directly or after enrich-
ment of the phytoplankton population by incubation in the presence of added nitrate, phosphate and
light for 12 h. Test compounds (contained in acetone) were pipetted into each 300 ml B.O.D. bottleand
10 ml of boiling distilled water added immediately. The bottles then were filled with the test water and
incubated at ambient temperature for 12 h between 2 sets of flourescent lights, each having 8 light
bulbs. Bottles used for the dark reaction were painted black and covered with aluminum foil. Solvent
controls and controls containing 1000 ppm HgCl , were included in the tests. Dissolved oxygen (D.O.)
was determined with an Oxygen Meter Model YSI54.

Studies on Chemotaxis -

A gram negative rod-shaped motile bacterium was isolated from an estuarine environment (Bay
St. Louis, MS) on nutrient agar increased to 15 ppt salinity.


SECTION I
CONCLUSIONS

It is concluded from this study that: (1) essentially all of the mirex in aqueous solution will be
removed by adsorption to particulate matter (clay and dead bacterial cells) and will resist degrada-
tion; (2) only a small percentage (<15%) of carbofuran will be removed from aqueous solution by ad-
sorption but appreciable hydrolysis will occur; (3) there was no evidence of bioaccumulation of either
mirex or carbofuran; (4) a wide variety of physiological types of bacteria studied in both pure culture
and in microcosm systems were not adversely affected by mirex or carbofuran in concentrations up to
100 ppm although both compounds inhibited primary productivity and (5) a variety of degradation
products inhibit a number of different microorganisms and processes (particularly nitrification and
primary productivity) at concentrations as low as 1 ppm, but it is considered unlikely that these com-
pounds would ever reach the 1 ppm concentration in the estuarine environment.

Based upon the data obtained in this investigation it is concluded that mirex and carbofuran in
concentrations below 100 ppm would not have a deleterious effect on estuarine bacteria.


asparagine and 50 ppm mirex or carbofuran (Table 20). This positive chemotactic response was
nullified when 10 ‘ M asparagine was employed along with 50 ppm mirex or carbofuran (Table 21). In
tests employing nutrient broth (0.08%) as the attractant, no chemotactic response was observed in the
presence of 10 ppm mirex or 10 or 50 ppm carbofuran (Table 22 & 23). A positive chemotactic response
was observed with nutrient broth (0.08%) in the presence of 50 ppm mirex. This positive response to 50
ppm mirex could be neutralized by incubating the mirex and nutrient broth in combination for a
period of 3.5 h.

Microcosm Studies with Mirex and Carbofuran

The systems, termed here as microcosms, were not true microcosms or mini-ecosystems in that
they were static with no input of light energy or detrital organic matter which is constantly fed into
the natural sediment ecosystem. During construction of the microcosms, the sediments were dis-
turbed which undoubtably altered their nature and possibly the niche of some of the microorganisms
present in the system. Even with these drawbacks it was felt that static microcosm studies were im-
portant in establishing the effect of pesticides upon the mixed microbial flora within the sediments.

Effect on Bacterial Populations-

In microcosm studies the numbers of aerobic heterotrophic bacteria, anaerobic heterotrophic
bacteria and nine biochemical groups of microorganisms were followed for a period of four weeks after
the microcosm sediments were amended with various concentrations of mirex or carbofuran. The
results are summarized in Tables 24 through 27 (mirex) and Tables 28 through 31 (carbofuran).
Changes in the microbial populations at the 0.1 ppm level could not necessarily be linked to the
pesticide because acetone was added as a diluent.

Table 20. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD 1076M ASPARAGINE IN THE
PRESENCE OR ABSENCE OF 50 PPM MIREX OR CARBOFURAN.

Sample No. 1 2 3 4
Contents
in vial Celle in Rila Celle in Rila Celle in Rila Celle in Rile
Acetone residue Acetone residue Acetone residue Acetone residue
Contents in
cepiiiery ween Mirex residue carbofuran residue
tube Rila Rila Rile Rile
— 10784 Asparagine 106M Asparagine 10°6M Asparagine
No. colonies coy a 63 47
on plage 14 Ave. 23 Ave. 7% Ave. 56 Ave.
at 10” 16 15 33°28 79 (76 5855
dilution 20 34 86 60



Table 14, EFFECT OF MIREX AND CARBOFURAN ON AMMONIFICATION BY FOUR PURE CULTURES OF BACTERTA.

Protein-N=

Test Culture Optical density of alture NH4-N (mg/1) (ag/1)

No. Days incubation ‘of incubation initial final

az 3 203 4

Blank 0,00 0,00 0.00 0,00 o 0 0 oO 1125 1125
control 223 0.06 0.12 0.28 0.35 0 0 35 97 1120 643
100 ppm
Mirex " 0.06 0.13 0.20 0.31 0 6.5 20 30 1125 648
100 ppm
Carbofuran =" 0.06 0.12 0.27 0.32 0 0 2 28 1120 650
Control 276 0.26 0.32 0.50 0.62 10 130 23° 62 1120 275
100 ppm
Mirex ” 0.25 0.34 0.47 0.56 yo 10 37 «557 i210 553
100 ppm
Carbofuran =" 0.23 0.32 0.42 0,53 io 10 40 70 1130 325
Control 297 0,11 0.20 0,25 0.38 10 24 63 58 1120 558
100 ppm
Mirex ” 0.10 0.11 0.26 0.35 io 18 49 68 uo 555
100 ppm
Carbofuran =" 0.11 0,12 0.24 0.36 10 18 45 (56 1120 565
Control. 280 0.60 0.95 1.10 1.15 18 77 140 125 1120 593
100 pea
Mirex " 0.59 0.89 1.00 1.10 14-79 139.133 1130 583
100 ppm
Carbofuran =" 0.62 0.95 1-10 1.20 10 78 140 150 1130 583

ppm protein-N of medium after filtration chrough 0.45 y membrane filter. Conditions of test: medium: 15 ppt
Rila salts + peptone. Incubation: 30 C, static conditions. All values are the average of two determinations.

Effect of Mirex and Carbofuran on Nitrate Reduction

The effect of mirex and carbofuran on nitrate reduction by Achromobacter #274 showed that
nitrate utilization and nitrite production were essentially uneffected by concentrations of test com-
pounds up to 100 ppm after 18 h of growth (Table 15). The data do suggest, however, that increasing
concentrations of mirex and carbofuran delay the onset of nitrate reduction.

Table 15. EFFECT OF MIREX AND CARBOFURAN ON NITRATE-REDUCTION BY ACHROMOBACTER (#274).

Hours of incubation
Test system 6 iz 18 7 36 we
NO3-N NOZON |NO3-N NOp-N [weet NO]-N |NOy-N—NOy-N |NO3-N NOg-N ]NOj-N-NOg-N
(mg/l) (mg /1) | (mg/1)  (mp/1)] (me/1) (mg/d) | (mg/2) (mp / 1) | (mg/1) (mg /1) | (mg /1)_(mg/1))
Control 101 al 0.75 70 1.48 66 1.66 60 1.75 5S 1.75
Mirex 1 ppm 110 92 0.60 66 1.70 631.75 60 1.90 621.75
| wirex 10 ppm 120 94 1.0 66 1.50 65 1.65 631.85 68 1.80
Mirex 100 ppm 130 140 80 1.25 60 1.58 63 1.80 68 Led
Carbofuran 1 ppm 132 437 72 1.30 66 1.50 70 1.70 nm 1.60
Carbofuran 10 ppm | 128 1000.77 68 1.58 651.58 65 1.80 70 1,75
i Carbofuran 100 ppm | 124 137 106 0.50 68 0.75 66 1.65 al 1.50

All values are the average of two determinations.

21


21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

TABLES (Continuted)

Page
Comparison of Chemotactic Response of the Estuarine Bacterium Toward 10°4M
Asparagine in the Presence or Absence of 50 ppm Mirex or Carbofuran . 25.
Comparison of Chemotactic Response of the Estuarine Bacterium Toward 10 ppm
Mirex or Carbofuran in the Presence of 0.08% Nutrient Broth ..................00.000000 25
Comparison of Chemotactic Response of the Estuarine Bacterium Toward 50 ppm
Mirex or Carbofuran (as residue) in the Presence of 0.08% Nutrient Broth ............... 26
Microbial Counts on Sediment Samples from Microcosm Tanks
Immediately after Amending with Mirex ............0..00 0.00 cc eee eee cece tees eee e eee 26
Microbial Counts on Sediment Samples from Microcosm Tanks
One Week after Amending with Mirex ........ 0.20.00... 00 0e0 secs ese ee eee et eee nett eens 27
Microbial Counts on Sediment Samples from Microcosm Tanks
Two Weeks after Amending with Mirex............ 0.0. 00ccceeeeeeee eee ceeeeeeteeteeeees 27
Microbial Counts on Sediment Samples from Microcosm Tanks
Four Weeks after Amending with Mirex ............0...0.. 00 0c ccc cece cece ence eee ees 28
Microbial Counts on Sediment Samples from Microcosm Tanks
Immediately after Amending with Carbofuran. 28
Microbial Counts on Sediment Samples from Microcosm Tanks
One Week after Amending with Carbofuran ............00..6 0c cece ec ee eect eee 29
Microbial Counts on Sediment Samples from Microcosm Tanks
Two Weeks after Amending with Carbofuran .............6 0.000000 e cece eee eee eee 29
Microbial Counts on Sediment Samples from Microcosm Tanks
Four Weeks after Amending with Carbofuran...........0...6 0.200000 0 cee ce cee eee e eee 30
Mean Bacterial Counts (x 10‘) in Sediments Amended with 100 ppm Mirex and
Held Under Various Conditions of Salinity and Temperature ..............0....002.. 2005 31
Mean Bacterial Counts (x 10‘) in Sediments Amended with 100 ppm Carbofuran and
Held Under Various Conditions of Salinity and Temperature ...........--...-..00+.0-005 32
Dehydrogenase Activity in Sediments Treated with Different Preparations and
Concentrations of Mirex...... 2.00.00 0.0 0000 e cece ees eect nett t tenner ees 32

Dehydrogenase Activity in Sediments Treated with Different Preparations and
Concentrations of Carbofuran . .

Proteolytic Activity of Microorganisms in Sediment Samples
Amended with Carbofuran and Mirex ........... 222000000 eee reece eects 34


10

11

12

13

14

15

16

17

18

19

20

TABLES

Adsorption of Mirex on Montmorillonite Clay..

Adsorption of Mirex on Kaolinite Clay ...........000ee eee e eter eee e ener eee e ences 14
Adsorption Studies of Carbofuran on Montmorillonite Clay ... 15
Carbofuran Adsorption on Kaolinite Clay ..............000 eee e eee cence eee e eens 15
Fate Studies - Mirex 16
Fate Studies - Carbofuran ........... 6. ce epee cere cece eee eee neers 16
Hydrolysis of Mirex ......6.. 6000.00 cece cece eee eee entree nner n nner e eee ee nee 17
Hydrolysis of Carbofuran in 15 ppt Salinity ..............0 cece eee teeter ence ne es 17
Hydrolysis of Carbofuran in 0 ppt Salimity 02.0.0... 0c ccc cece eee teen ee ee 17
Effect of Varying Concentrations of Mirex on Growth of

Pure Cultures of Bacteria in Broth Medium .............-- 56.00 sees eee eee eee eee eee 19
Effect of Varying Concentrations of Carbofuran on Growth of

Pure Cultures of Bacteria in Broth Medium ........... 000.600 cs esee eee e eee e eee eee eee es 19
Effect of Mirex and Carbofuran on Methane Consumption by

Microflora in Estuarine Sediments ................ 000 c ccc cece ese ee eee e eee eee enee ees 20
Effect of Mirex and Carbofuran on Methane Consumption by

Methanomonas methanooxidans «0.2.66... 6 06.6 ccc ec eceeen ene enn en eee en es 20
Effect of Mirex and Carbofuran on Ammonification by

Four Pure Cultures of Bacteria .........0... 000200 c ccc ee eee e eee eee eee ee tees teeter ones 21
Effect of Mirex and Carbofuran on Nitrate-Reduction by Achromobacter (#274).......... 21
Effect of Mirex and Carbofuran on Nitrification by

Microorganisms in Estuarine Sediments ................... 0000 eee eee cette eee eeeeeeteees 22
Effect of Mirex and Carbofuran on the Metabolic Activity and

Primary Productivity of Estuarine Pond Water ................ 00 ee eceee eee e eee e ee ee ee 22
Effect of Mirex and Carbofuran on the Metabolic Activity and Primary

Productivity of Estuarine Pond Water Enriched for Phytoplankton...................555 23
Comparison of Chemotactic Response of the Estuarine Bacterium Toward 10°°M.
Asparagine in the Presence or Absence of 10 ppm Mirex or Carbofuran .................5 23
Comparison of Chemotactic Response by Estuarine Bacterium Toward 10°°M

Asparagine in the Presence or Absence of 50 ppm Mirex or Carbofuran . . 24

iv


The isolate was inoculated into an Erlenmeyer flask containing Bacto-nutrient broth having a
salinity of 7.5 ppt and incubated at ambient temperature under shake conditions for 15 to 18 h. Cells
were harvested by centrifugation at 20 C at 4500 rpm for 10 min, washed 3 times in physiological
saline, and resuspended to the desired concentration in a solution having a salinity of 7.5 ppt. Acetone
containing mirex or carbofuran at 10 ppm or 50 ppm was pulled into capillary tubes by capillary ac-
tion until the tubes were % full. The tubes were then placed in an oven at 80 C, thus leaving a residue of
the pesticide on the internal walls of the capillary tube. Acetone “residue” tubes were prepared in a
similar manner except no pesticide was contained in the acetone.

Chemotaxis was determined by use of the capillary tube technique, and the procedures employed
were a modification of those described by Mitchell, Fogel and Chet#?.

Capillary tubes were filled % full with the desired test solution; subsequently the top ends of the
tubes were plugged with silicone grease and the outsides of the tubes were washed in a stream of water
and wiped dry with a sterile Kimwipe®. The tube was inserted into a small vial containing 0.4 ml cell
suspension of approximately 3.0 x 10° viable cells/ml for 12 min. Subsequently, the tube was removed,
washed in a small stream of water and broken into 3 or 4 sections (excluding upper \% section) into
physiological saline diluent. Dilutions in physiological saline were prepared, plated, and spread on
the surface of nutrient agar plates containing 7.5 ppt salinity.

Microcosm Studies-

Microcosms consisted of all glass vessels (usually 4 liter wide mouth jars) containing a mixture of
estuarine sediment and sea water. This mixture was prepared by homogenizing sediment from the top
11.6 cm of the estuary bottom with artificial sea water in a ratio of 2:1 (w/w) using a large Waring
blender. Within 24 h after the sediment mixture was placed in the vessel, settling occurred and abouta
3 cm deep layer of clear water appeared over about 9 cm of sediment, with the top sediment layer ox-
idized and the bottom layer reduced as indicated by the color. The microcosm thus mimiced the es-
tuarine sediment system. Some infauna forms of polychaetes survived the homogenizing and became
active in the microcosms.

Sediments in these studies were obtained from Davis Bay and were generally described as “sandy
course silt” Sediments were checked for mirex and it was not detected.

A single lot of technical grade carbofuran (Niagara Chemical Division, FMC Corporation,
Middleport, N.Y., ME-L514, 4717-54-A, 8/9/73) was used for all the sediment microcosm studies. Two
lots of technical grade mirex were used with lot number SN-16-6988-43 (Allied Chemical Corp.,
Baltimore, Md.) being used for studies on the effect of various mirex concentrations on the
heterotrophic bacterial populations and the various biochemical groups of organisms. Lot number
SN-3-100-36 (Mirex Technical, Allied Chemical Corp., Baltimore, Md.) was used for the temperature-
salinity studies and for all other studies. The purified mirex and carbofuran were prepared by
recrystallization, and the water washed carbofuran prepared by placing in a filter funnel and
washing with distilled water until the filtrate was colorless.

Pesticides in concentrations of 1 ppm and greater were added as the dry powder by weight during
the sediment mixing procedure. The amount added thus represented the addition to the whole system.
However, after the microcosm settled, most of the pesticides were probably in the sediment. For a
pesticide concentration of 0.1 ppm, 10 mg of the pesticide were dissolved in 100 ml of acetone and 0.1
ml of this solution added to each 1000 g of the sediment water mixture.

10


ACKNOWLEDGMENTS

The contribution of the following individuals is acknowledged with sincere appreciation: Mr.
Gary W. Childers and Mr. Dinesh D. Vaishnav of the Microbiology Department of Mississippi State
University; Mrs. Terri Gray, Mrs. Ming Ming Fang, Mr. Larry Lane and Dr. B. R. Layton of the Mis-
sissippi State Chemical Laboratory; Miss Sandra Lofton of the Gulf Coast Research Laboratory; and
Dr. C. M. Ladner of the Mississippi State University-National Space Technology Laboratory.


The numbers listed for mirex are all the result of averaging three replicates. The recovery by this
method is 70-80%, and thus the data show that no significant decrease in mirex concentration oc-
curred during the duration of the experiment. No indication of the presence of dehalogenated
derivatives was found. The samples were held in gallon jars during the experiment and had normal
exposure to sunlight.

Table 5. FATE STUDIES - MIREX

Spiked 6-10-74 with 0.01 ppm Mirex

Time/days % Mirex recovered

0 5

1 79

2 88

4 83
8 76
16 79
31 78
40 2
53 68
63 7
70 79
81 72
88 71
100 70
113 62
123 68
130 64

Fate Studies with Carbofuran

The analyses were conducted by the heptafluorobutyric anhydride derivitization procedure
described in the method section. The coefficient of variation for this method is about 50%. Recoveries
were very poor when preliminary hydrolysis to free conjugated residues were employed and so the
reported data are for free carbofurans. No detectable residues of 3-hydroxy- or 3-ketocarbofuran were
found in any of these samples. The phenols would not be detected by this method. Table 6 is a compila-

Table 6. FATE STUDIES  CARBOFURAN

Time/days % Carbofuran

0 81
a2 50

1 86

2 33

4 61

6 55
10 64
13 54
16 67
a 33
49 24.7

tion of the data for disappearance of carbofuran from an estuarine sediment-water system. Theinitial
concentration of carbofuran was 10 ppm. The data are more variable than for mirex. Occasionally one
of the replicates showed no detectable carbofuran. These results were sometimes obtained on identical
samples run in the same set on the same day. The reason for this erratic behavior has not been deter-
mined.

16


Ecological Research Series



Effect of Mirex and Carbofuran Derivatives on Methane Consumption

by Microflora in Estuarine Sediments

Table 42 shows the results of the effect of mirex and carbofuran derivatives on the process of
methane consumption by the microflora in estuarine sediments after 12 days of incubation at 30 C un-
der static conditons. No inhibition was noted with any of the test compounds; instead, there are in-
dications of a stimulatory effect on the rate of methane consumption by several of the derivatives.
Since the test system (sediments) is so complex in nature, it is difficult to properly assess this
phenomenon. One possible explanation could be that these compounds are retarding certain groups
of bacteria that might otherwise compete with the methane comsumers.

Table 42, EFFECT OF MIREX AND CARBOFURAN DERIVATIVES ON METHANE CONSUMPTION BY MICROFLORA IN
ESTUARINE SEDIMENTS,

Teot system 2 3 & S$ 6 7 8 9 10 WL 42
Blank o 0 0 0 oOo 2 4 )
Control 4 9 WL 18% 8 35 a2 53 HS
I-tlydroxyearbofuran-10 ppp, 4 = 8 sLS 202835 HDT
Lppm 4 899 «11 5] 922 CSS 87

3-Ketocarbofuran-10 ppm 4 15m RM 42 51 59° 70 «79 86
1 ppm 4 13 18 2% «637 «4B 5975 BLOG

Kepone-10 ppm 2 9 MW 18 2 2% 35 4 530 6475,
1 ppm 4 9 13 22 «29 «40 «646 5773 BBG
Reduced kepone-10 ppm 4 UM 13 % 33° be 53 6877 BBG
1 ppm 7 11 18 26 35 46 55 68 81 86 86

2,8-Dihydrogen mirex-10 ppm 4 1 13 22 29 40 48 59 73° 79 8
lppm 4 Il 15 24 35 48 57 70 79 79 84

10-Hydrogen 10 ppu 7 «915 20 31 44 59° 73 84 BB BBB
and 5,10-dihydrogen-1 ppp 4 9 13 20 29 40 46 59 73° 81 86
mirex

® ml of gas consumed.

Conditions of test: Sdhagen unit apparatus, medium 15 ppt salinity with mineral ealts incuba-
Ted under gee ataosphere of 65% CH,, 30% 0, and 5% COp, static conditions. ALL values are
the average of three determinations.

Effect of Mirex and Carbofuran Derivatives on Ammonification

A pure culture, #280, was used to study the effect of mirex and carbofuran derivatives on the
process of ammonification. After 3 days of incubation at 30 C under static conditions no difference
was noted between the tests containing the derivatives and the control when monitoring substrate
disappearance (protein-N) and product formation (NH,-N). These results are illustrated in Table 43.

Effect of Mirex and Carbofuran Derivatives on Nitrate Reduction

The results of the effect of mirex and carbofuran derivatives on nitrate reduction are found in
Table 44. Cultures containing the test compounds (concentration 1 and 10 ppm) showed the same rate
of substrate utilization (NO,-N) and product formation (NO,-N) as did the control.

39


SECTION IIT
INTRODUCTION

The primary utilization of mirex as an insecticide is in the control of the western harvester and
the imported fire-ant. The latter species infest most of the southeastern United States and millions of
acres are treated annually in this area by aerial application of baits containing this toxicant ata rate
of 0.20 to 1.66 g per hectare. There are few areas in the southeast that do not have some history of
mirex application.

Carbofuran is registered for utilization on field corn, rice, peanuts, alfalfa and sugar cane (EPA’).
It is applied to and incorporated into the soil at rates from 45.9 to 1725.5 g per hectare.

With the history and rate of application of these pesticides to the greater part of the southeast, itis
obvious that some portion, both of mirex and carbofuran, will enter the estuaries of the Gulf of Mexico.
In these estuaries, microorganisms are a vital link in the overall ecological system. There arenodata
available on the effect of either mirex or carbofuran on this estuarine microbial ecosystem.

In order to fill this gap in our existing knowledge, the Environmental Protection Agency awarded.
Mississippi State University Contract No. 68-03-0288, “The Effect of Mirex and Carbofuran on Es-
tuarine Microorganisms”.

The objective of this investigation was to supply a sufficient amount of scientifically valid data
on the fate and effects of mirex and carbofuran in the estuary in such a form that these data can be
employed in deriving water quality standards for these pesticides. Specifically, the work was design-
ed to establish the chemical, physical and microbiological fate of these pesticides in the estuarine en-
vironment and determine meaningful effect(s) on important estuarine microorganisms and their ac-
tivities. It is extremely important to maximize the transferability of scientific results to the environ-
ment, and thus the experimental approach described involves the use of microcosm systems as well as
pure culture studies.


SECTION II
RECOMMENDATIONS

It is recommended that studies be conducted to identify the microbiologically toxic material(s) oc-
curring in some batches of mirex as well as the products resulting from UV radiation of moist mirex.

The toxicological and chemical behavior of conjugates of carbofuran metabolites should be per-
formed to completely delineate the effect of this pesticide.

Since mirex and probably carbofuran will partition into an organic phase from an aqueous
system and because oil is a common contaminant in estuaries, it is recommended that the impact of
oil-mirex and oil-carbofuran systems should be examined for their deleterious impact on estuarine
organisms, biodegradability and chemical and physical fate.

These investigations should be followed by studies of more complex systems which would include
higher members of the food chain, both in the laboratory and in the field studies.


at the above concentration, carbon dioxide production was reduced by 87.5% and 29.2%, respectively.
No significant alteration in the carbon dioxide evolution rate was detected at lower concentrations of
carbofuran or at any concentration of mirex tested.

Respirometric Studie:

With the rate of glucose mineralization being decreased in sediments amended with carbofuran,
it was expected that the oxygen consumption during glucose utilization may also be affected. As
shown in Table 38, the amount of oxygen consumed during the breakdown of glucose was reduced in
amended sediments. This reduction was comparable percentagewise to the reduction in glucose
mineralization.

Table 38. OXYGEN CONSUMPTION DURING GLUCOSE UTILIZATION BY ORGANISMS IN SEDINENTS
AMENDED WITH 1000 PPM CARBOFURAN.

Total oxygen Oxygen consumption Reduction

consumption corrected for in oxygen
endogenous consumption
w) respiration of amended
(al) sediment

9}

--- Five days after sediments amended ---

Untreated sediment
(endogenous respiration) 224% - -

Untreated sediment
with glucose 303 79

Amended sediments
(endogenous respiration} 228 - --

Amended sediments
with glucose 236 ul 86.1

Twelve days after sediments amended ------------------

Untreated sediment
(endogenous respiration) 195 -- _

Untreated sediment
with glucose 223 28 --

Amended sediments
(endogenous respiration) 176 -

Amended sediments
with glucose 188 12 S7.1

* Average of two replicate samples over a six hour period.

Hydrogen Sulfide Production-

Microcosms prepared with beach sand were amended with 1000 ppm mirex, 1000 ppm carbofuran
or 100 ppm carbofuran, and were incubated in the dark. After seven days, the control microcosm and
the one amended with mirex developed a dark gray-colored reducing layer below the sand water inter-
face. This layer was absent in the carbofuran amended samples. Subsequent studies showed a concen-
tration greater than 10 ppm carbofuran was required to produce this effect.

Hydrogen sulfide, which causes the blackening effect, is produced either from organic sulfur com-
pounds or from sulfate reduction. Previous microcosm studies have shown that the addition of car-
bofuran does not reduce the number of organisms capable of making these transformations in
sediments. Thus it is speculated that carbofuran in some way prevents the production of hydrogen
sulfide without killing the organisms.

35


Effect of Mirex and Carbofuran on the Chemotactic Response of an Estuarine Bacterium

Table 18. EFFECT OF MIREX AND CARBOFURAN ON THE METABOLIC ACTIVITY AND
PRIMARY PRODUCTIVITY OF ESTUARINE POND WATER ENRICHED FOR

PHYTOPLANKTON.
Test syeten Metabolic Primary
activity productivity
(mg 0, coneumed/1 (mg 0, produced/1
in 12h) in 12h)
Controt 1.40 4.65
Acetone control 1.30 4.85
BgClz control 0.00 0.00
Mirex 1 ppm 1.30 5.00
10 ppm 1.10 5.00
carbofuran 1 pa 1.25 4.05
10 ppa 1.30 3.05

All values are the average of two determinations.

Preliminary experiments indicated that concentrations of 10 and 50 ppm of mirex and carbofuran.
were not toxic to this bacterium while a concentration of 250 ppm was definitely lethal. Additionally,
it was demonstrated that no significant increase in bacterial numbers occurred during an incubation
period of 54 min. With asparagine as the attractant, a positive chemotactic response toward this com-
pound occurred with increasing concentrations from 10°* M to 10°? M. The bacterium failed to
demonstrate a chemotactic response in tests employing 10° 6 M asparagine in the presence of 10 ppm
mirex or carbofuran (Table 19), but gave a positive chemotactic response in the presence of 10-§ M

‘Table 19. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERTUM TOWARD 10”°y ASPARAGINE IN THE
PRESENCE OR ABSENCE OF 10 PPM MIREX OR CARBOFURAN.

Sample

No 1 2 3 4
Contents
in vial Cells in Rila Celis in Rile Cells in Rile Cells in Rila

Contents in

Acetone residue

Acetone residue

Acetone residue

Acetone residue

capillary Mirex residue Carbofuran residue
tube
Rila Rila Rila Rila
107° Asparagine 107°" Asparagine 107° Asparagine
30 40 SL 56
@) 34 Ave. 50 Ave. 62 Ave. 66 Ave.
3635 50 50 1 65 67 70
39 58 76 90
No.colonies
on plate 51 19 86 84
at_, (a) 57 Ave. 82 Ave. 109 Ave. 118 Ave.
10-9 66 © 60 96 89 130 114 1g 112
dilution 68 99 133 128
30 56 74 72
(a) 30 Ave. 66 Ave. 83 Ave. 87
50 41 66 (66 8482 87
53 76 87 98

’a) Replicates of separate experiments.

23


Table 43. EFFECT OF DERIVATIVES OF MIREX AND CARBOFURAN ON AMMONIFICATION BY A PURE
CULTURE OF AN ESTUARINE BACTERIUM ~ #280 (BENECKA).

Way=N_(mg/1) Protein-N@ (mg/i)

Test_gstem Initial Day 1 Day 2 Day 3 | titial | Final

Blank 0 o ° o 1200 | 1200

Control ° ug 195270 1200 698

3-Hydroxycarbofuren 10 ppm Cy 121-200-272 1200 710

1 ppm 0 125-230-310 1200 no

3-Ketocarbofuran 10 ppm ° 125192280 1200 727

2 ppm 0 125227292 1200 70

Kepone 10 ppm ° 75 100-240 1200 698

1 ppm 0 127210297 1200 715

Reduced kepone 10 pea 0 120-200-282 1200 690

1 ppm 0 133235302 1200 715

2,8-Dihydrogen 10 ppm o 125200275 1200 698

mirex 1 ppm 0 127-220-300 1200 70

10-Hydrogen 10 ppm ) 123 202,277 1200 710

and 5,10-dihydrogen- 1 ppm 0 123260300 1200 727
mirex

* Protein-N of medium after filcration through 0.45 » membrane filter: Conditions
of test: Medium: 15 ppt Rila sea salts, mineral salts, Incubation: 30 C, sta~
tic conditions. All values are the average of two determinations.

Table 44. EFFECT OF MIREX DERIVATIVES AND CARBOFURAN DERIVATIVES ON NETRATE-REDUCTION BY
AN ESTUARINE BACTERIUM #280 (BENECKA).

Hours of incubation

Test system 0 é 12 24
Wg <WNOy =NWOg-NNOZ=N—NO,=N-NODNNOG=NNOL=W
(mg li) (og/ 1) (ug/3) (mg /2) (me /1)__(mg/1) (og /2) (mg /1)

Blank 100 o | 100 oO | 100 0 {100 0

Control 100 0 43 0.7 | 7.5 2.4 [elo 2.8

3-Ketocarbofuran 10 ppm 100 0 7 65 [24.0 2.4 J<1.0 3,2

1 ppa 100 0 64 63 J12.0 24 fe1.0 | 3.2

3-Hydroxycarbofuran 10 ppm | 100 o 72.66] 5.0 2.2 [<1.0 0 3.2

2ppn | 100 ° 69 175] 7.5 2.1 fata 3.2

Kepone 10 ppa oe 0 ee 4731 7.0 2.1 [<1.0 3.0

1 ppm 100 0 90 62] 24.0 2.1 J<l.o 3.2

Reduced kepone 10 ppm 100 ° 84] 7.0 24 [10 3.2

1 ppm 100 ° 7873 | 7.5 2.3 [cdo 3.2

2,8-Dihydrogen mirex 10 ppm} 100 ° 56.77 /10.0 2.3 J2,0 3.0

1 ppm! 100 0 2.70) 7.5 2.1 |<1.0 3.0

10-Hydrogen 10 ppm] 100 0 93 68] 7.5 2.4 [<1.0 3.0

and 5,10- d4tydrogen- 1 ppm! 100 0 88 70] 6.5 2.4 J <t.0 3.0
mirex

411 values are the average of two determinations.

Effect of Mirex and Carbofuran Derivatives on Nitrification

Three of the test compounds, 3-hydroxycarbofuran, kepone and reduced kepone at concentrations
of 1 and 10 ppm retarded nitrification as illustrated in Table 45 by reduction in both disappearance of
substrate (NH,-N) and product formation (NO,-N and NO,-N). No effect on nitrification was found
with 3-ketocarbofuran, 2,8-dihydrogen mirex and the mixture of 10-hydrogen and 5,10-dihydrogen
mirex.

40


Effect of Mirex and Carbofuran on Nitrification

To determine if mirex and carbofuran would affect nitrification, estuarine sediments were
employed rather than pure cultures. Within the limits of the tests employed, no deleterious effect on
the process was observed in terms of substrate disappearance (ammonia) or product formation (nitrite
and nitrate) even after 20 days of incubation (Table 16).

Table 16. EFFECT OF MIREX AND CARBOFURAN ON NITRIFICATION BY MICROORGANISMS IN ESTUARINE SEDIMENTS.
Days of incubation
° 5 10 15 20

Itest system

NHG=NNOZ-N NOg-N [Nig-W NOQ-N NOs-N [NH3-N NO)-W NO3-N [NH-N HON HON] NHg-N NO2-N NOg—W

(ag/1) (me/2) (mg/1) | (mg/L) (mg/L) (mg /1)| (mg/2) (me/1) (me/1)| (ag/1) (mg/L) (me/1)| (me/1) (we/2) (ae /1)
Blank as 00 10 0 0 | 10 o o | 13 0 o | 12 © 0
control 128 0000 105 0 0 | 205 trace trace | 100 trace trace} 105 .065 ~.5
Mirex
1 ppo 15 0 0 93 0 0 aon oN 83" <,025} 92.05 +.025
10 ppm 0 0 0 aoz 0 0 | 100 " TM | 100" <.025} 110.01 <.5
100 ppm 19 0 0 15 0 0 | 105 " TM | 100 " <.025} 110.01 <.5
carbofuran
1 ppm 1200 0 0 9% 0 0 ao" of ae" <,025) 93 <.01
20 ppm 1g 00 103 0 «©6©0 | 106 0" "| 190" 5.025] 105.02
100 ppa 12000 (0 105 0 ©6©0 (| too 0" "| oo 025} 205 <.02
Conditions of test: Medium: 15 ppt Rile salts + NH,Cl + mineral salts, pl 7.0, Incubation: 30 C, shake

conditions.

All values are the average of two determinations.

Effect of Mirex and Carbofuran on Primary Productivity

As shown in Table 17, neither mirex nor carbofuran inhibited the metabolic activity of estuarine
pond water, but did inhibit primary productivity. These experiments were repeated employing pond
water enriched with phytoplankton. The results (Table 18) confirmed the lack of inhibition of
metabolic activity by both compounds, confirmed the inhibition of primary productivity by car-
bofuran, but differed in the fact that mirex failed to inhibit primary productivity.

Table 17. FFFECT OF MIREX AND CARBOFURAN ON THE METABOLIC ACTIVITY AND PRIMARY
PRODUCTIVITY OF ESTUARINE POND WATER.

Test system Metabolic activity Primary
(ag 0, consumed/1 productivity
fa 12h) (mg 09 produced /1
in 12 h)
Control 0.95 2.35
Acetone control 0.95 2.10
HgCl, control 0.00 0.00
Mirex (1 ppm) 1.00 1.30
(10 pa) 1.05 1.70
Carbofuran (1 ppm) 1.20 1.35
(20 ppa) 1.05 11s

All values are the average of two determinations.

22


37

38

39
40

41

42

43

44

47

48

TABLES (Concluded)

Page
Mineralization of Glucose by Microorganisms in Sediments Amended with
Various Concentrations of Carbofuran and Mirex.. . 34
Oxygen Consumption During Glucose Utilization by Organisms in
Sediments Amended with 1000 ppm Carbofuran ............. 00. ccee eee eee een cece eeeee 35
Bioaccumulation Studies Using Carbofuran .............. 0c cece cece ee ee ee en ee ee enee 36
Bioaccumulation Studies of Mirex by Culture #280 (Benecka)...........0-.00ceeeeeeeee ee 37
Results of “Disc Assays” of UV Irradiated Hexachlorobenzene and
Mirex Against Culture #274 (Achromobacter) .......... 0000s ceee cece eee en eee ee nee eeees 38
Effect of Mirex and Carbofuran Derivatives on Methane Consumption by
Microflora in Estuarine Sediments ............... 0000 ceee ete e eee eee eee eee e ene ee tenes 39
Effect of Derivatives of Mirex and Carbofuran on
Ammonification by a Pure Culture of an Estuarine Bacterium - #280 (Benecka).......... 40
Effect of Mirex Derivatives and Carbofuran Derivatives on
Nitrate-Reduction by an Estuarine Bacterium - #280 (Benecka) ........... 00.000 ceeeeee ee 40
Effect of Mirex and Carbofuran Derivatives on Nitrification by
Microorganisms in Estuarine Sediments ..............000.00cceeeeeeeeeeee eens ee enees 41

The Effect of Mirex and Carbofuran Derivatives on the Metabolic Activity and
Primary Productivity of Estuarine Pond Water ..

The Effect of Mirex and Carbofuran Derivatives on the Metabolic Activity and
Primary Productivity of Estuarine Pond Water Enriched for Phytoplankton ..

Dehydrogenase Activity in Sediments Treated with Derivatives of Mirex..............-- 43


were erratic. There is some evidence to support the hypothesis that some of the growth that was
observed and some of the results obtained through manometry were the action of the organism on
either mirex or a compound formed from mirex. Once again, failure to obtain a mirex-utilizing
organism does not prove the non-existence of an organism possessing this characteristic, but rather
reflects only the fact that under the conditions and materials used in this investigation none was ob-

tained.

13


Table 31- MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS FOUR WEEKS APTER AMENDING WITH CARBOFURAN.

SS
Amended with carbofuran
Control 0.1 ppm 10 pp 1000 ppm 10,000 ppm
“ tour #/m. “Tank fank Tank Tank ‘Tank  ‘Tenk ‘Tank ‘Tank ‘Tank ‘Tank
x 25 2627. 28 293g x uu 34
Nitrate reducers oS 7.9 7.0 2.6 0.4 0.3 4.02400 17.0 24.0 28.0
Ammonifers 105 1.713.049 49 3.3 3.3 33.0 140.0 170.0 170.0
H,S Producers 105 0.300338 FOS ae oS Pe
Sulfate reducers yoo 328 083 0.8 0.8 0.22 0.5
Lipid hydrolyzers 10° 4 22.0 3u3 19.0 2250. 2s4 24.0 19.0 32.0 33,0.
Casein hydrolyzers 10° 46 Aaa 9.5 18.0. 3.5 2.0 33.0 33.0 49.9 49.0
Gelatin hydrolyzers 10° 7.9 33,0 4.9 749 4.0 33.0 240.0 240.0 49.0 oO.
Starch hydrolyzers 10° 1.7 2.1 3.3 1.11.3 2.4 2.4 Ll 1 7.9
Chitin hydrolyzers 10% Lil 1.3 Ll 1.7 2.6 1.3 0 9.7 0.6 0.2
Aerobic Plate count 10° 13.1 16.7 20.7 17.2 15.5 17.5 34.0 39.0 23.4 27.0

6
Anaerobic plate count 10 8.9 13.3 19.6 18.1 13.7 7.3 13.1 20.0 18.2 18.4

In the studies with mirex, it was noted that concentrations in excess of 10 ppm caused a slight but
significant reduction in the two and four week aerobic plate counts as compared to the control.
Morphological characteristics of the colonies on plates from all treated samples were similar to those
on plates from the control samples. Anaerobic plate counts were not significantly different from the
contro] at any concentration of mirex. Statistical analysis of the data gathered on the other nine
biochemical groups of organisms was conducted without showing any difference between treatments
and controls.

After two weeks incubation, the samples amended with 1000 ppm and 10,000 ppm carbofuran
showed an increase in the number of aerobic bacteria, anaerobic bacteria and some of the other
groups of heterotrophic bacteria over numbers found in control sediments. Colonies on the plates from
both treated and control samples were morphologically the same. At a concentration of 10 ppm, pop-
ulations in treated and control samples were about the same.

Since the aerobic heterotrophic bacterial plate counts were easy to perform and could be
replicated with a minimum of effort to produce a direct number rather than a statistically derived one
as produced by the MPN counts, it was decided to use the plate count as the measure of effect of the
pesticide treatments in further studies.

Salinity-Temperature Studies-

The effect of various salinities and incubation temperatures on the bacterial population of the
sediment was measured in the presence and absence of both mirex and carbofuran in a concentration
of 100 ppm. The temperatures and salinities employed were approximately the maximum and
minimum values recorded in the estuary.

Overall, the bacterial populations in mirex-amended sediments did not differ from the controls at
any of the salinities and temperatures tested. Statistical analysis of the data verified this observation
(Table 32). The previous study showed that mirex suppressed the bacterial populations. However, this
difference was attributed to lot-to-lot variability in the mirex since in the previous study a different lot
of mirex was employed.


The addition of carbofuran to samples produced significantly higher bacterial counts as com-
pared to the control under all conditions of salinity and temperature tested (Table 33). None of these
temperature-salinity conditions produced results noticeably different from the other.

Table 32, MEAN BACTERIAL COUNTS (x 104) IN SEDIMENTS AMENDED WITH 100 PPM MIREX AND HELD
UNDER VARIOUS CONDITIONS OF SALINITY AND TERMPERATURE.

Incubation 15 ppt - 10 C 15 ppt -21 C 15 ppt - 30 5 ppt - 21 C 30 ppt - 21 C

period
after
pesticide _, a a a ¢
added 2 “ 2 x £ “ £ ‘ 2
ayy) EBB bp £ ¢@ &
8 z 8 z 8 = 8 z 8 z
0 uF -143¢ 91 98 96 168° | 56 54 lll 10
4 442 420 662651 722 772, | 645648 532 487
9 743 697 942843 933 878 {1066 938 807695
16 802 792 892 845 | 1155 907? | 798 755 748 682
22 868 824 1177 1046 | [3885] [10,136)* | 710 668 966 952
30 680 679 980 1136 608 645 | 745-769 1080 ae?
44 972 926 | [8300] 4485 918 802 {1236 977 |: [5638] 1370°

a Mean of three replicates with five plates counted on each replicate
{J Poor agreement among replicates which differed by one log in count.

Treatment counts significantly different from control counts at 95% level as deter-
mined using t-test.
Treatment counts significantly different from control counts at 99% level as deter-
mined using t-test.

Dehydrogenase Activity-

Dehydrogenase activity is used as an indication of anaerobic metabolism in sediments. Pamat-
mat and Bhagwat" pointed out that anaerobic metabolism may greatly exceed aerobic metabolism
in sediments. Therefore, a measure of this activity was used to evaluate the effect of pesticides on
microbial activity.

Since previous studies had indicated variability among lots of technical grade pesticides, purified
preparations of the pesticides as well as the technical grade material were employed in these studies.

As shown in Table 34, preparations of mirex added to sediments produced no alteration in the
dehydrogenase activity when compared to untreated sediments. At some incubation times, the
dehydrogenase activity was increased slightly and in others it was decreased slightly when compared
to the control. Differences in levels of mirex did not alter this phenomenon.

All preparations of carbofuran at a concentration of 100 ppm significantly depressed the
dehydrogenase activity (Table 35). At concentrations of 10 and 1 ppm carbofuran, depression of
dehydrogenase activity was noted in only a few cases and in some cases, the activity was higher in
amended sediments than in untreated ones. Overall, there appeared to be little difference among the
different preparations of carbofuran.

31


10.

ll.

12.

13.

SECTION VIII
REFERENCES

EPA. 1972 Summary of Registered Agricultural Pesticide Chemical Uses. III-C.1-4.2, 1972.

Kennedy, M., M. Holliman and B. R. Layton. (Personal communication) Identification of the Ma-
jor Thermal Product of the Insecticide Mirex, to be published J. Ag. Food Chem.

Alley, E. G., D. A. Dollar, B. R. Layton and J. P. Minyard, Jr. Photochemistry of Mirex. J. Agr.
Food Chem. 21(1):138-139, 1973.

Alley, E. G., B. R. Layton and J. P. Minyard, Jr. Identification of the Photoproducts of the Insec-
ticides Mirex and Kepone. J. Agr. Food Chem. 22(3):442-445, 1974.

Dilling, W. L, H. P. Braendling and E. T. McBee. Pentacyclodecane, Chemistry II. Tetrahedron
23:1211-1224, 1967.

Metcalf, R. L., T. R. Fukato, C. Collins, K. Borck, S. A. El-Azie, R. Munoz and C. C. Cassil.
Metabolism of 2,2-dimethy]l-2,3-dyhydrobenzofuranyl-7-N-Methylcarbamate (Furadan) in
Plants, Insects, and Mammals. J. Ag. Food Chem. 16:300, 1968.

American Public Health Association (APHA). Standard Methods for the Examination of Water
and Wastewater. 13th ed. American Public Health Association, Inc., New York, 1971.

Cook, D. W. Metabolism of Carbohydrates by Hydrogenomonas eutropha. Ph.D. Dissertation,
Mississippi State University, 1966.

Hutton, W. E. and C. E. ZoBell. The Occurrence and Characteristics of Methane-Oxidizing
Bacteria in Marine Sediments. J. Bacteriol. 58:463-473, 1949.

Brown, L. R., R. J. Strawinski and C. S.McCleskey. The Isolation and Characterization of
Methanomonas methanooxidans (Brown and Strawinski). Can. J. Microbiol. 10:792-799, 1964.

Mitchell, Ralph, S. Fogel and I. Chet. Bacterial Chemoreception: An Important Ecological
Phenomenon Inhibited by Hydrocarbons. Water Res. 6:1137-1140, 1972.

Harrison, M. J., R. T. Wright and R. Y. Morita. Method for Measuring Mineralization in Lake
Sediments. Appl. Microbiol. 21:698-702, 1971.

Lowry, O. J., N. J. Rosenbrough, H. L. Farr and B. J. Randall. Protein Measurements with Folin.
Phenol Reagent. J. Biol. Chem. 193:265-275, 1951.

46


Table 10. EFFECT OF VARYING CONCENTRATIONS OF MIREX ON GROWTH OF PURE CUL-
TURES OF BACTERIA IN BROTH MEDIUM.

Culture Mirex (technical)

0.0 ppm 0.1 ppm_~—-1.0 ppm 10.0 pp

ob Bacterial counts after 72 h incubation
Wo/al | No/ul  No/alNo/al No/al

Bacillus (#222) 8.0 x 10° 2.0% 107 41x10" 1.2% 10° 2.4 x 10%
Achronobacter (#274) 1.6 x 10° 1.5 x10° 9.9x10° 7.8.x 10° 1.0.x 107
Vibrio (0276) 5.1107 4.2.x 10° 1.410" 7.8x 10° 9.5 410°
Benecka (#242) 3.3107 2.04107 7.9 x 10° 91x10" 6.2 x 10°
Pseudomonas (8297) 1.2 x 10° 6.4 410° 1.2 x 10% 1.3 x 10% 2.9 x 10°
Achromobacter (#223) 2.0.x 10° 3.3.x 10° 4.8.x 10° 1.6 x 10° 0.6 x 10°

4 8 8 2 8

Enterobacter (#189) 2.7 x 10° 12x10 0.5 x10" 1.3% 10° 1.6 x10

Acinetobacter (#173) 1.3.x 10° 7.3x10° 4.410% 4.8 x10° 3.1 x 10°

Conditions of test: Growth medium was nutrient broth prepared with 15 ppt
salinity seawater; temp of incubation was 25 C, Plate counts were deter-
mined in duplicate.

Table 11. EFFECT OF VARYING CONCENTRATIONS OF CARBOFURAN ON GROWTH OF PURE
CULTURES OF BACTERTA IN BROTH MEDIUM.

Culture carbofuran (technical)
0.0 ppm 0.1 ppm 1.0 ppm 10.0 ppm

oh _| Bacterial counts after 72 h incubation
No/nl | Wo/al  No/al  No/ml ——‘No/ml
Bacillus (6222) 8.0.x 10° 2.0x107 2.3% 107 1.0x%109 2,3 x 10°
Achromobacter (#274) 1.64 10° 1,5 x10? 7.1.x 10° 8.8 x 10° 8.7 x 10°
Vibrio (#276) 5.1.x 10° 4.220% 2.3x108 --- 8.5 x 108
Benecka (#242) 3.3.x 10° 2.0% 107 5.2 x 10° 11x10? 3.2 x 108
Pseudomonas (297) 1.2.x 10° 6,4 x 10% 4.2 x 108 9.4 x 10% 8.2 x 10°
Achronobacter (223) 2.0 x 10° 3.3 x 108 2.6 x 108 4.7 x 108 4.8 « 108
Enterobacter (#189) 2.7 x 10° 1.1% 10° 0.8x 10° 0.8 x 10° 1,2 x 10°

Acinetobacter (#173) 1.3.x 10° 7.3 x 108 8.5 x10 2.8x 10% 5.6 x 108

Conditions of tests: Growth medium was nutrient broth prepared with 15 ppt
salinity seavater; temp of incubation was 25 C. Plate counts were deter~
mined in duplicate.

Effect of Mirex and Carbofuran on Methane Utilization

No inhibition by mirex or carbofuran of methane utilization by estuarine sediments was observed
as determined by the total volume of gas consumed (Table 12). Although not of estuarine origin, a pure
culture of M.methanooxidans was employed in a similar study (Table 13). In this case, however, all
concentrations (0.1, 1.0, 10, and 100 ppm mirex and carbofuran) tested retarded gas consumption for
several days, but after 10 days of incubation, the gas consumption in the presence of test compounds
was the same as that in the absence of test compounds.

19


ABSTRACT

The purpose of this investigation was to help establish the chemical, physical and micro-
biological fate of mirex and carbofuran in the estuarine environment and determine the effect(s) on
important estuarine microorganisms and their activities. Chemical studies on the adsorption, fate
and hydrolysis were conducted. The microbiological studies involved the use of both pure cultures and
mixed cultures in a microcosm system and included twelve distinct physiological groups of micro-
organisms.

It was concluded that neither mirex nor carbofuran would have a deleterious effect on estuarine
bacteria under normal conditions, and there was no evidence of bioaccumulation. Degradation
products of both compounds were shown to be toxic to some microorganisms.

This report was submitted in fulfillment of Contract No. 68-03-0288 by Mississippi State Universi-
ty under the sponsorship of the Environmental Protection Agency. Work was completed as of 31
December 1974.

ii


All attempts to isolate the specific compound(s) responsible for the inhibitory activity of the
technical mirex failed.

In another series of experiments water-moistened mirex alone and in combination with HCB
were exposed to UV irradiation (20 min under high pressure mercury lamp, 1800-6000 A) and
employed in disc assay tests. The data (Table 41) clearly illustrate that irradiation of the mirex
resulted in a material which was toxic to Achromobacter #274. Curiously, irradiated mirex plus HCB
appeared to have a greater toxicity than irradiated mirex alone, even though nonirradiated mirex
plus HCB and irradiated HCB were nontoxic. Unfortunately, no record is available on the storage
conditions under which the batches of mirex were kept, and it could well be that they had a history of
exposure to moisture and UV irradiation over a period of time.

Table 41. RESULTS OF "DISC ASSAYS" OF UV IRRADIATED
HEXACHLOROBENZENE AND MIREX AGAINST CULTIRE

#274 (ACHROMOBACTER) .

Test eysten Zone of inhibition
w Wo WW
treatment treatment
Purified mirex # -
Technical mirex + -
CB - -
HCB + purified mirex (2:100) +H -
HB + purified mirex (2:10) HH -

® Increase in "#"'s represents an increase in the diameter of
the zone of inhibition

Since no organisms capable of utilizing either mirex or carbofuran were obtained, tests on the im-
pact of metabolites were impossible. Under the circumstances, the above named photoproducts of
mirex and 3-hydroxycarbofuran and 3-ketocarbofuran (known degradation products of carbofuran)
were employed in subsequent tests.

Results of Qualitative Studies on Effect of Mirex and Carbofuran Derivatives

Qualitative studies on the effect of derivatives of mirex and carbofuran on the hydrolysis of
starch, lipid, chitin, casein, and gelatin by several pure cultures of estuarine bacteria were conducted.
Four cultures, #222, #276, #242 and #280, were tested for chitin hydrolysis. Only cultures #242 and #280
showed the ability to hydrolysis chitin and neither of these cultures was affected by any of the test
compounds, Six cultures were utilized for the tests involving starch hydrolysis. Kepone and reduced
kepone inhibited growth of three out of six while the remaining compounds showed no inhibition.
None of the test compounds affected the growth of seven casein hydrolyzers. Growth of two out of six
gelatin hydrolyzers was inhibited by kepone, and one out of six by reduced kepone. Only one culture,
#242, illustrated the ability to hydrolyze lipid and it was not affected by any of the test compounds.


Bioaccumulation Studies on Mirex and Carbofuran-

Table 39 lists the results of a 48 h study on the bioaccumulation of carbofuran by culture #280.
Cells in the test system were removed by centrifugation and the carbofuran in the liquid was analyzed
by liquid chromatography. Peak heights were compared to an internal standard (carbaryl) in each
sample and the carbofuran to internal standard ratio was established for each test. Relative values
were obtained by making sample #2 equal to unity since it should not have a loss of carbofuran. The
relative values found in Table 39 are not significantly different with respect to tests with live cells,
dead cells and test with no cells. Sample #7, which contained the live cells + 1% glucose, had the lowest
relative value, but it also has a 27% increase in total solids due to increase in cell mass. These data in-
dicate that the carbofuran was removed from the test system by adsorption to the cells and not by
bioaccumulation.

Table 39, BIOACCUMULATION STUDIES USING CARBOFURAN.

Sample # Test system Ratio Relative value
carbofuran/internal 2 = 1)
standard
Controls
(no cells)
“a 100 ml Mineral salts
2 100 ml Mineral salts + 150 ppm carbofuran 1.22 1.0
#3 100 ml Mineral salts + 1% glucose
% 100 ml Mineral salts + 150 ppm carbofuran + 1% glucose a4 0.97
Live cells
#5 100 ml Mineral salts + 5% cells
% 100 ml Mineral ealts + 5% cells + 150 ppm carbofuran 1s 0.93
” 100 ml Mineral salts + 5% cells + 150 ppm carbofuran 1.09 0.89
+ 1% glucose
Dead cells
#8 100 ml Mineral salts + 5% celle
49 100 mi Mineral salts + 5% cells + 150 ppm carbofuran 1 0.92
#10 100 ml Mineral salts + 5% celle + 150 ppm carbofuran Las 0.96

+1% glucose

= No carbofuran detected, all values are the average of two determinations.

The protocol for the bioaccumulation studies with mirex was the same as that for the studies with
carbofuran. After 48 h incubation, the cells were removed by centrifugation and the mirex remaining
in the liquid was determined by standard techniques outlined by the official method of the Food and
Drug Administration (Anonymous"®) using electron capture gas liquid chromatography. The results
are listed in Table 40. By considering the amount of mirex found in the liquid of samples #7 & 8 to be
100%, it can be noted that the amount adsorbed by the dead cells (samples #9 & 10) was about 95% of
the total present. The live cells (samples #11 & 12 adsorbed only 88% of the mirex even though there
was a 27% increase in total mass of cells due to multiplication. Within the limitations of the test
system used, it is concluded that there was no bioaccumulation of mirex occurring using culture #280.

36


Figure 2. Structural configuration of mirex and derivatives of mirex.

Ry
2

Rs,

Ry
Ry

For mirex, R, ~ R, = R3=R,=R,=Cl

For kepone, R +R = double bond oxygen; R3~ R,~ R, = Cl
For 8-hydrogen derivative, 1, R,=H;R,~R2=R3=R,=Cl

For 2,8-dihydrogen derivative, 2, R,4=R,=H;R,~ R,2=R3;-C
For 10-hydrogen derivative, 3, R, = H; R,=R3=R,=R,=C1
For 5,10-dihydrogen derivative, 4, R, = Rg ~ H; Rp = R,=Rs5=Cl
For reduced kepone, 5, R3 = OH; R, = R2 = Rg =R5=Cl

METHODS

Chemical Methods

Analysis of Carbofuran and its Derivatives-

Anumber of problems was encountered in attempts to develop good analytical methods for these
compounds. Direct analysis by gas chromatography was extensively studied utilizing a conductivity
detector that is specific for nitrogen. Despite numerous approaches that were tried, decomposition
during the analysis turned out to be an insurmountable problem.

Direct determination by ultraviolet absorption (Metcalf, et al.5) was not suitable for the
hydrolysis studies because it was shown that the absorption observed by these workers was not dueto
either the carbofuran or any of its identified derivatives, including the phenol.

Several other methods were tried and failed, but two methods were found to provide satisfactory
results. The gas-liquid chromatography (glc) method is most suitable for low levels and where car-
bofuran and its 3-hydroxy- and 3-keto-derivatives are expected to occur together. The high pressure
liquid chromatography (hplc) method is best suited for higher concentrations and where only thecar-
bofuran is expected to occur along with its phenolic hydrolysis product.


TECHNICAL REPORT DATA .
(Please read Instructions on the reverse before completing)

REPORT NO. 2 3. RECIPIENT’S ACCESSIONMNO,
EPA~660/3-75-024

TITLE AND SUBTITLE

5. REPORT DATE

March 1975

=

The Effect of Mirex and Carbofuran on Estuarine
Microorganisms

6. PERFORMING ORGANIZATION CODE

AUTHOR(S) Ig. PERFORMING ORGANIZATION REPORT NO.

Lewis R. Brown, Earl G. Alley, David W. Cook

=

. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO.

Mississippi State University

ro

1_FAQ77
Mississippi State, Mississippi 39762 77. CONTRACT/GRANT NO.

68-03-0288
13, TYPE OF REPORT AND PERIOD COVERED

12. SPONSORING AGENCY NAME AND ADDRESS

U.S. Environmental Protection Agency
National environmental Research Center
Office of Research and Development

Corvallis, Oregon 97730
15, SUPPLEMENTARY NOTES

14. SPONSORING AGENCY CODE

ROAP/TASK NO. 1O0AKC/33

16, ABSTRACT

The purpose of this investigation was to help establish the chemical, physical
and micro-biological fate of mirex and carbofuran in the estuarine environment and
determine the effect(s) on important estuarine microorganisms and their activities.
Chemical studies on the adsorption, fate and hydrolysis were conducted. The micro-
biological studies involved the use of both pure cultures and mixed cultures in a
microcosm system and included twelve distinct physiological groups of microorganisms.

It was concluded that neither mirex nor carbofuran would have a deleterious
effect on estuarine bacteria under normal conditions, and there was no evidence of
bioaccumulation. Degradation products of both compounds were shown to be toxic to
some microorganisms.

This report was submitted in fulfillment of Contract No. 68-03-0288 by Missis-
sippi State University under the sponsorship of the Environmental Protection Agency.
Work was completed as of 31 December 1974.

7. KEY WORDS AND DOCUMENT ANALYSIS
b.IDENTIFIERS/OPEN ENDED TERMS |c.

a. DESCRIPTORS cosati Field/Group

Estuarine Environment Chemical Studies

Toxicity

Mirex - Fate Microcosm
Carbofuran - Fate Adsorption Fate
Degradation Products Hydrolysis

Physiological Groups

18. DISTRIBUTION STATEMENT.

19. SECURITY CLASS (This Report)

21. NO. OF PAGES

Unclassified 69
20. SECURITY CLASS (This page) 22. PRICE
Unclassified

EPA Form 2220-1 (9-73)

f US GOVERNMENT PRINTING OFFICE 1975-698.639 1163 REGION 10



tant decanted into a 1 liter separatory funnel. The petroleum ether layer was washed twice with water,
the organic layer dried and introduced into a florisil column. After this point in the procedure, the
water method was followed exactly.

Other Chemical Methods-

Nitrate, nitrite and ammonia were determined by standard procedures (APHA’). Protein-
nitrogen was determined according to the modification of Lowry’s procedure (Cook®).

Microbiological Methods
Dise Assay Techniques-

A number of variations of the disc assay procedure was employed to test the effect of mirex on
bacteria. Tests involved dissolving test compounds in acetone and placing the material on number
740E, 12.7-mm paper discs (Scheicher and Schnell, Inc., Keene, N.H.). The saturated discs were kept
overnight to allow the acetone to evaporate. In other tests, solid discs of test compounds were prepared
by using an hydraulic press with a 12.7-mm die and 137,880,000 N/m? (14,062,000 kg/m; 1 kg =9,805
newtons) pressure. In other tests, loose particles of test compounds were applied directly on the sur-
face. In all cases, seeded nutrient agar plates were employed with incubation at 30 C for 24 h.

Growth Curve Studies-
Nutrient broth medium was employed and growth was monitored by plate count or changesin op-

tical density of the broth using a B&L Spectronic-20 colorimeter (520 nm). Studies were conducted in
duplicate at 30 C for 57h.

Qualitative Physiological Studies-

Qualitative observations of test-tube reactions were employed to evaluate the effect of varying
concentrations of test compounds on glucose fermentation and hydrogen sulfide production. Glucose
fermentation studies were conducted in Durham fermentation tubes using 1% glucose broth with
phenol red indicator. Lead acetate semisolid agar was employed to test for hydrogen sulfide produc-
tion. For these tests, the compounds were added aseptically to the test media after sterilization. In-
cubation was at 30 C for 5 days.

Bacto-gelatin agar, Bacto-spirit blue agar with cottonseed oil, Bacto-starch agar, skim milk, and
finely ground purified chitin in % strength Bacto-marine agar were employed for the studies on
gelatin-, lipid-, starch-, casein-, and chitin-hydrolysis, respectively. Test compounds were applied to
the plates using three different techniques: (1) test compounds (in acetone) were spread over the en-
tire surface of the agar, the acetone was allowed to evaporate, and the test bacteria were streaked ina
single line across the surface; (2) the test compounds (in acetone) were applied in a single line on the
surface of the agar and the test culture was streaked evenly over the entire plate; and (3) filter paper
strips impregnated with test compounds were placed in the agar for streak plates or on the surface of
pour plates.


EPA~660/3-75-024
JUNE 1975

THE EFFECT OF MIREX AND CARBOFURAN
ON ESTUARINE MICROORGANISMS

By

Dr. Lewis R. Brown
Associate Dean Arts and Sciences
and Professor of Microbiology
Mississippi State University
Mississippi State, Mississippi 39762

Dr. Earl G. Alley
Director Research and IAS Division
Mississippi State Chemical Laboratory
Mississippi State, Mississippi 39762

Dr. David W. Cook
Assistant Director and Head
Microbiology Section
Gulf Coast Research Laboratory
Ocean Springs, Mississippi 39564

Contract No. 68-03-0288
Program Element No. 1-EA077
ROAP/TASK No. 10AKC/33

Project Officer

Dr. Al W. Bourquin
Gulf Breeze Environmental Research Laboratory
Sabine Island
Gulf Breeze, Florida 32561

National Environmental Research Center
Office of Research and Development
US. Environmental Protection Agency
Corvalis, Oregon 97330

For eale by the Superintendent of Documents, U.S, Government
Printing Office, Washington, D.C. 20402


Table 29. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS ONE WEEK AFTER AMENDING WITH CARBOFURAN.

Amended with carbofuran

Control 0.1 ppm 10 ppm 1000 ppm 10,000 ppm

Microbial #/ml Tank ‘Tank Tank Tank Tank Tank Tank Tank Tank Tank
jerobiet Sroup x 25 26 27 28 29 30 3 32 33 34
Nitrate reducers 10> 49.0 1.7 _—79.0—-79,.0 240.0 49.0 7.0 13,0 2.8 2.0
‘Ammonifers 10° 7.9 4.9 13.0 3.37.9 14,0 33,0 24.0 33.0 14.0
H2S producers 10° 0.5 2.4 1.3 1.7 7.9 14 0.03 0.3 3.3 0.5
Sulfate reducers 104 3.3 3.3 2.2 2.8 2.4 2.8 4.9 3.3 0.95 0.8
Lipid hydrolyzers 105 3.3 11.0 22.0 7.9 7.94.9 49.0 7.9 33.0 33.0
Casein hydrolyzers 105 4.9 2.4 3.3 3.3 2.2 1.30 11.0 13.0 49.0 13.0
Gelatin hydrolyzers 105 4.9 7.9 17.0 7.9 13.0 4.9 2.4 49.0 24.0 2.4
Starch hydrolyzers 10> 2.22.2 4.9 4.9 0.8 13 4.9 2.2 79 1.9
Chitin hydrolyzere 104 4.9 4.6 3.3, 2.2 27 2.2 14 0.8 0.8 0.5
Aerobic plate count 10° 10.4 11.2 12.6 11.7 6.7 8.5 3.8 4A 5.9 6.1
Anaerobic plate count 10® 6.6 6,2 10.0 9.2 4.6 5.4 3.3 3.8 5.8 4.1

Table 30, MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS TWO WEEKS APTER AMENDING WITH CARBOFURAN.

Amended with carbofuran

Control 0.1 po 10 po 1000 ppm 10,000 ppm

uicrobia? group #/ol Tank Tank Tank Tank Tank Tank Tank Tank Tank Tank

x 25 26 27 28 29 30 31 32 33 34

Nitrate reducers 10° 79.0 49.0 4.9 170.0 4.5 21.0 79.0 240.0 130.0 240.0
Ammonifere 105 1.3 4.9 7.9 13.0 4.9 14.0 66,0 13.0 17.0 0.8
B28 producers 10° 08 080.3 0S SSB
Sulfate reducers 104 337.9 3.3 24 330
Lipid hydrolyzers 105 3.3 13.0 4.9 33.0 22.0 7.9 79.0 33.0 79.0 33.0
Casein hydrolyzers 10° 43 3 4.6 2.9 7.0 49 17.0 13.0 17.0 17.0
Gelatin hydrolyzers 105 3.3769 24 13.0 4.9 1.0 130,0 22.0 240.0 130.0
Starch hydrolyzers 10° 1300012 2.2 49 103 3B 8
Chitin hydrolyzers 104 0.9 3.3) 0.5 4.9 3.3 49 0.8 ° ° 0.4
Aerobic plate count 1o® = 14.0 19.2 22.4 = 19.5 20.5 14.5219) 26.3 35.0320
Anaerobic plate count 10° 7.8 10,8 12.0 12,4 10.8 9-8 12,5146 AS11352

29


Hydrolysis and Aqueous Photochemical Studies with Mirex

Hydrolysis of mirex was studied in four systems: Distilled water, saline water 15 ppt, distilled
water with 1 ppm arginine added, and distilled water with 1 ppm arginine added and exposed to
sunlight. Table 7 summarizes the results.

Table 7. HYDROLYSIS OF MIREX

Concentrations of mirex in ug/l

Time Distilled 15 ppt 1 ug/t 1 ve/t
Days 20 Salinity Arginine Arginine plus

Light

0 0.23 0.20 0.13 0.23

30 0.33 0.14 0.12 0.40

60 0.13 0.06 0.38 0.13

90 O.uL 0.03 0.05 0.04

The numbers in Table 7 are for the most part the average of three replicates. The percent standard
deviation for these data is about 65. While the precision is not high, the data show a definite trend
towards disappearance of mirex with time.

Hydrolysis of Carbofuran

The hydrolysis of carbofuran was studied in 0.005 M sodium carbonate, buffered solution at 0 and
15 ppt, 25 C and pH 10. The initial concentration was about 300 ppm in all cases. Solutions were
prepared by adding the appropriate amount of carbofuran in 30 ml of ethanol to 220 ml of the buffer
solution. Aliquots were removed at various time intervals for analysis by liquid chromatography.
These data are shown in Tables 8 and 9.

Table 9, HYDROLYSIS OF CARBOFURAN
Table 8. HYDROLYSIS OF CARBOFURAN

0 ppt salinity pH 10

15 ppt salinity pH1O 0.005% NazCo3

Ta y cnben Time % Carbofuran
ime arbofuran (mt remainis
(win) remaining oe) in
4 100
4 100
10 89
9 4
20 83
25 80
35 3
35 7
45 n
45 72
38 64
55 70
65 62
65 69
85 37
5 65
55
85 63 *
105 35
95 64
us 35
105 60

17


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:

Environmental Health Effects Research
Environmental Protection Technology
Ecological Research

Environmental Monitoring
Socioeconomic Environmental Studies

aon

This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series. This series describes research on the effects of pollution

on humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine the
fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living
organisms in the aquatic, terrestrial and atmospheric environments.

EPA REVIEW NOTICE

This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval does
not signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or
recommendation for use.


Table 45, EFFECT OF MIREX AND CARSOFURAN DERIVATIVES ON NITRIFICATION BY MICROORGANISMS IN ESTUARINE SEDIMENTS.

Days of sncubgeion
rest system ° 5 10 15 20
TWO, cW WOPPAT MAN WD, NWO, NMG Wy NO." PAHS WopWW WO, WP WE Woy-W NO.
caghi) (og?1) (mgt) kmg?t) (agi) (mgt) {ng/1) (ogF1) (we71) |mgf1) (ag?) (ag?) |cmg)r) Cog ht) (we 1]
ark 130" 130 130 130 130
trol 330 128 125 trace trace] 100.02 6 | 90 12.9
l>-ketocarbofuran
10 pa 130 a8 w+ 9.02
1 ppm 130 130 7 + + 90.02
b-tiyaroxycarboturan
0 pps 130 130 ar + + fos ca | 120.02 <a
1 pe 130 128 us + +] 9 + <1 | 55° lor <'s
spone 10 ppm 130 128 124 + + 120 + <1 120 + <1
1 ppm 130 130 1s + + 110 + <1 100 + 4.5
Reduced tepone 10
20 poe 130 128 ws + 4 | 99 or <1 | 00.0% <a
1 pe 130 130 yo + 4 | 50 lo <i | 95 low wis
2, 8-pshyarogen
Birex 10 ppa | 130 130 w+) + | aos 102 <a | os a oe
1ppe | 130 128 y+ 4 | 90 lon <n | as az ‘8
ho-syarogen
and 5,10-dihydrogen —
drex 10 ppm 130 130 120 + + 1030.02) <1 85 12 8
1ppa | 130 130 yo + +! 9 lo <a | as liz Je

Conditions of test: Medium: 15 ppt Rila salts + MH, 1+ mineral salts, ph 7-0, Incubation; 30 C,shake conditions.
All values are the average of tvo determinations

Effect of Mirex and Carbofuran Derivatives on Metabolic Activity

and Primary Productivity

The results of the effect of mirex derivatives and carbofuran derivatives on the primary produc-
tivity of natural estuarine waters are reported in Table 46. In light of the low primary productivity,

Table 46. THE EFFECT OF MIREX AND CARBOFURAN DERIVATIVES ON THE METABOLIC
ACTIVITY AND PRIMARY PRODUCTIVITY OF ESTUARINE POND WATER.

Test system Metabolic activity Primary
(mg Oy consumed/l —_ productivity
in 12h) (ag 0) produced/1
in 12 h)

Control 0.95 2.35
Acetone control 0.95 2.10
HgC17 Control 0.00 0.00
Reduced kepone (1 ppm) 0.90 0.80
Qo ppm) 0.85 0.50
10-hydrogen der. of mirex (1 ppm) 1.25 1.80
(0 ppm) 1.10 1.50
B-hydrogen der. of mirex (1 ppm) 1.00 1.50
(19 ppm) 11s 1.70
2, B-d thydrogen der. of mirex (1 ppm) 1.15 1.65
(20 ppm) 1.05 1.65
3-ketocarbofuran (1 ppm) 1.10 2.20
(10 ppm) 0.80 0.95
3-hydroxycarbofuran (1 ppm) 1.20 1.60
(0 ppm) 1.10 1.35

All values are the average of two determinations.

41


Table 21. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD 107M ASPARAGINE IN THE
PRESENCE OR ABSENCE OF 50 PPM MIREX OR CARBOFURAN.

Sample No. 1 2 3 4
Contents
in vial Cells in Rila Cells in Rila Cells in Rila Celle in Rila
Acetone residue Acetone residue Acetone residue Acetone residue
Contents in
capillary Mirex residue Carbofuran residue
tube Rila Rila Rila
10~4m Asparagine 10744 Asparagine 1074M Asparagine
36 49 53 80
(a) 41 Ave. 58 Ave. 62 Ave. 93° Ave.
No.colonies 4h G2 65 60 73 (66 9893
on plage 45 66 74 102
at 10
dilution
36 65 80
(b) 43 Ave. 65 Ave. 84 Ave.
48 47 70 70 -- 90-88
60 81 - 96
ae 37 76 56
(a) 14 Ave. 62 Ave. 85 Ave. 67 Ave.
15 16 77 69 86 83 70 68
22 79 86 79
Overall ave. 35 66 14 83

(a) Replicates obtained from experiment in which time sequence began with sample no, 1 followed sequen-
tially by samples no. 2,3, and 4.

(b) Replicates obtained from experiment in which time sequence began with sample no, 4 followed sequen-
tially by samples no. 3,2, and 1.

Table 22. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD 10 PPM MIREX OR CARBOFU-
RAN IN THE PRESENCE OF 0.08% NUTRIENT BROTH.

Sample No. 1 2 3 4

Contents
in vial Cells in Rila Cells in Rila Cells in Rila Cells in Rila
Acetone residue Acetone residue Acetone residue Acetone residue

Contents in

capillary oo Mirex residue Carbofuran residue
tube Rila Rila Rila
oon Nutrient broth Nutrient broth Nutrient broth
No.colonies 69 107 140 96
on plate 72 Ave. 125 Ave. 153 Ave. 110 Ave.
at 10TM 730°«72 1310-124 160 156 111109
dilution 74 131 173 118

25


Table 36, PROTEOLYTIC ACTIVITY OF MICROORGANISMS IN SEDIMENT SAMPLES AMENDED WITH CARBOFURAN AND MIREX.

Concentration
of pesticide Sediment incubation period after pesticides added
added to
sediment
(ppm) Initial 1 week 2 weeks 3 weeks 4 weeks
wares ert r eer r ster eens -- Mirex amended sediments
0 81.0" 85.0 75.6 82.0 76.0
1000 79.0 82.0 77.0 75.0 74.0
100 80.0 86.0 77.1 74.0 72.0
10 79.0 87.0 80.6 78.0 74.0
1 73.0 87.5 83.7 76.0 75.0
alae Carbofuran amended sediments ---------------------
0 81.7 90.3 82.7 74.7 82.0
100 —_ 71.0 81.3 69.9 80.3
10 75.2 72.6 83.7 69.9 83.5
1 80.8 77.4 81.3 70.6 80.0

@- Percent of added peptone protein utilized during 22 hour incubation period.

Average of four replicates,

Table 37. MINERALIZATION OF GLUCOSE BY MICROORGANISMS IN SEDIMENTS AMENDED WITH VARIOUS CONCENTRATIONS OF
CARBOFURAN AND MIREX.

Concentration . . .
of pesticide Sediment incubation period after pesticide added
added to
sediment
(ppm) Initial 1 week 2 weeks 3 weeks 4 weeks
wenn ee eee teen eee Mirex amended sediments ---------------------+----+-------0--
0 11588 1715 1328 941 1040
1000 922° -21,0> 222% +29.7 1277, - 3.8 877 - 6.8 890 -14.4
100 1033° -10.8 1690 - 1.5 1317 - 0.8 829 -11.9 1123 + 7.9
10 918° -21.0 1818 + 5.9 1246 - 6.2 855 - 9.1 910 -12.6
1 g97° -22.5 = 1583 - 7.7 1369 + 3.1 gol - 5.3 894 -14.0

Carbofuran amended sediments

0 518 1165 1022 719 1758
1000 228° -56.6 233° -80.3 230° -77.5 169° -76.5 219° -87,5
100 3758 -27.6 904° -22.4 763° -25.3 554° -22.9 1244¢ -29,2
10 298 -43.1 1149 - 1.4 987 - 3.4 721 + 0.3 1789 + 1.8
1 2a -45.8 1261 + 8.2 991 - 3.0 603° -16.1 1728 - 1.7

4~ Level of radioactivity (CPM) in the C02 produced from the mineralization of C14 -
glucose. Average of four replicates.

b. Percent difference between control and treated sediments.

© - Significantly different from control (P>.05). t-test.

34


Table 12. EFFECT OF MIREX AND CARBOFURAN ON METHANE CONSUMPTION BY
MICROFLORA IN ESTUARINE SEDIMENTS.

Time in Days
Text system 304 5 6 7 8 9 1 W 12
Control 9 18 29 37 48 59° 70 73 73
Mirex 0.1 ppm ie 26 40 48 «57 64 75079777
Mirex 1.0 ppm 1s 2% 35 44 SL 59 66 «6730-7977
Mirex 10.0 ppm 15 240 37 4859 6B 7S 777.
Mirex 100.0 ppm 2029 «37 «48 55 66 7777

carbofuran 0.1 ppm 11. «L206 —« 33. 4255 6H HBO.
carbofuran 1.0 ppm «15. 22,3 4G HCCC 777,
carbofuran 10.0 pn 11 «15 24 «37 4455 6675S

Carbofuran 100.0 ppm 11 11 22 33 42 53 66 73° «77 77

*ml of gas consumed,

Conditions of test: Sohngen unit apparatus, medium 15 ppt salinity with
mineral salts, incubated under gas atmosphere of 65% CH,, 30% 0» and
5% CO,, static conditions. All values are the average Of three deter
ninations.

Table 13. EFFECT OF MIREX AND CARBOFURAN ON METHANE CONSUMPTION BY
METHANOMONAS METHANOOXIDANS .

Time in days

Test system 2 3 4 5 6 7 10 12
Coatrol 4? 15 29° 4666707375 BL
Mirex 0.1 ppm 4 W Ym 33 4 70 «730-77
Mirex 1.0 ppm 4 13° 20 29° «42 536877
Mirex 10.0 ppm 4 15 22 29 42 4B HSCS?
Mirex 100.0 ppm 7 13 18 2% 33 42 70 73° «77

Carbofuran 0.1 ppm 4 «= 18S 29 44 GZ 707375 BO
Carbofdran 1.0 ppm = @ «01S 22 LST 8D
Carbofuran 10.0 ppm 7 «15 2637, 57 62s 70s BL

Carbofuran 100.0 ppm 4 15 24 35 53 64 68 70 7

ml of gas consumed ,,

Condition of test: Sohngen unit apparatus, medium - mineral salts, incu-
bated under gas atmosphere of 65% CH,, 30% 0, and 5% CO,, atatic condi-
tions. All values are the average of ‘three determinatians.

Effect of Mirex and Carbofuran on Ammonification

The data in Table 14 demonstrate that mirex and carbofuran (100 ppm) did not inhibit the produc-
tion of ammonia from protein by four cultures of bacteria [Benecka (#280), Pseudomonas (#297), and
two species of Achromobacter (#223 and #274)]. In the case of Achromobacter #274, substrate (pro-
tein) disappearance was decreased by the mirex even though the amount of ammonia produced was
not decreased.


SECTION IV
MATERIALS AND METHODS

MATERIALS

Carbofuran

Technical carbofuran (98% purity) and analytical standards (99% purity) were obtained from
FMC Corporation. Analytical standards of 3-hydroxy- and 3-ketocarbofuran as well as the three
phenol metabolites were obtained from the same source. Technical carbofuran was further purified by
recrystallization from toluene in one case, and ethanol in another. (See Figure 1)

Mirex

One batch of mirex (batch 1) was obtained from the FDA Laboratory at Perrine, Florida. All other
batches of mirex (technical) were obtained from Allied Chemical Company. This material was
purified by chromatography on a silica gel column with cyclohexane as the solvent. Examination of
the technical material and some analytical standards showed the presence of hexachlorobenzene and
other less abundant compounds of similar volatility. Hexachlorocyclopentadiene was usually present
in very small quantities; and, while the other compounds could not be identified, it was shown that
they were not hexachlorofulvene or 1,2,3,4,5-pentachloro-5-trichloromethyl cyclopentadiene. The
total of these impurities was estimated to be about 0.1%. The chromatographic purification effectively
removed these impurities when only the early eluting fractions were utilized.

The major product of the pyrolysis of mirex is hexachlorobenzene (Kennedy et al.”), so the
presence of this impurity is not surprising.

Kepone

Technical kepone was obtained from Allied Chemical Company and purified by azeotropic dis-
tillation of water and crystallization from benzene.

Mirex and Kepone Photoproducts

1,2,3,4,5,5,6,7,9,10,10 undecachloro [5.3.0.0?-°.03 9.04. 8] pentacyclodecane, 1, 1,3,4,5,5,6,7,9,10,10-
decachloro [5.3.0.02: ®.0%. °.04. 8] pentacyclodecane 2, 1,2,3,4,5,5,6,7,8,9,10 undecachloro
{5.3.0.02- 6.0%. °.04- 8] pentacyclodecane 3 and 1,2,3,4,5,6,7,8,9,10-decachlora [5.3.0.0?: 6.0% 9.04 8] pen-


this experiment was rerun using a water previously enriched in phytoplankton population. Table 47
lists the results of this experiment. No growth inhibition was observed with the 10-hydrogen mirex or
8-hydrogen mirex. Both concentrations (1 and 10 ppm) of the reduced kepone, kepone, 3-
ketocarbofuran, 3-hydroxycarbofuran reduced the primary productivity. A reduction in the primary
productivity also was noted with the 2,8-dihydrogen mirex, but the reduction was not as great as for
the above compounds.

Effect of “Wing Derivatives” of Mirex (Mixture of 10-hydrogen and
5,10-dihydrogen mirex) on the Dehydrogenase Activity in Sediments

Table 48 lists the results of the effect of a mixture of “wing derivatives” of mirex (10-hydrogen and
5,10-dihydrogen mirex) on dehydrogenase activity in sediments. No reduction in dehydrogenase ac-
tivity was observed with any of the concentrations of the test compounds. On the other hand, several
of the concentrations showed a stimulatory effect on dehydrogenase activity of sediments sampled up
to 28 days after application of the test compound.

Table 47, THE EFFECT OF MIREX AND CARBOFURAN DERIVATIVES ON THE METABOLIC
ACTIVITY AND PRIMARY PRODUCTIVITY OF ESTUARINE POND WATER EN-
RICHED FOR PHYTOPLANKTON.

Test system Metabolic Primary
activity productivity
(mg 0p consumed/1 (mg 0) produced /1
in 12h) in 12h)

Control 1.40 4.65
Acetone control 1.30 4.85
HgCly control 0.00 0.00
Reduced kepone 1 ppm 1.25 1.35
10 ppm 1,00 0.30
Kepone 1 ppm 1.20 3.60
10 ppm 1.20 1.80
10-Hydrogen mirex 2 ppm 1.20 5.15
10 pen 1.20 4.60
Betydrogen mirex 1 ppm 1.35 5.20
10 ppm 1.35 4.85
2,8-Dihydrogen mirex 1 ppm 1.35 4.40
10 ppm 1.40 4.05
3+Ketocarbofuran 1 ppm 1.45 4.10
10 ppm 1.45 3.05
3-Hydroxyearbofuran 1 ppm 1.40 3.90
10 ppm 1.10 2.70

All values are the average of two determinations.

42


SECTION VI
SUMMARY

At least 95% of the mirex in aqueous solution is removed by adsorption to organic matter (dead
bacterial cells), kaolinite clay and montmorillonite clay within 2, 7 and 30 days, respectively. Only 8-
15% of the carbofuran is removed from aqueous solution by adsorption to these materials.

No significant decrease in mirex concentration occurred in a sediment-water system exposed to
sunlight for 130 days, but there was evidence of some disappearance in purely aqueous solutions. Con-
trarywise, approximately 75% of the carbofuran disappears within 49 days in sediment-water system,
but no detectable residues of 3-hydroxy- or 3-ketocarbofuran were found.

In disc assay tests, carbofuran failed to inhibit the growth of any of the 20 bacterial isolates tested
while the results with mirex varied from batch to batch with one batch producing zones of inhibition
on 18 of the 20 cultures. Gas chromatographic analyses demonstrated that different batches of mirex
contained varying amounts of unidentified impurities. For the most part, the remaining studies were
conducted with purified mirex.

In pure culture studies, mirex and carbofuran failed to inhibit gelatin hydrolysis, starch
hydrolysis, lipid hydrolysis, casein hydrolysis, chitin hydrolysis, glucose fermentation, H,S produc-
tion, nitrate reduction, ammonification and methane utilization. These concentrations also failed to
inhibit methane utilization and nitrification by estuarine sediments. Ten ppm mirex and carbofuran
did not inhibit the metabolic activity of estuarine pond water, but did inhibit primary productivity.
Concentrations of 1000 ppm mirex and carbofuran did not effect the growth curves of bacterial
cultures in broth.

Microcosm studies confirmed the lack of inhibition by mirex. A concentration of 1000 ppm did not
significantly alter the mixed microbial populations or the microbial activities of estuarine sediments.
On the other hand, the addition of carbofuran to sediments in concentrations of 100 ppm or greater
caused an increase in the numbers of aerobic heterotrophic bacteria, but did not favor the develop-
ment of any single type of bacteria. Further, the addition of carbofuran to sediments, in concen-
trations of 100 ppm or greater, significantly reduced the sediment dehydrogenase activity, the ability
of the sediment microflora to mineralize glucose, the rate of oxygen utilization during glucose
assimilation by sediment microorganisms, and the production of hydrogen sulfide by sediment
bacteria.

Degradation products of mirex and carbofuran were shown to be far: more toxic to
microorganisms than were the parent compounds. Of the mirex degradation products, kepone and
reduced kepone exhibited the greatest toxicity. The 3-hydroxycarbofuran was more toxic than either
carbofuran or 3-ketocarbofuran.

Ultraviolet irradiation of moist mirex resulted in a bacteriologically toxic material.

44


Analysis of Carbofuran in Water by hple-

Analyses were performed directly on the aqueous solutions by injecting the samples into a 1.8m
by 1.5 mm i.d. column packed with Corasil II. The packing was deactivated by equilibrating it with
diisopropyl ether saturated with water and the same solvent system was used for the mobile phase.
Carbaryl was used as an internal standard and the quantitation done by measuring peak heights.

Analysis of Carbofuran in Sediments-

Ten grams of sediment and water were shaken with 50 ml of methylene chloride in a centrifuge
bottle. The sediments were removed by centrifugation and filtration through glass wool. The sedi-
ment was treated twice more with 25 ml of methylene chloride. The combined extracts were placed ina
separatory funnel, and the methylene chloride layer was removed. The aqueous phase was washed
with 25 ml of methylene chloride, the combined extracts dried with sodium sulfate and the solvent
removed with a Kuderna-Danish evaporator. The residue was taken to dryness under a stream of
nitrogen and then redissolved in 5 ml of 15% diethyl ether in petroleum ether. The entire sample was
introduced into a 1 cm diameter column containing 4 g af activated florisil which had been prepared
with 10 ml of petroleum ether. The column was eluted with 70 ml of 15% diethyl ether in petroleum
ether followed by 100 ml of 50% diethyl ether. The 50% fraction was reduced to a small volume and
transferred to a Miniaktor (Applied Science Laboratories, Inc.) and the solvents removed with a
stream of nitrogen. Ethyl acetate (0.2 ml) and heptafluorobutyric anhydride (0.1 ml) were added to the
reactor, and it was tightly capped, wrapped with aluminum foil and heated at 45 C for 16 h. The con-
tents were diluted with 10 ml of petroleum ether, washed three times with water, and the organic layer
dried and made to an exact volume for electron capture gas chromatographic analysis. The
parameters for this analysis were: a 1.5m by 4mmi.d. all glass column utilizing on-column injection
was used; the column temperature was 165 C, the injector 245 C, and the detector 210 C. The column
was packed with 3% diethylene glycol succinate on Gas-Chrom Q. With these conditions, carbofuran
had a retention time of 1.2 min, 3-hydroxycarbofuran 1.8 min, and 3-ketocarbofuran 2.4 min.

Analysis of Mirex and its Derivatives-

Mirex and the photoproducts described in the materials section can all be quantitated by the
following procedures.

Analysis of Mirex in Water-

A sample of the water to be analyzed was extracted three times with 75 ml portions of petroleum
ether. The combined extracts were dried and passed through a florisil column. Petroleum ether was
used to elute the column and the eluate concentrated to 1-10 ml and then quantitated by electron cap-
ture gas chromatography. This method was shown to produce high recoveries (90-100%) of the com-

pounds.

Analysis of Mirex in Sediments-

Sediments were analyzed in the same manner as waters except that the samples were shaken
with two volumes of 1:1 acetone-petroleum ether. The samples then were centrifuged and the superna-