Toxicology, 4 (1975) 183--194 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
UPTAKE, ELIMINATION, AND BIOTRANSFORMATION O F THE LAMPRICIDE 3-TRIFLUOROMETHYL-4-NITROPHENOL (TFM) BY L A R V A E OF THE AQUATIC MIDGE CH IR O NO MU S T E N T A N S *
JOSEPH A. KAWATSKI and MARY A. BYrrNER**
Department of Biology, Viterbo College La Crosse, Wis. 54601 (U.S.A.) (Received September 25th, 1974)
SUMMARY
Accumulation of [ 14C] 3-trifluoromethyl-4-nitrophenol ([ 14C] TFM) by chironomid larvae from sublethal aqueous concentrations is rapid and dependent on hardness of exposure water. TFM is readily biotransformed to at least two more polar derivatives, including sulfated TFM and TFM-glucuronide or galacturonide. Some TFM is also reduced to the phenol amine. Chironomids can quickly eliminate all TFM derivatives as well as the parent [14 C/TFM.
INTRODUCTION
The lampricide TFM is employed to control the parasitic sea lamprey (Petromyzon marinus). The effects of this toxicant on non-target species
must be determined if continued use of the c o m p o u n d is to be permitted in the United States [1]. In field applications, TFM is used in a 98 : 2 combination with 2',5-dichloro-4'-nitrosalicylanilide {Bayer 73) because of apparent synergistic action of the t w o toxicants [2]. The 98 : 2 mixture is referred to as TFM-2B. The acute laboratory toxicities of TFM, TFM-2B, and Bayer 73 to the aquatic midge Chironomus tentans Fabricius and effects of the chemicals on chironomid oxygen consumption are known [3,4]. We report here on the TFM residue dynamics (rate and magnitude of uptake, elimination, and biotransformation) in laboratory populations of C. tentans. * This work was supported by contract 14-16-008-644 of the Bureau of Sport Fisheries and Wildlife, Fish and Wildlife Service, U.S.A. Department of the Interior. ** Present address: Department of Pharmacology, University of Michigan, Ann Arbor, Mich. 48104 (U.S.A.). Abbreviations: GLM, glucuronide-like TFM metabolite; NMM, non-migrating material; RTFM, 3-trifluoromethyl-4-aminophenol;TFM, 3-trifluoromethyl-4-nitrophenol.
183
METHODS The Bureau of Sport Fisheries and Wildlife, Fish Control Laboratory, La Crosse, Wis., provided the following materials: T F M ( A ) ( 9 5 . 7 % 3-trifluoromethyl-4-nitrophenol; Aldrich Chemical Co.); [ 14C] TFM {99.5% uniformly ring-labeled; 3.66 mCi/mmole; Mallinckrodt Chemical Works); Bayer 73 (70% 2-aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide; Chemagro Corp.); and reconstituted water (hardnesses as mg/1 of CaCO3 : 40--48, soft; 160--180, hard; and 280--320, very hard) [5]. J.J. Lech, Medical College of Wisconsin, provided reduced TFM [6] (RTFM; 3-trifluoromethyl-4-aminophenol).
Laboratory rearing system C. tentans, obtained from S.K. Derr, Michigan State University, was propagated in our laboratory in 5-, 20-, and 400-1 glass or fiberglass aquaria under a 16-h photoperiod of mixed fluorescent and incandescent light in soft water at 22 ± 1.5 °. A food and substrate mixture, composed of ground Trainers Dog Rewards ® (Horlick's Corp.) and macerated paper hand towels {Crown Zellerback Corp.), was added periodically to sustain growth and reproduction. This mixture contained no detectable TFM or Bayer 73 residues as determined by gas-liquid chromatographic analysis. Experimental test system For all experiments, exposure/recovery chambers were 1.5- or 2-1 Pyrex beakers containing 800 ml of pre-aerated reconstituted water (22 ± 1 °) and about 30--50 fourth instar chironomids, which had been acclimated to the system for 24 h prior to exposures. Toxicants were introduced via acetone solution, with the a m o u n t of acetone in test systems never exceeding 1 ml/1. Quantitative analysis of water and test animals Residues of [14 C] TFM were quantitatively measured by liquid scintillation spectrometry. Samples were counted for 10 min with a Nuclear-Chicago Mark II spectrometer; the external standard channels ratio m e t h o d of quench correction was used to obtain corrected DPM which were then converted directly to pg of TFM or TFM-metabolite (in terms of the parent compound). The scintillation cocktail consisted of toluene--Triton X-100 (2 : 1) with 4 g PPO and 0.1 g POPOP per 1. Test waters were sampled in triplicate by transferring 1-ml aliquots directly to scintillation vials, and 15 ml of cocktail were then added. Chironomids were sampled in triplicate also, using 2--3 larvae per sample. Larvae were removed from test systems, blotted dry, weighed, and transferred to individual counting vials. To these vials, 0.5 or 1.0 ml of NCS tissue solubilizer (Nuclear-Chicago Corp.) was added, and the vials were allowed to stand, capped, at room temperature for 2 h before adding 15 ml of cocktail. TFM residue accumulation was expressed in terms of total dry weight of larval 184
tissue, by converting wet to dry weights using a predetermined dry weight factor.
Qualitative analysis o f water and test animals Thin-layer chromatography (TLC), coupled with liquid scintillation counting of separated residues, was the principal qualitative tool. The following commercially prepared p r e ~ o a t e d silica gel plates were used: Polygram SIL IV-HR (Brinkman Instrument Co.) and 6061 Silica gel (Eastman Kodak Co.). Several eluting solvent systems were employed, including n-butanol-acetic acid--water (8 : 2 : 1); chloroform--acetic acid--acetone (14 : 3 : 2); benzene--acetone--3% ammonium hydroxide (150 : 50 : 1); and three systems of Lech [7] : benzene--diethyl ether--acetic acid (100 : 50 : 1); chlorof o r m - m e t h a n o l - 3 0 % ammonium hydroxide (8 : 4 : 1}; and n-butanol-acetic acid--water (35 : 3 : 10}. Water samples for TLC were spotted directly or were extracted first with organic solvents. To prepare tissue samples, 20--100 larvae were homogenized, with or without added water or aqueous buffer, for 3 to 5 min with a motor-driven Teflon pestle. When solvent extraction was desired, the appropriate solvent was then added to the grinding vessel and the tissue was further homogenized. Tissue samples were spotted as aqueous preparations or as solvent extracts. Immediately after performing TLC, the region of plate from 0.5 cm below to 10 cm above each spotting point was cut into 8--12 pieces, which were then placed into separate scintillation vials. It was assumed that the sum of radioactivity above any spotting point represented 100% of the radioactivity for a given sample. Non-radioactive TFM and RTFM, [14C]TFM, and tissue controls were run simultaneously with samples on each TLC plate. Further details of the TLC procedure have been described elsewhere [ 8]. Enzyme-, acid-, and base-hydrolysis o f TFM metabolites To determine whether TFM metabolites were conjugates of the TFM molecule, tissue homogenates were subjected to hydrolysis by fl-glucuronidase and sulfatase, under conditions similar to those used by Dodgson et al. [9], for up to 24 h at 37 ° followed by hydrolysis with acid (0.25 N HCI) and base (0.25 N NaOH) at 80 ° for up to 24 h. Aliquots of homogenate were removed at various times during hydrolysis, neutralized if necessary, and analyzed b y TLC. The enzyme reaction medium consisted of 0.3 ml of whole animal tissue homogenate (representing a b o u t 20 larvae) in sodium acetate--acetic acid buffer (0.5 M, pH 5.2) and 0.2 ml of appropriate enzyme in acetate buffer. The enzyme concentrations in these mixtures were ~-glucuronidase (Sigma Chemical Co. type V-A from E. coli), 4000 units/ml; and sulfatase, phenol and aryl (Sigma Chemical Co. type III from limpets, low in ~-glucuronidase activity), 80 units/ml. On occasion, these hydrolyses were also performed on exposure and postexposure test waters. 185
RESULTS
Gross uptake and elimination o f TFM Accumulation of residue from water containing sublethal concentrations of TFM was biphasic, being dependent largely on hardness of exposure water and the concentration of toxicant in exposure systems. Initial uptake was rapid. Maximum accumulation occurred within 24 h after which no significant increase in total b o d y burden was realized (Fig. 1). The plateau level of total body residue of toxicant was directly related to exposure concentration. As exposure water hardness increased, less TFM was accumulated by chironomid larvae. The rate of TFM uptake by larvae exposed to TFM alone was n o t significantly different from the rate of TFM uptake from similar solutions of TFM-2B. There was a consistent trend for less TFM to be accumulated in the presence of 2% of Bayer 73 (Table I). Presence of various substrates in exposure vessels reduced the rate of initial toxicant accumulation and, in most instances, resulted in a lesser total body residue during similar exposure periods (Table II). Presence of substrate did not cause significant removal of toxicant from the solution by absorption. However, the effect of substrate on rate of toxicant absorption by test animals was a secondary effect in t h a t the different substrates (paper toweling, sand, silt) altered hardness and pH. This, in turn, resulted in a lesser accumulation of toxicant by chironomids. When test animals were transferred to toxicant-free water after exposure to TFM, accumulated residue was rapidly eliminated, and the elimination occurred more completely when post-exposure water was periodically renewed (Fig. 2). Under different conditions, the biological half life of TFM residue (time required for disappearance of 50% of accumulated residue) varied from 3.6 to 15.3 h (Table III). Statistically, there was no correlation between biological half life (TI/2) and total accumulated burden of toxicant.
140"
o~ 100-p
8O
/
]'
6040
200
.f.
24 48 Exposwe t,me (hours)
72
Fig. 1. A c c u m u l a t i o n o f T F M residue by C h i r o n o m u s tentans f o u r t h instar larvae f r o m w a t e r at t h r e e hardnesses ( o s o f t w a t e r ; c, hard w a t e r ; +, very hard water) at 22 e 1 ° during 72 h o f c o n t i n u o u s e x p o s u r e to 75 pg/l o f TFM.
186
TABLE I ACCUMULATION OF TFM RESIDUE (IN /~g/g) BY CHIRONOMUS T E N T A N S FOURTH INSTAR LARVAE FROM SOFT WATER AT 22-+1° DURING 72 h OF CONTINUOUS EXPOSURE TO SUBLETHAL CONCENTRATIONS OF TFM AND TFM-2B (IN /~g/1) Hour of exposure
Exposed to TMF
0 1 2
4 7 11 24 48 72
Exposed to TFM-2B
Exposure water
Tissue
Exposure water
89.6 (5.6) a 93.8 (2.4) 94.2 (1.2)
-56.8 (10.2) 103.1 (3.2)
94.6 (1.0) 90.0 (0.1) 93.3 (2.2)
93.5 92.3 91.9 93.2 95.0 97.0
192.5 253.0 306.9 366.9 299.9 276.3
90.5 91.8 90.5 92.7 93.4 97.9
(0.2) (1.2) (1.3) (1.2) (0.7) (1.1)
(36.9) (41.1) (17.0) (244.3) (75.2) (56.8)
(2.2) (2.5) (1.9) (2.3) (2.1) (2.0)
Tissue -58.3 (1.3) 95.0 (16.2)
167.0 219.2 289.4 322.1 257.6 253.1
(31.3) (9.5) (33.3) (28.9) (30.6) (118.6)
a Standard deviations given in parentheses,
Also t h e r e was n o significant d i f f e r e n c e b e t w e e n t h e 7n/~ o f T F M residue in test animals e x p o s e d t o T F M - 2 B c o m p a r e d t o t h o s e e x p o s e d t o similar a m o u n t s o f T F M alone. When o t h e r factors were equal and the hardness o f e x p o s u r e w a t e r was t h e same as t h a t o f p o s t - e x p o s u r e water, t h e rate o f e l i m i n a t i o n was m o r e rapid in h a r d e r p o s t - e x p o s u r e waters. T h e ~/~ for T F M in test animals e x p o s e d t o i m m o b i l i z i n g c o n c e n t r a t i o n s o f t o x i c a n t (1.65
~oo" 5O
C0
24
4B
72
96
~ecovery t,me (,hours)
Fig. 2. E l i m i n a t i o n o f T F M residue by ,':,hironomus tentans f o u r t h instar larvae during 96 h after an 8-h exposure t o 90 pg/1 o f T F M . E l i m i n a t i o n occurred in soft w a t e r at 22 ± 1 ° w i t h ( ) and w i t h o u t ( . . . . . ) periodic changing o f recovery water.
187
Hardness (mg/l as CaCO3) at 48 h
pH at 48 h
Type of substrate
95.0 221.4 356.6 402.6 394.5
40
7.2
None
(4.5) a (27.9) (31.4) (27.1) (30.8)
a Standard deviations given in parentheses.
2 5 12 24 48
Hour of exposure
74.1 (9.2) 111.8 (12.4) 180.8(16.3) 231.1(20.6) 384.5 (41.9)
35
6.8
Artificial (paper toweling)
70.0 (8.0) 124.2 (5.1) 211.0 (51.6) 276.0 (29.5) 202.7 (70.1)
110
7.4
Silt No. 30 mesh
63.5(16.2) 123.9 (3.0) 177.5 (6.3) 189.0 (68.9) 182.5 (23.1)
108
7.4
Silt No. 80 mesh
67.4 (5.2) 158.6 (30.3) 308.2(13.0) 437.4 (15.4) 402.1 (31.5)
50
7.5
Sand No. 30 mesh
78.5 (3.4) 180.3(17.2) 269.9(28.3) 301.1(24.2) 266.8 (22.1)
56
7.5
Sand No. 80 mesh
64.5 (7.6) 149.7 (6.2) 179.6 (23.2) 194.9 (3.5) 187.4 (25.3)
8O
7.4
Sand No. 200 mesh
ACCUMULATION OF TFM RESIDUE (IN //g/g) BY C H I R O N O M U S T E N T A N S FOURTH INSTAR LARVAE FROM SOFT WATER AT 22+1 ° CONTAINING 15 g OF DIFFERENT SUBSTRATES DURING 48 h OF CONTINUOUS EXPOSURE TO 85 //g/I OF TFM
TABLE II
T A B L E III E L I M I N A T I O N O F T F M R E S I D U E BY CHIRONOMUS T E N T A N S F O U R T H I N S T A R L A R V A E A T 22+1 o A F T E R E X P O S U R E T O T F M OR T F M - 2 B U N D E R D I F F E R E N T CONDITIONS Results o f individual e x p e r i m e n t s are g r o u p e d a n d s e p a r a t e d b y h o r i z o n t a l lines. Exposure conditions Toxicant in pg/l
Total exposure t i m e (h)
Water hardness
pg T F M residue/g larval tissue at end of exposure
Hardness of recovery water
Biological half life of T F M residue (h)
TFM TFM TFM TFM-2B TFM-2B TFM-2B
85 85 85 85 85 85
8 8 8 8 8 8
soft hard very h a r d soft hard very h a r d
229.2 79.6 85.5 230.2 83.9 74.9
soft hard very h a r d soft hard very h a r d
15.3 9.1 6.4 12.7 8.5 8.1
TFM TFM TFM TFM-2B TFM-2B TFM-2B
85 85 85 85 85 85
8 8 8 8 8 8
soft soft soft soft soft soft
271.6 271.6 271.6 234.9 234.9 234.9
soft hard very h a r d soft hard very h a r d
8.0 6.3 5.6 7.3 8.8 8.5 11.5 10.6 9.5 14.6 8.9 12.7
.
TFM TFM TFM TFM-2B TFM-2B TFM-2B
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
165 165 165 165 165 165
8 8 8 8 8 8
soft soft soft soft soft soft
471.6 471.6 471.6 359.2 359.2 359.2
soft hard very h a r d soft hard very h a r d
TFM TFM
90 90
8 8
soft soft a
194.6 194.6
soft soft a
TFM TFM TFM
1650 1650 1650
2 2b 6b
soft soft soft
554.6 493.9 499.2
soft soft soft
5.5 3.6 8.3 8.2 13.6
a R e c o v e r y w a t e r was r e n e w e d w i t h t o x i c a n t - f r e e w a t e r periodically d u r i n g t h e eliminat i o n period. b Test a n i m a l s were i m m o b i l i z e d at t h e e n d of e x p o s u r e period, b u t were m o b i l e t h r o u g h o u t t h e r e c o v e r y period.
mgfl) was greater for those organisms which had been immobilized for 6 h as compared to 2 h, even though immobilization resulted in cessation of toxicant accumulation. All immobilized test animals regained activity when placed in toxicant-free post-exposure water.
Biotransformation In initial experiments, aqueous homogenates of test animals were extracted with various organic solvents prior to TLC, but such extraction re-
189
suited in highly variable recovery of TFM and its metabolites. Analysis with five different TLC systems of solvent extracts of larvae, exposed for 24 h to 95 pg/l of TFM, yielded the following percent recoveries (S.D. in parentheses) of authentic TFM: acetone, 33.2 (5.8); ethyl ether, 63.9 (9.1); acetone/ ether, 37.4 (4.5); benzene, 88.4 (2.2); and methanol, 13.4 (2.1). Aqueous homogenates, spotted directly on thin-layer plates, yielded 58.4 (9.8)% of TFM. When larvae were exposed similarly b u t for a period of 72 h, examination of aqueous homogenate at 12-h intervals by four TLC systems revealed that the relative recovery of authentic TFM varied only slightly after the first 12 h of exposure. The mean percent recoveries (S.D. in parentheses) during the 72-h period were: TFM, 59.8 (4.1); RTFM, 2.9 (1.4); other, 38.5 (3.5). Much of the material identified as " o t h e r " was material which did not migrate on thin-layer plates. When homogenates were centrifuged (2000 rev./min) prior to TLC and supernatant fractions were analyzed, the percent recovery of NMM in supernatant did not differ from that of the uncentrifuged homogenate. Upon closer examination of tissue homogenates by TLC, we observed the presence of a GLM, i.e. a material which migrated in different systems as though it were TFM-glucuronide. However, when the homogenate containing GLM was subjected to hydrolysis by ~-glucuronidase, we found that only one-third of the total GLM was TFM-glucuronide; a large portion of the remaining GLM appeared to be a sulfated TFM derivative since it was hydrolyzable with sulfatase (Table IV). In addition, some of the material identified as GLM and NMM was not hydrolyzable by/~-glucuronidase or by sulfatase but was hydrolyzed by 0.25 N HCI and 0.25 N NaOH, with the resultant recovery of virtually 100% authentic TFM. Generally, during the initial exposure phase (i.e. the accumulation phase) of larvae to TFM, the total accumulated was composed of progressively less authentic TFM (down to about 50%) and a concurrent increase in GLM was observed with time. When larvae were exposed to higher concentrations of TFM, neither TFMglucuronide nor sulfated TFM was recovered from tissue homogenates, but significant quantities of GLM and NMM were still produced (Table V). When these larvae were allowed to recover in toxicant-free water, they eliminated several c o m p o u n d s including TFM, RTFM, GLM (part of which was TFMglucuronide and sulfated TFM), and unidentified NMM. In an effort to determine whether NMM was composed of authentic TFM or other material which had been b o u n d to or trapped in or by membranes and protein, we conducted TLC of successive 20-min benzene extracts of aqueous larval homogenates. Both aqueous and benzene phases were analyzed by TLC as were concurrent non-extracted aqueous homogenates. Our conclusions are as follows (Fig. 3). NMM was composed of 61% TFM, 22% RTFM, and 17% highly polar, hydrophilic, non-diffusable, non-extractable material.
190
b-a ¢D t-a
54.2 (10.9)
52.5 (4.7)
16
34
0.6 (0.7)
0.8 (0.6)
4.0 (1.9)
RTFM
32.7 (3.6)
31.6 (6.8)
(15.6)
21.6
GLM
a Standard deviations given in parentheses.
60.7 (12.4) a
TFM
13.5 (1.2)
13.4 (4.0)
12.9 (3.7)
NMM
Percent of 14C-residue in aqueous control homogenates recovered as
8
(h)
Exposure time
1.2 (0.6)
0.2 (0.4)
1.7 (1.4)
Other
none
none
~-Glucuronidase
64.0% TFM; 23.1% GLM 78.1% TFM; 10.9% GLM ........................
67.1% TFM; 19.9% GLM
none
Sulfatase
Significant changes after hydrolysis by
RECOVERY OF 14C-RESIDUE FROM AQUEOUS HOMOGENATES OF CHIRONOMUS T E N T A N S FOURTH INSTAR LARVAE EXPOSED CONTINUOUSLY TO 116 pg/l OF TFM IN SOFT WATER (22-+1 °) AND RESULTS OF 24-h INCUBATION OF HOMOGENATES WITH ~-GLUCURONIDASE AND SULFATASE
TABLE IV
~D t~
54.3 (4.5) 39.5 (3.6)
Water
38.1 (6.6)
Water
Larvae
59.5 (12.0)
Larvae
91.2 (5.8)
Water
--
2.5 (1.6)
3.9 (6.8)
2.0 (2.1)
2.4 (4.2)
70.3 1.1 (2.7) a (1.6)
26.3 (8.2)
32.0 (6.5)
45.5 (3.8)
28.8 (11.5)
3.5 (4.2)
18.5 (4.5)
33.0 (5.6)
10.0 (3.5)
12.6 (5.4)
8.0 (3.2)
2.8 (3.2)
7.7 (0.2)
NMM
2.5 (4.3)
2.4 (0.0)
--
3.8 (1.1)
0.2 (0.4)
--
Other
55.1% TFM; 0% GLM: 27.8% NMM
none
76.3% T F M ; 23.7% GLM 0% NMM
none
none
none
48.3% G L M ; 20.1% NMM
none
46.9% T F M ; 34.9% GLM
none
none
none
~-Glucuronidase
Sulfatase
GLM
TFM
RTFM
Significant changes after hydrolysis by
Percent of 14C.residu e in sample recovered as
Larvae
Sample
a Standard deviations given in parentheses.
12 + 16
12+8
12+0
(h)
Exposure and recovery time
Results of 4-h incubation of residue samples with ~-glucuronidase and sulfatase.
R E C O V E R Y O F 1 4 C - R E S I D U E F R O M A Q U E O U S H O M O G E N A T E S OF CHIRONOMUS T E N T A N S F O U R T H I N S T A R L A R V A E A F T E R A 12-h E X P O S U R E TO 224 pg/l O F T F M IN S O F T W A T E R (22-+1°), F R O M L A R V A E D U R I N G P O S T - E X P O S U R E P E R I O D S IN T O X I C A N T - F R E E WATER, A N D F R O M E X P O S U R E / R E C O V E R Y W A T E R
TABLE V
[ ] GLM [ ] ~TFM
Do~h~
~I0 ~ 8 6 S 4
I 2 SuccessLve
3 extractlons
4
Fig. 3. Recovery of TFM and TFM metabolites from aqueous h o m o g e n a t e s and four successive benzene extracts o f aqueous h o m o g e n a t e s of Chironomus tentans fourth instar larvae which had been exposed for 8 h to 335 pg/! of TFM in soft water (22 + 1°). C, aqueous control h o m o g e n a t e ; B, benzene phase; W, a q u e o u s phase. DISCUSSION
Others have demonstrated that larvae of C. tentans possess a remarkable ability to recover after short-term exposure to TFM [3]. We repeatedly have observed that animals which had been rendered immobile and rigid after several hours of TFM exposure readily became mobile again when transferred to toxicant-free water. It is not surprising, then, to find that chironomids are both able to rapidly eliminate and to partially biotransform accumulated TFM. The rate of elimination, like rate of uptake, is in part related to water hardness. As water hardness increases, TFM is accumulated less rapidly but eliminated more rapidly by chironomids. This observation is consistent with the physical properties of TFM and with its toxicity to chironomids. In soft water (at low pH's), a greater proportion of the TFM molecules exist as the free phenol than as phenolate ion; the reverse is true in harder water. The phenol is the more readily absorbed form. Consequently, in soft waters TFM is more toxic than in hard waters containing similar total toxicant concentration. However, since chironomids can excrete unchanged TFM, as well as biotransformed TFM, hard waters tend to promote rapid elimination of accumulated residue. Under continuous exposure to sublethal concentrations of TFM, chironomids will consistently accumulate only finite amounts of toxicant, after which total body residue levels may vary slightly or even decline. This undoubtedly is a reflection n o t only of the solubility equilibria of TFM and its products between external water and lipoid tissue and membranes but also of the intrinsic ability of the organism to protect itself via detoxication. In soft water, TFM and Bayer 73 appear to be slightly synergistic in toxicity [3], but the rate of TFM accumulation in the presence of Bayer 73 is virtually identical to the rate of TFM accumulation in the absence of Bayer 73. The observed synergism is therefore not the result of increased
193
a b s o r p t i o n or a c c u m u l a t i o n o f T F M , b u t is likely d u e to physiological e f f e c t s at a n o t h e r level. Because t h e synergistic a c t i o n is related t o e x p o s u r e w a t e r hardness and t o e x p o s u r e time, it is c o n c e i v a b l e t h a t t h e presence o f T F M e n h a n c e s B a y e r 73 a b s o r p t i o n , t h e r e b y p r o d u c i n g synergistic t o x i c i t y . T h e f o r m a t i o n o f m o r e - p o l a r T F M derivatives is o n e o f t h e m a j o r r o u t e s o f t o x i c a n t e l i m i n a t i o n b y c h i r o n o m i d larvae. T F M - g l u c u r o n i d e or galactur o n i d e and s u l f a t e d - T F M are t w o o f t h e b i o t r a n s f o r m a n t s . It appears t h a t at least o n e o t h e r , m o r e - p o l a r derivative is also p r o d u c e d and e x c r e t e d , alt h o u g h it is possible t h a t this p r o d u c t is an artifact o f i n c o m p l e t e e n z y m a t i c hydrolysis. Much o f t h e a c c u m u l a t e d 1 4C.residue b e c o m e s associated with cell f r a g m e n t s , and it appears t h a t b i o t r a n s f o r m a t i o n o c c u r s just p r i o r to or d u r i n g t h e process o f elimination. REFERENCES I Rosalie A. Schnick, A review of literature on TFM (3-trifluormethyl-4-nitrophenol) as a lamprey larvicide, U.S. Bur. Sport Fish. Wildl., Invest. Fish Contr., 44, 1972, 31 p. 2 J.H. Howell, E.L. King Jr., A.J. Smith and L.H. Hanson, Synergism of 5,2'-dichloro4'-nitro-salicylamilide and 3-trifluormethyl-4-nitrophenol in a selective lamprey larvicide, Great Lakes Fish. Comm. Tech. Rept. No. 8, 1964, 21 p. 3 J.A. Kawatski, Margaret M. Ledvina and C.R. Hansen Jr., Acute toxicities of TFM and Bayer 73 to larvae of the midge Chironomus tentans, U.S. Bur. Sport Fish. Wildl., Invest. Fish Contr., 57, 1974, 7 p. 4 J.~. Kawatski, V.K. Dawson and Marlys L. Reuvers, Effect of TFM and Bayer 73 on in vivo oxygen consumption of the aquatic midge Chironomus tentans, Trans. Am. Fish. Soc., 103 (1974) 551. 5 L.L. Marking, Juglone (5-hydroxy-l,4-naphthoquinone) as a fish toxicant, Trans. Am. Fish. Soc., 99 (1970) 510. 6 J.J. Lech, Metabolism of 3-trifluoromethyl-4-nitrophenol (TFM) in the rat, Toxicol. Appl. Pharmacol., 20 (1971) 216. 7 J.J. Lech, Isolation and identification of 3-trifluoromethyl-4-nitrophenol glucuronide from bile of rainbow trout exposed to 3-trifluoromethyl-4-nitrophenol, Toxicol. Appl. Pharmacol., 24 (1973) 114. 8 J.A. Kawatski and Mary J. McDonald, Effect of 3-trifluoromethyl-4-nitrophenol on in vitro tissue respiration of four species of fish with preliminary notes on its in vitro biotransformation, Comp. Gen. Pharmacol., 5 (1974) 67. 9 K.S. Dodgson, J.I.M. Lewis and B. Spencer, Studies on sulphatases, the arylsulphatase and ~-glucuronidase of marine molluscs, Biochem. J., 55 (1953) 253.
194
UPTAKE, ELIMINATION, AND BIOTRANSFORMATION O F THE LAMPRICIDE 3-TRIFLUOROMETHYL-4-NITROPHENOL (TFM) BY L A R V A E OF THE AQUATIC MIDGE CH IR O NO MU S T E N T A N S *
JOSEPH A. KAWATSKI and MARY A. BYrrNER**
Department of Biology, Viterbo College La Crosse, Wis. 54601 (U.S.A.) (Received September 25th, 1974)
SUMMARY
Accumulation of [ 14C] 3-trifluoromethyl-4-nitrophenol ([ 14C] TFM) by chironomid larvae from sublethal aqueous concentrations is rapid and dependent on hardness of exposure water. TFM is readily biotransformed to at least two more polar derivatives, including sulfated TFM and TFM-glucuronide or galacturonide. Some TFM is also reduced to the phenol amine. Chironomids can quickly eliminate all TFM derivatives as well as the parent [14 C/TFM.
INTRODUCTION
The lampricide TFM is employed to control the parasitic sea lamprey (Petromyzon marinus). The effects of this toxicant on non-target species
must be determined if continued use of the c o m p o u n d is to be permitted in the United States [1]. In field applications, TFM is used in a 98 : 2 combination with 2',5-dichloro-4'-nitrosalicylanilide {Bayer 73) because of apparent synergistic action of the t w o toxicants [2]. The 98 : 2 mixture is referred to as TFM-2B. The acute laboratory toxicities of TFM, TFM-2B, and Bayer 73 to the aquatic midge Chironomus tentans Fabricius and effects of the chemicals on chironomid oxygen consumption are known [3,4]. We report here on the TFM residue dynamics (rate and magnitude of uptake, elimination, and biotransformation) in laboratory populations of C. tentans. * This work was supported by contract 14-16-008-644 of the Bureau of Sport Fisheries and Wildlife, Fish and Wildlife Service, U.S.A. Department of the Interior. ** Present address: Department of Pharmacology, University of Michigan, Ann Arbor, Mich. 48104 (U.S.A.). Abbreviations: GLM, glucuronide-like TFM metabolite; NMM, non-migrating material; RTFM, 3-trifluoromethyl-4-aminophenol;TFM, 3-trifluoromethyl-4-nitrophenol.
183
METHODS The Bureau of Sport Fisheries and Wildlife, Fish Control Laboratory, La Crosse, Wis., provided the following materials: T F M ( A ) ( 9 5 . 7 % 3-trifluoromethyl-4-nitrophenol; Aldrich Chemical Co.); [ 14C] TFM {99.5% uniformly ring-labeled; 3.66 mCi/mmole; Mallinckrodt Chemical Works); Bayer 73 (70% 2-aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide; Chemagro Corp.); and reconstituted water (hardnesses as mg/1 of CaCO3 : 40--48, soft; 160--180, hard; and 280--320, very hard) [5]. J.J. Lech, Medical College of Wisconsin, provided reduced TFM [6] (RTFM; 3-trifluoromethyl-4-aminophenol).
Laboratory rearing system C. tentans, obtained from S.K. Derr, Michigan State University, was propagated in our laboratory in 5-, 20-, and 400-1 glass or fiberglass aquaria under a 16-h photoperiod of mixed fluorescent and incandescent light in soft water at 22 ± 1.5 °. A food and substrate mixture, composed of ground Trainers Dog Rewards ® (Horlick's Corp.) and macerated paper hand towels {Crown Zellerback Corp.), was added periodically to sustain growth and reproduction. This mixture contained no detectable TFM or Bayer 73 residues as determined by gas-liquid chromatographic analysis. Experimental test system For all experiments, exposure/recovery chambers were 1.5- or 2-1 Pyrex beakers containing 800 ml of pre-aerated reconstituted water (22 ± 1 °) and about 30--50 fourth instar chironomids, which had been acclimated to the system for 24 h prior to exposures. Toxicants were introduced via acetone solution, with the a m o u n t of acetone in test systems never exceeding 1 ml/1. Quantitative analysis of water and test animals Residues of [14 C] TFM were quantitatively measured by liquid scintillation spectrometry. Samples were counted for 10 min with a Nuclear-Chicago Mark II spectrometer; the external standard channels ratio m e t h o d of quench correction was used to obtain corrected DPM which were then converted directly to pg of TFM or TFM-metabolite (in terms of the parent compound). The scintillation cocktail consisted of toluene--Triton X-100 (2 : 1) with 4 g PPO and 0.1 g POPOP per 1. Test waters were sampled in triplicate by transferring 1-ml aliquots directly to scintillation vials, and 15 ml of cocktail were then added. Chironomids were sampled in triplicate also, using 2--3 larvae per sample. Larvae were removed from test systems, blotted dry, weighed, and transferred to individual counting vials. To these vials, 0.5 or 1.0 ml of NCS tissue solubilizer (Nuclear-Chicago Corp.) was added, and the vials were allowed to stand, capped, at room temperature for 2 h before adding 15 ml of cocktail. TFM residue accumulation was expressed in terms of total dry weight of larval 184
tissue, by converting wet to dry weights using a predetermined dry weight factor.
Qualitative analysis o f water and test animals Thin-layer chromatography (TLC), coupled with liquid scintillation counting of separated residues, was the principal qualitative tool. The following commercially prepared p r e ~ o a t e d silica gel plates were used: Polygram SIL IV-HR (Brinkman Instrument Co.) and 6061 Silica gel (Eastman Kodak Co.). Several eluting solvent systems were employed, including n-butanol-acetic acid--water (8 : 2 : 1); chloroform--acetic acid--acetone (14 : 3 : 2); benzene--acetone--3% ammonium hydroxide (150 : 50 : 1); and three systems of Lech [7] : benzene--diethyl ether--acetic acid (100 : 50 : 1); chlorof o r m - m e t h a n o l - 3 0 % ammonium hydroxide (8 : 4 : 1}; and n-butanol-acetic acid--water (35 : 3 : 10}. Water samples for TLC were spotted directly or were extracted first with organic solvents. To prepare tissue samples, 20--100 larvae were homogenized, with or without added water or aqueous buffer, for 3 to 5 min with a motor-driven Teflon pestle. When solvent extraction was desired, the appropriate solvent was then added to the grinding vessel and the tissue was further homogenized. Tissue samples were spotted as aqueous preparations or as solvent extracts. Immediately after performing TLC, the region of plate from 0.5 cm below to 10 cm above each spotting point was cut into 8--12 pieces, which were then placed into separate scintillation vials. It was assumed that the sum of radioactivity above any spotting point represented 100% of the radioactivity for a given sample. Non-radioactive TFM and RTFM, [14C]TFM, and tissue controls were run simultaneously with samples on each TLC plate. Further details of the TLC procedure have been described elsewhere [ 8]. Enzyme-, acid-, and base-hydrolysis o f TFM metabolites To determine whether TFM metabolites were conjugates of the TFM molecule, tissue homogenates were subjected to hydrolysis by fl-glucuronidase and sulfatase, under conditions similar to those used by Dodgson et al. [9], for up to 24 h at 37 ° followed by hydrolysis with acid (0.25 N HCI) and base (0.25 N NaOH) at 80 ° for up to 24 h. Aliquots of homogenate were removed at various times during hydrolysis, neutralized if necessary, and analyzed b y TLC. The enzyme reaction medium consisted of 0.3 ml of whole animal tissue homogenate (representing a b o u t 20 larvae) in sodium acetate--acetic acid buffer (0.5 M, pH 5.2) and 0.2 ml of appropriate enzyme in acetate buffer. The enzyme concentrations in these mixtures were ~-glucuronidase (Sigma Chemical Co. type V-A from E. coli), 4000 units/ml; and sulfatase, phenol and aryl (Sigma Chemical Co. type III from limpets, low in ~-glucuronidase activity), 80 units/ml. On occasion, these hydrolyses were also performed on exposure and postexposure test waters. 185
RESULTS
Gross uptake and elimination o f TFM Accumulation of residue from water containing sublethal concentrations of TFM was biphasic, being dependent largely on hardness of exposure water and the concentration of toxicant in exposure systems. Initial uptake was rapid. Maximum accumulation occurred within 24 h after which no significant increase in total b o d y burden was realized (Fig. 1). The plateau level of total body residue of toxicant was directly related to exposure concentration. As exposure water hardness increased, less TFM was accumulated by chironomid larvae. The rate of TFM uptake by larvae exposed to TFM alone was n o t significantly different from the rate of TFM uptake from similar solutions of TFM-2B. There was a consistent trend for less TFM to be accumulated in the presence of 2% of Bayer 73 (Table I). Presence of various substrates in exposure vessels reduced the rate of initial toxicant accumulation and, in most instances, resulted in a lesser total body residue during similar exposure periods (Table II). Presence of substrate did not cause significant removal of toxicant from the solution by absorption. However, the effect of substrate on rate of toxicant absorption by test animals was a secondary effect in t h a t the different substrates (paper toweling, sand, silt) altered hardness and pH. This, in turn, resulted in a lesser accumulation of toxicant by chironomids. When test animals were transferred to toxicant-free water after exposure to TFM, accumulated residue was rapidly eliminated, and the elimination occurred more completely when post-exposure water was periodically renewed (Fig. 2). Under different conditions, the biological half life of TFM residue (time required for disappearance of 50% of accumulated residue) varied from 3.6 to 15.3 h (Table III). Statistically, there was no correlation between biological half life (TI/2) and total accumulated burden of toxicant.
140"
o~ 100-p
8O
/
]'
6040
200
.f.
24 48 Exposwe t,me (hours)
72
Fig. 1. A c c u m u l a t i o n o f T F M residue by C h i r o n o m u s tentans f o u r t h instar larvae f r o m w a t e r at t h r e e hardnesses ( o s o f t w a t e r ; c, hard w a t e r ; +, very hard water) at 22 e 1 ° during 72 h o f c o n t i n u o u s e x p o s u r e to 75 pg/l o f TFM.
186
TABLE I ACCUMULATION OF TFM RESIDUE (IN /~g/g) BY CHIRONOMUS T E N T A N S FOURTH INSTAR LARVAE FROM SOFT WATER AT 22-+1° DURING 72 h OF CONTINUOUS EXPOSURE TO SUBLETHAL CONCENTRATIONS OF TFM AND TFM-2B (IN /~g/1) Hour of exposure
Exposed to TMF
0 1 2
4 7 11 24 48 72
Exposed to TFM-2B
Exposure water
Tissue
Exposure water
89.6 (5.6) a 93.8 (2.4) 94.2 (1.2)
-56.8 (10.2) 103.1 (3.2)
94.6 (1.0) 90.0 (0.1) 93.3 (2.2)
93.5 92.3 91.9 93.2 95.0 97.0
192.5 253.0 306.9 366.9 299.9 276.3
90.5 91.8 90.5 92.7 93.4 97.9
(0.2) (1.2) (1.3) (1.2) (0.7) (1.1)
(36.9) (41.1) (17.0) (244.3) (75.2) (56.8)
(2.2) (2.5) (1.9) (2.3) (2.1) (2.0)
Tissue -58.3 (1.3) 95.0 (16.2)
167.0 219.2 289.4 322.1 257.6 253.1
(31.3) (9.5) (33.3) (28.9) (30.6) (118.6)
a Standard deviations given in parentheses,
Also t h e r e was n o significant d i f f e r e n c e b e t w e e n t h e 7n/~ o f T F M residue in test animals e x p o s e d t o T F M - 2 B c o m p a r e d t o t h o s e e x p o s e d t o similar a m o u n t s o f T F M alone. When o t h e r factors were equal and the hardness o f e x p o s u r e w a t e r was t h e same as t h a t o f p o s t - e x p o s u r e water, t h e rate o f e l i m i n a t i o n was m o r e rapid in h a r d e r p o s t - e x p o s u r e waters. T h e ~/~ for T F M in test animals e x p o s e d t o i m m o b i l i z i n g c o n c e n t r a t i o n s o f t o x i c a n t (1.65
~oo" 5O
C0
24
4B
72
96
~ecovery t,me (,hours)
Fig. 2. E l i m i n a t i o n o f T F M residue by ,':,hironomus tentans f o u r t h instar larvae during 96 h after an 8-h exposure t o 90 pg/1 o f T F M . E l i m i n a t i o n occurred in soft w a t e r at 22 ± 1 ° w i t h ( ) and w i t h o u t ( . . . . . ) periodic changing o f recovery water.
187
Hardness (mg/l as CaCO3) at 48 h
pH at 48 h
Type of substrate
95.0 221.4 356.6 402.6 394.5
40
7.2
None
(4.5) a (27.9) (31.4) (27.1) (30.8)
a Standard deviations given in parentheses.
2 5 12 24 48
Hour of exposure
74.1 (9.2) 111.8 (12.4) 180.8(16.3) 231.1(20.6) 384.5 (41.9)
35
6.8
Artificial (paper toweling)
70.0 (8.0) 124.2 (5.1) 211.0 (51.6) 276.0 (29.5) 202.7 (70.1)
110
7.4
Silt No. 30 mesh
63.5(16.2) 123.9 (3.0) 177.5 (6.3) 189.0 (68.9) 182.5 (23.1)
108
7.4
Silt No. 80 mesh
67.4 (5.2) 158.6 (30.3) 308.2(13.0) 437.4 (15.4) 402.1 (31.5)
50
7.5
Sand No. 30 mesh
78.5 (3.4) 180.3(17.2) 269.9(28.3) 301.1(24.2) 266.8 (22.1)
56
7.5
Sand No. 80 mesh
64.5 (7.6) 149.7 (6.2) 179.6 (23.2) 194.9 (3.5) 187.4 (25.3)
8O
7.4
Sand No. 200 mesh
ACCUMULATION OF TFM RESIDUE (IN //g/g) BY C H I R O N O M U S T E N T A N S FOURTH INSTAR LARVAE FROM SOFT WATER AT 22+1 ° CONTAINING 15 g OF DIFFERENT SUBSTRATES DURING 48 h OF CONTINUOUS EXPOSURE TO 85 //g/I OF TFM
TABLE II
T A B L E III E L I M I N A T I O N O F T F M R E S I D U E BY CHIRONOMUS T E N T A N S F O U R T H I N S T A R L A R V A E A T 22+1 o A F T E R E X P O S U R E T O T F M OR T F M - 2 B U N D E R D I F F E R E N T CONDITIONS Results o f individual e x p e r i m e n t s are g r o u p e d a n d s e p a r a t e d b y h o r i z o n t a l lines. Exposure conditions Toxicant in pg/l
Total exposure t i m e (h)
Water hardness
pg T F M residue/g larval tissue at end of exposure
Hardness of recovery water
Biological half life of T F M residue (h)
TFM TFM TFM TFM-2B TFM-2B TFM-2B
85 85 85 85 85 85
8 8 8 8 8 8
soft hard very h a r d soft hard very h a r d
229.2 79.6 85.5 230.2 83.9 74.9
soft hard very h a r d soft hard very h a r d
15.3 9.1 6.4 12.7 8.5 8.1
TFM TFM TFM TFM-2B TFM-2B TFM-2B
85 85 85 85 85 85
8 8 8 8 8 8
soft soft soft soft soft soft
271.6 271.6 271.6 234.9 234.9 234.9
soft hard very h a r d soft hard very h a r d
8.0 6.3 5.6 7.3 8.8 8.5 11.5 10.6 9.5 14.6 8.9 12.7
.
TFM TFM TFM TFM-2B TFM-2B TFM-2B
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
165 165 165 165 165 165
8 8 8 8 8 8
soft soft soft soft soft soft
471.6 471.6 471.6 359.2 359.2 359.2
soft hard very h a r d soft hard very h a r d
TFM TFM
90 90
8 8
soft soft a
194.6 194.6
soft soft a
TFM TFM TFM
1650 1650 1650
2 2b 6b
soft soft soft
554.6 493.9 499.2
soft soft soft
5.5 3.6 8.3 8.2 13.6
a R e c o v e r y w a t e r was r e n e w e d w i t h t o x i c a n t - f r e e w a t e r periodically d u r i n g t h e eliminat i o n period. b Test a n i m a l s were i m m o b i l i z e d at t h e e n d of e x p o s u r e period, b u t were m o b i l e t h r o u g h o u t t h e r e c o v e r y period.
mgfl) was greater for those organisms which had been immobilized for 6 h as compared to 2 h, even though immobilization resulted in cessation of toxicant accumulation. All immobilized test animals regained activity when placed in toxicant-free post-exposure water.
Biotransformation In initial experiments, aqueous homogenates of test animals were extracted with various organic solvents prior to TLC, but such extraction re-
189
suited in highly variable recovery of TFM and its metabolites. Analysis with five different TLC systems of solvent extracts of larvae, exposed for 24 h to 95 pg/l of TFM, yielded the following percent recoveries (S.D. in parentheses) of authentic TFM: acetone, 33.2 (5.8); ethyl ether, 63.9 (9.1); acetone/ ether, 37.4 (4.5); benzene, 88.4 (2.2); and methanol, 13.4 (2.1). Aqueous homogenates, spotted directly on thin-layer plates, yielded 58.4 (9.8)% of TFM. When larvae were exposed similarly b u t for a period of 72 h, examination of aqueous homogenate at 12-h intervals by four TLC systems revealed that the relative recovery of authentic TFM varied only slightly after the first 12 h of exposure. The mean percent recoveries (S.D. in parentheses) during the 72-h period were: TFM, 59.8 (4.1); RTFM, 2.9 (1.4); other, 38.5 (3.5). Much of the material identified as " o t h e r " was material which did not migrate on thin-layer plates. When homogenates were centrifuged (2000 rev./min) prior to TLC and supernatant fractions were analyzed, the percent recovery of NMM in supernatant did not differ from that of the uncentrifuged homogenate. Upon closer examination of tissue homogenates by TLC, we observed the presence of a GLM, i.e. a material which migrated in different systems as though it were TFM-glucuronide. However, when the homogenate containing GLM was subjected to hydrolysis by ~-glucuronidase, we found that only one-third of the total GLM was TFM-glucuronide; a large portion of the remaining GLM appeared to be a sulfated TFM derivative since it was hydrolyzable with sulfatase (Table IV). In addition, some of the material identified as GLM and NMM was not hydrolyzable by/~-glucuronidase or by sulfatase but was hydrolyzed by 0.25 N HCI and 0.25 N NaOH, with the resultant recovery of virtually 100% authentic TFM. Generally, during the initial exposure phase (i.e. the accumulation phase) of larvae to TFM, the total accumulated was composed of progressively less authentic TFM (down to about 50%) and a concurrent increase in GLM was observed with time. When larvae were exposed to higher concentrations of TFM, neither TFMglucuronide nor sulfated TFM was recovered from tissue homogenates, but significant quantities of GLM and NMM were still produced (Table V). When these larvae were allowed to recover in toxicant-free water, they eliminated several c o m p o u n d s including TFM, RTFM, GLM (part of which was TFMglucuronide and sulfated TFM), and unidentified NMM. In an effort to determine whether NMM was composed of authentic TFM or other material which had been b o u n d to or trapped in or by membranes and protein, we conducted TLC of successive 20-min benzene extracts of aqueous larval homogenates. Both aqueous and benzene phases were analyzed by TLC as were concurrent non-extracted aqueous homogenates. Our conclusions are as follows (Fig. 3). NMM was composed of 61% TFM, 22% RTFM, and 17% highly polar, hydrophilic, non-diffusable, non-extractable material.
190
b-a ¢D t-a
54.2 (10.9)
52.5 (4.7)
16
34
0.6 (0.7)
0.8 (0.6)
4.0 (1.9)
RTFM
32.7 (3.6)
31.6 (6.8)
(15.6)
21.6
GLM
a Standard deviations given in parentheses.
60.7 (12.4) a
TFM
13.5 (1.2)
13.4 (4.0)
12.9 (3.7)
NMM
Percent of 14C-residue in aqueous control homogenates recovered as
8
(h)
Exposure time
1.2 (0.6)
0.2 (0.4)
1.7 (1.4)
Other
none
none
~-Glucuronidase
64.0% TFM; 23.1% GLM 78.1% TFM; 10.9% GLM ........................
67.1% TFM; 19.9% GLM
none
Sulfatase
Significant changes after hydrolysis by
RECOVERY OF 14C-RESIDUE FROM AQUEOUS HOMOGENATES OF CHIRONOMUS T E N T A N S FOURTH INSTAR LARVAE EXPOSED CONTINUOUSLY TO 116 pg/l OF TFM IN SOFT WATER (22-+1 °) AND RESULTS OF 24-h INCUBATION OF HOMOGENATES WITH ~-GLUCURONIDASE AND SULFATASE
TABLE IV
~D t~
54.3 (4.5) 39.5 (3.6)
Water
38.1 (6.6)
Water
Larvae
59.5 (12.0)
Larvae
91.2 (5.8)
Water
--
2.5 (1.6)
3.9 (6.8)
2.0 (2.1)
2.4 (4.2)
70.3 1.1 (2.7) a (1.6)
26.3 (8.2)
32.0 (6.5)
45.5 (3.8)
28.8 (11.5)
3.5 (4.2)
18.5 (4.5)
33.0 (5.6)
10.0 (3.5)
12.6 (5.4)
8.0 (3.2)
2.8 (3.2)
7.7 (0.2)
NMM
2.5 (4.3)
2.4 (0.0)
--
3.8 (1.1)
0.2 (0.4)
--
Other
55.1% TFM; 0% GLM: 27.8% NMM
none
76.3% T F M ; 23.7% GLM 0% NMM
none
none
none
48.3% G L M ; 20.1% NMM
none
46.9% T F M ; 34.9% GLM
none
none
none
~-Glucuronidase
Sulfatase
GLM
TFM
RTFM
Significant changes after hydrolysis by
Percent of 14C.residu e in sample recovered as
Larvae
Sample
a Standard deviations given in parentheses.
12 + 16
12+8
12+0
(h)
Exposure and recovery time
Results of 4-h incubation of residue samples with ~-glucuronidase and sulfatase.
R E C O V E R Y O F 1 4 C - R E S I D U E F R O M A Q U E O U S H O M O G E N A T E S OF CHIRONOMUS T E N T A N S F O U R T H I N S T A R L A R V A E A F T E R A 12-h E X P O S U R E TO 224 pg/l O F T F M IN S O F T W A T E R (22-+1°), F R O M L A R V A E D U R I N G P O S T - E X P O S U R E P E R I O D S IN T O X I C A N T - F R E E WATER, A N D F R O M E X P O S U R E / R E C O V E R Y W A T E R
TABLE V
[ ] GLM [ ] ~TFM
Do~h~
~I0 ~ 8 6 S 4
I 2 SuccessLve
3 extractlons
4
Fig. 3. Recovery of TFM and TFM metabolites from aqueous h o m o g e n a t e s and four successive benzene extracts o f aqueous h o m o g e n a t e s of Chironomus tentans fourth instar larvae which had been exposed for 8 h to 335 pg/! of TFM in soft water (22 + 1°). C, aqueous control h o m o g e n a t e ; B, benzene phase; W, a q u e o u s phase. DISCUSSION
Others have demonstrated that larvae of C. tentans possess a remarkable ability to recover after short-term exposure to TFM [3]. We repeatedly have observed that animals which had been rendered immobile and rigid after several hours of TFM exposure readily became mobile again when transferred to toxicant-free water. It is not surprising, then, to find that chironomids are both able to rapidly eliminate and to partially biotransform accumulated TFM. The rate of elimination, like rate of uptake, is in part related to water hardness. As water hardness increases, TFM is accumulated less rapidly but eliminated more rapidly by chironomids. This observation is consistent with the physical properties of TFM and with its toxicity to chironomids. In soft water (at low pH's), a greater proportion of the TFM molecules exist as the free phenol than as phenolate ion; the reverse is true in harder water. The phenol is the more readily absorbed form. Consequently, in soft waters TFM is more toxic than in hard waters containing similar total toxicant concentration. However, since chironomids can excrete unchanged TFM, as well as biotransformed TFM, hard waters tend to promote rapid elimination of accumulated residue. Under continuous exposure to sublethal concentrations of TFM, chironomids will consistently accumulate only finite amounts of toxicant, after which total body residue levels may vary slightly or even decline. This undoubtedly is a reflection n o t only of the solubility equilibria of TFM and its products between external water and lipoid tissue and membranes but also of the intrinsic ability of the organism to protect itself via detoxication. In soft water, TFM and Bayer 73 appear to be slightly synergistic in toxicity [3], but the rate of TFM accumulation in the presence of Bayer 73 is virtually identical to the rate of TFM accumulation in the absence of Bayer 73. The observed synergism is therefore not the result of increased
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a b s o r p t i o n or a c c u m u l a t i o n o f T F M , b u t is likely d u e to physiological e f f e c t s at a n o t h e r level. Because t h e synergistic a c t i o n is related t o e x p o s u r e w a t e r hardness and t o e x p o s u r e time, it is c o n c e i v a b l e t h a t t h e presence o f T F M e n h a n c e s B a y e r 73 a b s o r p t i o n , t h e r e b y p r o d u c i n g synergistic t o x i c i t y . T h e f o r m a t i o n o f m o r e - p o l a r T F M derivatives is o n e o f t h e m a j o r r o u t e s o f t o x i c a n t e l i m i n a t i o n b y c h i r o n o m i d larvae. T F M - g l u c u r o n i d e or galactur o n i d e and s u l f a t e d - T F M are t w o o f t h e b i o t r a n s f o r m a n t s . It appears t h a t at least o n e o t h e r , m o r e - p o l a r derivative is also p r o d u c e d and e x c r e t e d , alt h o u g h it is possible t h a t this p r o d u c t is an artifact o f i n c o m p l e t e e n z y m a t i c hydrolysis. Much o f t h e a c c u m u l a t e d 1 4C.residue b e c o m e s associated with cell f r a g m e n t s , and it appears t h a t b i o t r a n s f o r m a t i o n o c c u r s just p r i o r to or d u r i n g t h e process o f elimination. REFERENCES I Rosalie A. Schnick, A review of literature on TFM (3-trifluormethyl-4-nitrophenol) as a lamprey larvicide, U.S. Bur. Sport Fish. Wildl., Invest. Fish Contr., 44, 1972, 31 p. 2 J.H. Howell, E.L. King Jr., A.J. Smith and L.H. Hanson, Synergism of 5,2'-dichloro4'-nitro-salicylamilide and 3-trifluormethyl-4-nitrophenol in a selective lamprey larvicide, Great Lakes Fish. Comm. Tech. Rept. No. 8, 1964, 21 p. 3 J.A. Kawatski, Margaret M. Ledvina and C.R. Hansen Jr., Acute toxicities of TFM and Bayer 73 to larvae of the midge Chironomus tentans, U.S. Bur. Sport Fish. Wildl., Invest. Fish Contr., 57, 1974, 7 p. 4 J.~. Kawatski, V.K. Dawson and Marlys L. Reuvers, Effect of TFM and Bayer 73 on in vivo oxygen consumption of the aquatic midge Chironomus tentans, Trans. Am. Fish. Soc., 103 (1974) 551. 5 L.L. Marking, Juglone (5-hydroxy-l,4-naphthoquinone) as a fish toxicant, Trans. Am. Fish. Soc., 99 (1970) 510. 6 J.J. Lech, Metabolism of 3-trifluoromethyl-4-nitrophenol (TFM) in the rat, Toxicol. Appl. Pharmacol., 20 (1971) 216. 7 J.J. Lech, Isolation and identification of 3-trifluoromethyl-4-nitrophenol glucuronide from bile of rainbow trout exposed to 3-trifluoromethyl-4-nitrophenol, Toxicol. Appl. Pharmacol., 24 (1973) 114. 8 J.A. Kawatski and Mary J. McDonald, Effect of 3-trifluoromethyl-4-nitrophenol on in vitro tissue respiration of four species of fish with preliminary notes on its in vitro biotransformation, Comp. Gen. Pharmacol., 5 (1974) 67. 9 K.S. Dodgson, J.I.M. Lewis and B. Spencer, Studies on sulphatases, the arylsulphatase and ~-glucuronidase of marine molluscs, Biochem. J., 55 (1953) 253.
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