The toxic effects of trinitrotoluene (TNT) and its primary degradation products on two species of algae and the fathead minnow

Wj~e~ ~7"r~e:~-',~ VoL ~0. ~p 53-" it' .~a.3. P~gamon Press I ~ 0 . Printed in Great Brttam,

THE TOXIC EFFECTS OF TRINITROTOLUENE (TNT) AND ITS PRIMARY DEGRADATION PRODUCTS ON TWO SPECIES OF ALGAE AND THE FATHEAD MINNOW L. A. SMOCK

Department of Environmental Sciences and Engineering. School of PubEc Health. University of North Carolina, Chapel Hill. N.C. 27514. U.S.A. and D. L. SrONEBUR~'F,R National Park Service. 1895 Phoenix Boulevard, Atlanta, Geor~a 30349. U.S.A, and J. R. CLARK Virginia Polytechnic Institute and State University. Blacksburg, Vir~nia 24061, U.S.A.

(Receit~ed 8 January 1976) Ahstract--qhe effects or alpha trinitrotoluene (alpha TNT) and its primary degradation product ITNTcc), commonly referred to as "pink water", were determined on members of two trophie levels. The growth responses of the algae Selenastrum capricornutum and Microcystis aeruginosa were examined through static bioassays. Death and behavioral responses of the fathead minnow (Pimephales promelas) v,ere determined using a proportional diluter. Alpha TNT and TNTcc were both more toxic to the fathead minnow than to either species of alga. Five and 15 mg 1- t alpha TNT inhibited S. capricornutum and M. aeruginosa growth, respectively. TNTcc inhibited S. capricornutum growth at concentrations above 9 mg I- 1; it was lethal to M. aeruginosa at 50 mg 1- t, but stimulated growth at lower concentrations. The 96-h LC_~ovalues based on the death response of the fathead minnow to alpha TN'I" and TNTcc were 2.58 and 1.60 mg 1-t, respectively. The 96-h ~cso values based on the behavioral responses were 0.46 and 0.64 mg I- t respectively. There was no response to concentrations of 0.05 mg I ' ~ alpha TNT and 0.07 mg 1- I TNTcc. INTRODUCTION

tially treated condition. No federal standards exist for TNT. The U.S. Army has established limits of 1 mg The production and use of 2,4,6-trinitrotoluene (alpha 1-t alpha T N T in potable water and 5 mg I - t in TNT) is the integral component in the manufacture water used by fish and wildlife. The Soviet Union of munitions at ammunition plants. Significant quan- has set I mg 1- t as the maximum permissible concentities of wastes are generated during the processes tration in surface waters (Czerkinski, 1955, cited by necessary for the production, purification and loading McKee and Wolf, 1963). The intent of this study was of munitions. Among the various types of wastes to determine the effects of alpha T N T and TNTcc generated, alpha T N T and its breakdown products on members of two trophic levels. The growth reare some of the more significant wastes in terms of sponses of a green and blue-green alga were examvolume and impact on the aquatic environment. ined through static bioassays. The acute toxicities of The major wastewater problem originates from these wastes in terms of a 96-h median lethal concenT N T finishing processes, shell washout operations tration (LCs0) based on death and a 96-h median effecand equipment and building washdown, at which tive concentration (Ecs0) based on behavioral retime alpha T N T is dissolved in the wash water and sponses of the fathead minnow (Pimephales promelas undergoes a chemical transformation to a complex Rafinesque) were determined using a proportional of various compounds and isomers, collectively diluter. referred to as the T N T colored complex f'FNTcc) or Most previous T N T research centered on its effects "pink water" due to its red color. Although alpha upon bacterial systems and also on the treatability T N T in solution is clear, photochemical irradiation of various T N T wastes (Sehott et al., 1943; Ruchhoft produces the TNTcc. Neither the nature of the reac- et al., 1945; Rogovskaya, 1951 ; Mathews et al., 1954; tion(s) or the exact product(s) formed is precisely Hudock and Gring, 1970). Hudock and Gring (1970) known. The rate of TNTcc formation is especially examined the toxicity of T N T wastes on the green dependent upon irradiation and pH, an alkaline pH alga Chlamydamonas reinhardi. In continuous flow enhancing the rate and intensity of color development bioassays, C. reinhardi exhibited little response to {Ruchhoft et al., 1945; Nay et al., 1972). Carbon alpha T N T concentrations up to 1.0 nag 1-1 ; concenadsorption is presently the favored final treatment trations above this completely inhibited growth. This method for T N T wastes, but it is as yet not used species was. however, substantially more resistant to at all munitions plants; therefore wastewater is dis- TNTcc, with no lethal effect noted until the concencharged to receiving streams in an untreated or par- tration was greater than 50 mg 1-i. 537

538

L.A.S.~toc~¢, D. L. STONEBURNERand J. R. CL.~atK

Studies have been performed on the toxicity of TNT wastes to fish. most of these being static bioassays lasting only 24h or less (Degani. 1943; Mathews et al.. 1954; Meinck et al., 1956, cited in McKee and Wolf. 1963: LeClerc. 1960). Peterson 11970) and Nay et al. (1974J performed static bioassays on blue~lls (Lepomis nutcrochirus) and determined 96-h t.cs0 values ranging from 2.3 to 2.8 mg 1-t of alpha TNT. Water hardness had no apparent effect on toxicity, but toxicity was greater at 10 than at 25:C IPeterson, 1970). Typical behavioral responses to alpha T N T include gasping at the surface, toss of equilibrium, rapid extension and contraction of gills, increased vibration of the pectoral fins and general lethargic actions iDegani, 1943: Mathews et al.. 1954). 3IETHOI)S

Alyal hioassays Two species of algae were used for the algal bioassays:

Selenastrum capricornurum Printz and Microcystis aeruginosu Kutz. emend Elenkin. Stock cultures were obtained from the National Eutrophication Research Program, Pacific Northwest Water Laboratory, Environmental Protection Agency, Covallis. Oregon. Cultures were maintained and tests performed according to the procedures described for the Algal Assay Procedure (National Eutrophication Research Program. 1971}. The nutrient medium was modified to insure that neither nitrates or phosphates limited growth by increasing the NaNO~ and K,,HPO.t concentrations to 51.0 and 10.4 mg I- ~, respectively. Continuous substage illumination was provided and the flasks were swirled twice daily. All culture flasks were 500 ml Erlenmeyer flasks with foam plugs. A stock solution of ILK)mg I- ~ alpha TNT was prepared by dissolving crystalline alpha TNT obtained from Eastman Organic Chemicals in distilled water. The stock solution of TNTcc was prepared by adding 100 mg NazCO3 to I I. of a stock solution of 100 mgl-~ alpha TNT prepared as above, thereby raising the pH of the solution to 9.7. The photochemical reaction was allowed to progress to completion under 400 ft-c illumination at 24~C. The S. capricornutum inoculum was sufficiently concentrated by centrifugation to enable a 5 ml transfer of inoculum per each test flask to result in an initial concentration of 103cells ml-~. The initial medium plus TNT volume per flask was 100 ml. All tests were run with three replicate flasks per concentration. For the respective tests, toxicant was added to result in concentrations of 9,7,5,3,1 and 0 (control) mgl - t alpha TNT and 40,25,9,5.3,1 and 0 (control) m g l - t TNTcc. All counts, reported as the number of cells ml- t were determined for each flask using a hemacytometer. Seven counts were made over a 17 day period for the alpha TNT bioassay and five counts were made over a 14 day period for the TNTcc bioassay. The M. aer,ujinosa inoeulum was sufficiently concentrated by centrifugation to enable a 10 ml transfer of inoculum per each test flask to result in initial biomass determinations of 60 and 51 mgl -~ in the alpha TNT and TNTcc flasks respectively. The initial medium plus TNT volume per flask was 150 ml. All tests were run with three replicate flasks per concentration. Concentrations of both alpha TNT and TNTcc were 50.25,15,10.5,1 and 0 (control) mg 1- t. The ~ o w t h of the cultures was assessed by determining changes in dry weight biomass. Ten millititres of culture were filtered through a 0.45/am Millipore filter which had previously been dried at 9ff~C for 24 h, cooled in a desiccator for 2 h and then weighed. Following filtration the filters were again dried, cooled and reweighed

as above. Corrections were made for filter weight loss. The difference between final and initial weights was reported as dry ~eight in rag l-~. Five dry weight determinations were made over a 14 day period for the alpha TNT bioassay and 6 determinations were made over a 15 day period for the -lNTcc hioassay. A growth curve for the algae at each concentration was plotted on semilogarithmic paper as biomass versus time for M. aeruginosa and cell counts versus time for S. capri° cornutum. The average value of the three replicate flasks at each concentration was used to determine the biomass o r cell count. The controls, with no toxicant, served as the basis of comparison to determine toxic effect. A two tailed T-test at the 95°~ confidence level was used to determine significant differences in growth response between the control cultures and the cultures at each toxicant concentration. Gas-liquid chromatography was used to determine the concentrations of TNT during the S. capricornutum bioassays. Concentrations of TNT were monitored during the M. aeru#inosa bioassays by following absorbance at 240nm Inear the band maximum tbr alpha TNT) on a Cary Model 15 double beam spectrophotometer.

Fish bioassays A stock population of I000 fathead minnows was purchased from a local fish hatchery and held for four weeks prior to use in the bioassays. Mortality was less than 5~ during this period. The average fish was 5.4 cm in length and weighed 2.5 g. Since the fish had been fed cool, cooked quaker oats prior to purchase, this food was continued during the holding period. Two days prior to the start of the bioassay 120 fish were randomly selected for the test and placed into 2 75-1. aquaria filled with aerated, carbon filtered water similar to that used during the bioassays. Upon initiating the bioassay 15 fish were placed into each of 8 aquaria. Hardness of the water was 68.8 mg las CaCO~. The aquaria were held in an environmental chamber set to maintain a constant temperature of 24 + I~C with constant illumination, The fish were not fed for two days prior to or during the bioassays, The dosing apparatus used for the bioassays was a modified design of the proportional diluter described by Mount and Brungs (1967) and had the advantage of "failingsafely," that is, if the dilution water decreased in flow the fish were not killed by an overdose of toxicant. Alpha TNT test concentrations were 44.9, 9.54, 6.64, 3.84, 1.78, 0.78 and 0.05 mg 1- t. TNTcc test concentrations were 44.9, 6.25, 5.20, 3.13. 1.55. 0.60 and 0.07 mgl -t. These dilutions and one of control water (no TNT) were delivered to eight test chambers at a rate of 500 ml every 15 rain. The chambers were rectangular boxes constructed of Plexiglass®; their volume to overflow was 15 1., hence the replacement rate was effectively 100°,o ifi 8 h. The flow rate was approximately 1.2 1. of water or diluted toxicant per g of fish per day. Concentrations of alpha TNT and TNTcc were initially determined and monitored throughout these tests by spectrophotometric analysis. Ninety-six hour Leto'S. median lethal concentrations, and v.Cso's, median effective concentrations, were determined for both alpha TNT and TNTce. Both death and behavioral responses were used as criteria for estimating toxicity. Observations were made for behavioral responses and dead fish were removed every two hours during the 96-h test period. Due to the immediate reaction of the fish to TNT, their response after the first 10 min was also included in the data analysis. A fish was considered dead if it failed to elicit any response to a tactile stimulus such as gentle prodding with a glass rod. The fish exhibited three specific behavioral responses which occurred before death. We called the entire sequence the moribund response. The number of fish exhibiting any of these responses in each test chamber was recorded every 2 h: how-

Effects of TNT ever, only those fish exhibiting the third phase of the moribund response sequence (and dying during the testi were used for the moribund response ~c,.o calculation. analysis of the response of fish to each specific test concentration was also performed, this essentially being a quantal bioassay tGaddum. 1953). The time at which 16, 30, 50 and 84°j mortality or behavioral response occurred was calculated at each concentration {Bliss, 1937). This information was then plotted on ~mi-logarithmic paper as percent mortality or behavioral response versus time with 9 5 ° confidence intervals. Fish exhibiting any phase of the moribund response sequence were included in these calculations. All concentrations in which death or a behavioral response occurred were individually plotted on the same graph. Changes in slopes and clustering of these curves have been used to differentiate different modes of toxicity [Bliss, 1937: Herbert and Merkins. 1952: Sheppard. 1955: Sprague and Ramsey. 19651. This information was then correlated with the behavioral responses observed for different times at each concentration.

539

all of the TN"I" had been transformed to compounds which did not absorb at 240 nm. the wavelength at which the concentrations were monitored. Algae were therefore no longer being affected strictly by the original forms of T N T . Due to the complexity of T N T reactions, the exact nature of these new products could not be determined. Figure 1 shows that concurrent with this transformation net mortality Ondicated by negative slopes and indicative of a toxic effect) ceased and the cell counts increased. This suggests that alpha T N T was toxic at higher concentrations, but that its transformation products did not inhibit growth. Using continuous flow bioassays which replenished alpha T N T and thus did not allow a net accumulation of its transformation products. Hudock and Gring (1970) found a threshold concentration of 1.0 mg 1-1 alpha T N T for C. reinhardi, above which growth was completely inhibited. Continuous flow bioassays with S. capricornutum might show similar complete growth inhibition above a threshold concentration of 3-5 mg t- t alpha TNT. The effects of alpha T N T on M. ae:',@losa were much less pronounced than they were on S. cupricornutum (Fig. 2). No growth inhibition was discernable thru 15 rag l - t alpha TNT. While the growth of the 25 mg 1-t cultures were initially inhibited, no significant differences existed between these and control cultures at day 7. Growth at 50 mg 1- t was significantly retarded throughout the duration of the bioassay. The possible effect of the transformation of alpha T N T and its concomitant decreased toxicity was again noticeable. At day 5 the production of gas vacuoles (pseudovacuoles) and a prominent gelatinous sheath led to the flotation, as an algal mat, of the majority of cells in

RESULTS AND DISCUSS/ON

Al.qal bioassays No effects on the growth of S. capricornutt~m were seen at concentrations up to 3 m g l - t alpha T N T (Fig. 1). Although a toxic effect was noticeable at concentrations greater than this, growth inhibition was not permanent, there being no significant difference (P > 0.05, d.f. = 4) between counts at all concentrations by day 17. At 5 rag I - t alpha T N T growth began at day 3: there was no significant difference between these and control cultures by day 7. The 7 and 9 mg I- 1 cultures were still significantly different from 0 to 5 mg 1-, cultures at day 13. The chemical analysis performed during this and other bioassays indicated that during the first three days there was a rapid decrease of both alpha T N T and TNTcc concentrations. By the 5th or, at most, 7th day nearly

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Fig. I. Growth curves of S. capricornutum exposed to various concentrations of alpha TNT. - - x - - x - ling 1-~; - - O - - O - - 3rag I - t ; - - l - - l - 5rag I-t; - - I ' 1 - - [ ] - - 7mg I-t: - - A - - A - - 9 mg l- t.

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Fig. 2. Growth curves of M. aeru~jinosa exposed to various concentrations of 'alpha TNT. - - x - - x - 0mg 1-1; - - O - - O - - 1 mg 1- t ; --O---O--- 5 mg 1- t ; ~ l l - - I I - 10mg 1 - 1 ; - - r - l - - ~ - - 15mg 1-'; - - & - - & - - 25mg 1-1; - - A - - A - - 50 mg 1- t, all flasks with alpha TNT. Algae in the control flasks were homogeneously mixed throughout the medium and showed no signs of vacuole or sheath production. The degree of vacuole and sheath production, and thus matting was proportional to the alpha T N T concentration. This condition occurred throughout the I0.0

remainder of the bioassay. Blue-~een algae, including M. aeru.qinosa, are known to produce gas vacuoles and sheaths which ,are thought to serve as a density. and thus depth regulating mechanism (Fogg, 1941; 1965). Canabaeus (1929, cited in Hutchinson, 1967) suggested that gas vacuoles may serve as a means of raising an organism from an anaerobic or toxic region of a lake. Since vacuole and sheath production only occurred in those flasks containing some form of T N T and in proportion to the T N T concentration, it is possible that this was a reaction to a toxic environment. The gelatinous sheath could also serve as a protective mechanism by sealing the algal cells from a toxin, which must then diffuse through the sheath before reaching the cells. Aggregation of cells, due to the adhesive nature of the sheath, could also serve to inhibit a toxin from reaching the inner cells within the mass. TNWcc was not as toxic to S. c'apricorntttttm as was alpha TNT. There was no significant difl'erence in growth response between the control and toxicant cultures below concentrations of 9 m g l - ~ TNTcc (Fig. 3). The 9 and 2 5 m g 1-~ cultures showed no significant difference with the controls at day 14 and 9, respectively. TNTcc was toxic at 40 mg 1-1 completely inhibiting growth. Hudock and Gring H970) reported similar results with C. reinhardi, 50 mg 1TNTcc completely inhibiting growth, but with little effect below that concentration. M. aeruyinosa growth was generally stimulated by TNTcc except at 50 mg 1-~, which was toxic (Fig. 4).

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Fig. 5. Death response of the fathead minnow at specific alpha TNT concentrations with 95°,/0confidence intervals. As early as days 3 and 5, the 15 and 10 mg I - t cultures, respectively, exhibited significantly greater growth than the controls. Growth was significantly greater in the 5, 10, 15 and 25mgl -~ cultures than in the controls at day 15 and the degree of stimulation was generally proportional to concentration. A likely cause of this response was production of a compound or compounds during the chemical changes of TNTcc during the first few days of the test which stimulated M. aeruginosa growth. This effect was not seen with S. capricornutunt Gas vacuole and sheath production again occurred as it did in the M. aeruginosa alpha TNT test, even though there was no toxic reaction to the TNTcc.

Fish bioassays The 96-h LCso for alpha TNT based on the death response was 2.58 _+_0.1 mg 1-~. This value is similar to the 96-h L¢~o and TLm values (2.3-2.8mgl - t )

reported by Peterson (1970) and Nay et al. (1974) for bluegills. A 96-h ECso of 0.46 ___0.1 mgl - t was found using the moribund response criteria, indicating the early behavioral reaction to alpha T N T before de.ath. The death response LCso for T N T c c was 1.60 + 0.1 mg 1-~, indicating that it was more toxic than alpha TNT, a reverse of the situation observed in the algal data. The moribund response rCso, 0.64 + 0.1 mg 1-t, indicated that the two TNT forms were not greatly different in terms of sublethal toxicity. The fish exhibited three behavioral reactions to both toxicants. We called the initial reaction the shock response, since it was evident within a few minutes of the onset of the test. The fish began gasping at the surface and then became lethargic. The response had a definite threshold concentration, appearing in an "all or none" manner as described below. Fish which went into the second response were characterized by a loss of motor control. The fish

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Fig. 7. Death response of the fathead minnow at specific TNTcc concentrations with 95% confidence intervals. "'skittered" or swam in a jerking motion at the surface, rapidly extending and contracting their gills. At low toxicant concentrations this was the last response noted. Had the length of the bioassays been extended, these fish would probably have exhibited the third behavioral response observed at higher, more toxic, concentrations. The fish in the final behavior phase swam with a pitching, yawling, lethargic motion, responding only to tactile stimulus. This we named the "loss of equilibrium" response. Figures 5 and 6 summarize the death and behavioral responses at various alpha TNT concentrations. At least two modes of toxicity associated with the behavioral responses can be differentiated with a fair degree of confidence. All fish died within ten minutes when exposed to 44.9 mg I-t alpha TNT. The rapid death, as evidenced by the vertical response curve and narrow confidence intervals (Fig. 5), indicated a specific mode of toxicity associated with the shock re-

sponsc. Alpha "INT concentrations of 9.54, 6.64 and 3.84 mg 1-t led to the shock response without immediate death; this response did not occur at lower concentrations. A second mode of toxicity is indicated by the death response curves for the 9.54 and 6.64 mg 1-t concentrations. These fish began skittering after several hours exposure, followed soon thereafter by the loss of equilibrium response. The median survival times for these fish were 8 and 11 h, respectively. Fish exposed to 1.78 and 0.78mg1-1 alpha TNT showed no signs of the shock response. No deaths occurred at these concentrations, nor did they exhibit the loss of equilibrium response. TNTcc produced similar results, except that the fish were more sensitive to this form of TNT (Figs. 7 and 8). Again, all fish died within 10rain exposure of 44.9 mg 1-L the curve being indicative of a specific mode of death not seen at lower concentrations. The shock response without immediate death occurred at

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Effects of TNT 6.25 and 5 . 2 0 m g l -~. These fish. and those at 3.t3 and 1.55 m g l - t which had not shown signs of the shock response, entered the second and third behavioral responses after two to four hours exposure. The median survival time of these fish ~ a s from 6 to 32 h. The clustering and slopes of their death response curves again indicate a similar, second mode of toxicity. The lo~est concentrations tested of both T N T l~rms are not graphed because the fish showed no response to them. These data indicate the concentrations at which no death or behavioral response occurred. Alpha T N T concentrations of 1.78 mg I -~ and less produced no death response: there was no behavioral response at 0 . 0 5 m g l - ~ , No deaths resulted at TNTcc concentrations of 0.60 mg I- t and less: there was no behavioral response at 0.07 mg I-t. Data from the fish bioassays should be used in establishing T N T standards since they were much more sensitive to both forms of T N T than were either of the algae tested. The no behavioral response concentrations are similar to the stream standard of 0.02 mg I- ~ alpha T N T which Nay er al. (19741 considered appropriate. Their value was determine by applying the application factor of 0.01 to their 96-h T L m for bluegills. O u r results indicate that concentrations of 0.05 mg I- 1 alpha T N T and 0.07 mg I- z TNTcc may be appropriate as necessary stream standards. However. this should be verified by long-term, chronic toxicity tests. Acknowledyements--Data published herein were derived from studies conducted at the Water Quality Engineering Division. U.S. Army Environmental Hygiene Agency. Aberdeen Proving Grounds, Maryland. The opinions or assertions contained herein are the private views of the authors and are not to be construed as offacial or as reflecting the views of the Department of the Army. Financial assistance aiding in the completion of this manuscript was provided by the National Park Service, Southeast Region. This assistance is gratefully acknowledged. The authors also acknowledge the help of William Dennis and especially Nell Jurinski with the chemical analyses; Michael Carothers for data processing: and Suellen Boop for typing the manuscript. REFERENCES

Bliss C. I. 11937) The calculation of the time-mortality curve. Appl. Biol. 24, 814-851. Cannabaeus L. (1929) Uber die Heterocysten and Gasvacuolen der Blaualgen und ihre Beziehung zueinander. PflanzenJbrschuny 13, 252-253. Czerkinski S. N. [19551 Protection of surface waters from pollution by industrial waste waters. Lit. Ber. ~l~ss. Ahu'ass. Lult Boden 4, 98.

543

Degani J. G. 11943) Studies of the toxicity of ammunition plant wastes to fishes. Trans. Ar~ Fish. Soc. 73, 4-%-51. Fogg G. E. (lC~41)The gas-vacuoles of the Myxophyceae /Cynanophyceae). Biol. Rer. 16, 205-217. Fogg G. E. 11965) Algal Cultures and Phytoplunkton Ecolog.v. Uni~. Wisconsin Press. Madison. 126pp. Gaddum J. H. 11953) Bioassays and mathematics. Ph~trmacol. Rev. 5, 87-134. Herbert D. W. M. & Merkins J. C. 11952) Toxicity of potassium cyanide to trout. J. Exp. Biol. 29, 632-649. Hudock G. A. & Gring D. M. 11970) Biolo~lical Effects of Trinitrotoluene. Naval Environmental Health Center Contract Number N00164-69-C0822. 74 pp. Hutchinson G. E. 11967) A Treatise on Limnology. Vol. II. l l l 5 p p . Wiley, New York. LeClerc E. 11960) The self-purification of streams and the relationship between chemical and biological tests. [laste Treatment, Proceedin.qs of the Second Symposiunz on the Treatment 0]' Waste Wat~'rs. Edited by C. G. Issac pp. 281-316. Pergamon, Oxford. Matthews E. R., Rex E. H. & Taylor R. L. 11954) Laboratory studies pertaining to the treatment of TNT wastes. AEC Report No. TID-6268 Los Alamos N. M. and Albuquerque N. M. 18 pp. McKee J. E. & Wolf H. W. 119631 Water Quality Criteria. 2nd Edition. 548 pp. State Water Quality Control Board. California. Meinck F., Stooff H. & Kohlschl.itter H. (1956) Industrial Waste Waters. (lndustrce-Abwasserl, 2nd Edition. 536 pp. Gustav Fischer Verlag. Stuttgart. Mount D. I. & Brungs W. A. 11967) A simplified dosing apparatus for fish toxicology studies. Water Res. I, 21-29. National Eutrophication Research Program 11971) Al#al Assay Procedure: Bottle Test, 82 pp. National Eutrophication Research Center. Environmental Protection Agency, Corvallis. Ore. Nay M. W.. Jr., Randall C. W. & King P. H. 11972) Factors affecting color development during treatment of TNT waste. Ind. Wastes (Sept/Oct). 20 29. Nay M. W.. Jr.. Randall C. W. & King P. H. 11974) Biological treatability of trinitrotoluene manufacturing wastewater. J. War. Polhm Control Fed. 46, 485-497. Peterson G. 11970) Evaluation of toxicity of selected TN-I" wastes on fish. Phase l--Acute toxicity of alpha TNT to bluegills. Sanitary Engineerin.q Special Study No. 24-007-70/71, U.S. Army Environ. Hygiene Agency. Edgewood Arsenal. MD. 35 pp. Rogovskaya T. I. (1951) The effect of trinitrotoluene on the microorganisms and biochemical processes of selfpurification. Mikrohiolo~tiya 20, 265-272. Ruchhoft C. C.. Le Bosquet M.. Jr.. and Meckler W. G. (1945) TNT wastes from shell-loading plants. Ind. Enyny Chem. 37, 937-943. Schott S.. Ruchhoft C. C. & Megregian S. 11943) TNT wastes. Ind. Engn9 Chem. 35, 1122-1127. Sheppard M. P. 11955) Resistance and tolerance of young speckled trout (SalrelinusJbntinah~s) to oxygen lack. with special reference to low oxygen acclimation. J. Fish. Res. Bd Can. 12, 387-446. Sprague J. B. & Ramsey A. B. 11965) Lethal levels of mixed copper-zinc solution for juvenile salmon. J. Fish. Res. Bd Can. 22, 425-432.