An Extended Pattern of Pathogen Induced Mortality of Fathead Minnows

Press Enter to show all options, press Tab go to next option

Philip A. Russell

Littleton/Englewood Wastewater Treatment Plant 2900 S Platte River Dr; Englewood, Colorado 80110


Since 1993 the Littleton/Englewood Wastewater Treatment Plant in Englewood, Colorado, has performed monthly Whole Effluent Toxicity analyses using the Fathead minnow (Pimphales promelas) and Ceriodaphnia dubia. Significant and persistent patterns of reduced survival of Fathead minnows were observed during the summers of 1995 and 1997. Analyses of this phenomenon implicate a unique, recurring pathogen. This presentation will review (1) Fathead minnow (FHM) survival data for the L/E WWTP from 1993 through 1997, (2) steps taken to identify possible sources that might impact FHM survival in the WET test, and (3) research conducted to identify the causative agent. The implications of our research, future research directions, and the need for regulatory modifications to the WET protocol to assure that toxicity is being measured, will be discussed.


Whole effluent toxicity, WET, pathogen, wastewater, toxicity, Fathead minnow


Since 1993, the Littleton/Englewood Wastewater Treatment Plant, located just south of metropolitan Denver, Colorado, has performed routine monthly Whole Effluent Toxicity analyses on the Fathead minnow (Promelas pimphales) and Ceriodaphnia dubia. Significant and persistent mortality of Fathead minnows was observed in 1995 and 1997. Ongoing and evolving analyses of this phenomena implicate a unique, recurring pathological effect resulting in reduced Fathead minnow survival. When strictly interpreted, the observed survival effects result in a significant pattern of WET failure and, subsequently, potential regulatory impact.

The L/E WWTP is located in Englewood, Colorado; it serves the south Denver metropolitan region including Englewood and Littleton. Treated effluent from the facility is discharged into the north flowing South Platte River just south of the City of Denver’s border. The service area produces mainly non-industrial wastewater from a population of 250,000; the average influent/effluent rate is ~24 MGD with a maximum design capacity of 32 MGD. The facility design utilizes a conventional activated sludge process for secondary treatment. The design also includes (1) the addition of a primary trickling filter after primary sedimentation and prior to the secondary aeration process, (2) a tertiary nitrifying trickling filter (NTF) system for the removal of ammonia just prior to chlorination and release into the South Platte river and (3) an odor control system that passes potential odor producing gasses through the NTF's prior to chemical scrubbing (Lutz 1994). An underground pipe ~1/4 mile in length delivers treated water to the S. Platte river after dechlorination.

This paper will (1) demonstrate an example of a long term, naturally occurring pathogenic impact on WET analyses, (2) show that a historical review of WET data can facilitate the understanding of long term patterns of WET test results and (3) provide a set of tools to identify possible pathogenic artifacts on WET tested wastewater.

Methods and Materials

WET Analyses

Short term chronic Whole Effluent Toxicity tests were conducted using the EPA standard method (Lewis 1994). For most of the WET testing, control/dilution water was reconstituted; this was necessary because river water infrequently caused a significant toxic response. A dilution series of 0% (Control),12.5%, 25%, 60% (Facility IWC), 75% and 100% effluent was analyzed from May 1993 to present. This design made it possible to always have a relatively large sample set which produced a complete dose-response curve. Three samples of an effluent composite were used in each test series. Tests were typically conducted within 24 hours of collection. For later TIE investigations, the WET test protocol was modified to simplify the test and ameliorate costs; only dilutions of 45% and 100% effluent were compared with a control sample.

Modified toxicity identification evaluations (TIE) were performed in accordance with EPA guidelines (Norberg-King 1992, Mount 1989). Special WET tests were conducted to compare filtered, UV treated, and sterilized effluent water with control samples.

Chemical and Physical Analyses

A wide suite of analyses were performed on effluent wastewaters and impacted river water including WET effluent water samples, a River Study program, routine facility NPDES and Quarterly Survey analyses, and special tests. Typical measurements included ammonia, nitrate and nitrite, pH, TSS, sample water temperature, hardness, alkalinity, conductivity, and a range of metals including Cu, Zn, Hg, Ag, Se, Mn, and Fe. Less frequently, organic chemicals including volatile, semi-volatile, and pesticide suites defined by 40 CFR 122 were measured to compare between normal and affected influent. One wastewater sample was also analyzed for herbicides during 1997 when fish survival was significantly impacted.

Biological Analyses

Concurrent biological assays were also conducted during periods when Fathead minnows were surviving and not surviving the WET analyses. These tests included (1) modified Microtox bacterial acute toxicity tests conducted using 90% effluent ( Bulich 1995); (2) a Xenometrics ProToxÒ stress gene assay (Orser 1995), and (3) a bioassay study of the S. Platte river (Denver 1997, Plafkin 1989).

Facility processes review

Processes inside the plant were examined in detail including general effluent quality, process control and operation, chemical usage, polymer usage, drinking water quality, and pretreatment program records.


Figure 1 is a graphic summary of the survival of P. pimphales in WET testing from 1993 through 1997. The vertical axis displays the starting date of sample collection, the horizontal axis displays the percentage effluent used in the WET test samples, and the relative darkness indicates the percentage survival of fish in the WET test sample (a dark color is a general indication that an unusual amount of fish died in a sample). Using this figure, it is possible to discern patterns of interest. In the Fall of 1993, an episode where ammonia caused an effect can be observed; this includes a typical dose-response relationship between effluent concentration and fish survival (samples containing more effluent have less fish surviving; the tone progresses predictably from light to dark as the relative concentration of effluent increases).

In 1995 and 1997, decreased survival for relatively long periods of time is apparent. A typical dose-response is not present (the relative percentage of fish that die are about the same regardless of the dilution of the sample water). The temporal patterns for 1995 and 1997 are remarkably similar: the effect on fish survival in both years starting in May-June and disappearing in October. The analysis of data in this graphic mode may present a potential model for looking at other long term WET effects. Figure 2 illustrates this information in a more specific manner using selected WET samples from 1997. The April sample illustrates a fairly typical, though slightly more noisy response than normal. The early May sample shows a moderate and significant survival response and the late May sample exhibits a strong response. It is notable that survival at any concentration of effluent never drops to zero. With the exception of the control sample, which is supposed to remain near a 100% survival, the relative survival response, regardless of effluent concentration, remains equal or maybe even more depressed at mid concentrations.

No correlation is observed between Fathead minnow mortality and (1) facility process, (2) chemical and physical parameters or (3) bioassays.

In 1995, Phase I TIE tests produced inconclusive results. These results, the lack of a dose-response, and lack of possible toxicants lead to the hypothesis that the cause of poor fish survival was due to either (1) a pathogen or (2) an unusual polymer, surfactant, or organic chemical that was specifically toxic to fish and did not produce a dose-response. Before further Phase II TIE investigations could be implemented, the survival of fish returned to normal through spontaneous remission. During, 1996 the problem did not reoccur but research and communications with fish pathologists and aquaculturists continued. The phenomena we observed was not reported in literature nor observed by numerous contacts. Most contacts agreed that a pathogen was the most likely cause of the reduced Fathead minnow survival. In the early Spring of 1997, decreased survival in the Fathead minnow returned with characteristics identical to those observed in 1995. A modified Phase II TIE demonstrated that the lethal effect could be removed by filtration.  Figure 3 shows a comparison of three samples illustrating the difference in survival produced by filtering the water through a 0.45 um thin film membrane filter. The filters used on the 6/4/97 and 7/4/97 samples were Gelman MetricelÒ , a mixed cellulose ester compound. The filter used on the 7/15/97 sample was Gelman VersaporeÒ , an acrylic co-polymer compound. Communication with the manufacturer indicated that the Versapore filter was not as efficient at removing bacteria from water as the Metricel.

After determining that micro filtration removed the toxic effect, additional WET tests were conducted using UV exposed and autoclaved (~105° C) effluent water samples. Both of these methods eliminated the lethal effect. Whole fish samples of deceased and living minnows were stained and sectioned for pathological examination. Results, to date, indicate no obvious characteristics attributable to an infection. Before further investigations could be implemented, the lethal phenomena spontaneously remitted.

In summary, the following observations characterize the significant decrease in survival of the Fathead minnow observed in 1995 and 1997 in the effluent from the L/E WWTP:

  1. A significant survival effect and temporal pattern are present.
  2. Only the Fathead minnow is effected; C. dubia exposed to various dilutions of effluent survive and reproduce as well as the control samples throughout the period of observations from 1993 through 1997.
  3. A dose-response is not observed.
  4. No potential toxic material has been identified or observed to co-vary among a large suite of potential agents.
  5. A bioassay of macro-invertebrate populations in the S. Platte River indicates no effect caused by plant effluent.
  6. Bacteria based toxicity tests do not indicate the presence of a toxicant.
  7. Filtration with a 0.45 um membrane filter removes the lethal effect.
  8. UV treatment removes the lethal effect.
  9. Sterilization at a temperature of ~105° C removes the lethal effect.
  10. No relation to plant processes or industrial discharges is observed.
  11. Surviving fish usually have the same average weight as control fish.
  12. Onset of the effect takes from 3-5 days.
  13. A seasonal pattern exists which may be related to effluent temperature; the effect generally starts in April and ends in October during the years when survival is effected.
  14. The causative agent survives, or is stimulated by, the chlorination process

The evidence leads to the conclusion that the most likely source for the decrease in survival, observed during WET testing, is attributable to a pathogen.  Observations of affected fish larva and the results produced by filtered samples suggest that the pathogen is bacterial in nature.


Two references have been published describing the potential impact of pathogens to Fathead minnow survival in WET analyses (Grothe 1996a, Kszos 1997). Grothe demonstrated that filamentous bacteria in cooling water contributed to reduced survival and an erratic dose-response of Fathead minnows by gross interference of respiration. Water treated with UV radiation or filtration eliminated the observed poor survival. Ksozos concluded that mortality of Fathead minnows subjected to WET testing of ambient water was pathogenic. The main evidence for this finding was based upon improved survival rates of larval fish raised in isolation versus larvae reared in communities. The study also observed that the effect was often seasonal and survival was enhanced by UV treatment of the water samples. Our observations present an even stronger argument that naturally occurring pathogens may cause false positive indications of toxicity in the Whole Effluent Toxicity test. The combination of (1) an observation of a no dose-response relationship and (2) the removal of a lethal effect by filtration, UV exposure, and sterilization of wastewater samples, provides a strong case for the occurrence of a natural pathogen that can produce a false positive indication of toxicity in the Whole Effluent Toxicity protocol.

References Cited

Bulich, A., and G. Bailey. 1995. Environmental toxicity assessment using luminescent bacteria. Pages 29-40 in M. Richardson (Ed.), Environmental Toxicology Assessment. Taylor and Francis Inc., Bristol, PA.

Denver, City and County of. 1997. 319 nonpoint source grant final report. City and County of Denver, Department of Environmental Health. Denver, CO.

Ford, D.L. (Ed.) 1992. Toxicity Reduction: Evaluation and control. Technomic Pub. Co. Inc., Lancaster, PA.

Grothe, D.R., and D.E. Johnson. 1996a. Bacterial interference in whole-effluent toxicity tests. Environ. Toxicol. Chem. 15: 761-764.

Grothe, D.R., K.L. Dickson, D.K. Reed-Judkins. 1996b. Whole effluent toxicity testing: an evaluation of methods and prediction of receiving system impacts. SETAC Press, Pensacola, FL.

Kszos, L.A., A.J. Stewart, J.R. Sumner. 1997. Evidence that variability in ambient fathead minnow short term chronic tests is due to pathogenic infection. Environ. Toxicol. Chem. 16: 351-356.

Lewis, P.A., D.J. Klemm, J.M. Lazorchak, J.J. Norberg-King, W.H. Peltier, M.A. Heber. 1994. Short term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms (Third Edition). EPA/600/4-91/002. Environmental monitoring systems laboratory-Cincinnati, USEPA, Cincinnati, OH.

Lutz, M.P., S.J. Davidson, D.W. Stowe. 1994. Control of odor emissions at the Littleton/Englewood wastewater treatment plant. Pages 10-29 - 10-39 in Proceedings: Odor and organic compound emission control for municipal and industrial wastewater treatment facilities. Water Environment Federation, Alexandria, Virginia. Order Number CP3402.

Norberg-King, T.J. et. al. 1992. Toxicity identification evaluation: characterization of chronically toxic toxic effluents, phase I. EPA/600/6-91/005F. ERL, USEPA, Duluth, MN.

Mount, D.I. et. al. 1989. Methods for aquatic toxicity identification evaluations: phase II toxicity identification procedures. EPA/600/3-88/035. ERL, USEPA, Duluth, MN.

Orser, C.S., et. al. 1995. Use of prokaryotic stress promoters as indicators of chemical toxicity. InVitro Tox. 8:71-85.

Plafkin, J.L., et. al. 1989. Rapid bioassesment protocols for use in streams and rivers: benthic macroinvertebrates and fish. EPA/444/4-89-001. Off. Water, USEPA, Washington, D.C.

USEPA. 1991. Technical support document for water quality-based toxics control.


USEPA. 1992. Introduction to water quality-based toxics control for the NPDES program. EPA 831-S-92-002


WET Historical Survival Record, Fat Head Minnow

Back to Figure1 reference in this paper


Figure 2. Fathead Minnow Survival - WET Analyses 1997

Percent Survival

Back to Figure 2 reference in this paper 


Figure 3. Fathead minnow survival in WET analyses:

Filtered (0.45 um membrane) versus unfiltered sample water.

Figure 3

Back to Figure 3 reference in this paper.

Copyright © 1998 by Littleton / Englewood WWTP. All rights reserved.