CONSIDERATIONS IN MOSQUITOFISH & PAPERMILL EFFLUENT STUDIES: ANALYSIS OF METHODOLOGY

JJ Noggle¹, WK Bradley², DS Ruessler³, MS Sepulveda¹, TS Gross^1,3

 ¹University of Florida, Gainesville, FL
²National Council for Air and Stream Improvement, Inc. (NCASI), Vanceboro, NC
³USGS, Florida Integrated Science Center, Gainesville, FL

Presented at the 22nd annual meeting of the Society of Environmental Toxicology
and Chemistry, Baltimore, MD, November 11-15, 2001.

ABSTRACT

Earlier studies have suggested masculinizing effects (anal fin elongation in females) of papermill effluents in Eastern mosquitofish, Gambusia holbrooki.  These effects have also been proposed as indicators of papermill effluent exposure in natural systems. The current investigation monitored the influence of preservation state, observer bias, and instrument bias on gender identification, anal fin masculinization, and body measurements in effluent-exposed mosquitofish. Eastern mosquitofish were collected using a backpack electroshocker and dip nets at five sites in and around Rice Creek, the receiving stream of effluent from the Georgia-Pacific Palatka, FL, operation.  Fish were sexed (presence/absence female urogenital papilla) and measurements made on fresh fish prior to fixation in formalin.  A subset was re-measured post-fixation then sent to a collaborating lab for gender identification (gross gonad examination) and measurements manually and by computer.  Fresh versus fixed values within the first lab as well as measurements between labs were both significantly correlated. However, formalin fixation decreased anal fin measurements compared to fresh tissues.  Sex identification between labs was significantly correlated, indicating the urogenital papilla is a reliable way to determine sex in this species.  Computer-aided measurements correlated with hand measurements in the collaborating lab, supporting the use of computerized measuring software for more efficient data collection.  Future analyses of methods will involve computer-aided measurements used to define the most appropriate anal fin characteristic associated with papermill effluent exposure.  These methodology studies will contribute to accurate assessment of mosquitofish potential as an indicator of papermill effluent exposure.
 

INTRODUCTION

• Eastern mosquitofish, Gambusia holbrooki, are sexually dimorphic with maturation-dependent secondary sexual characteristics:
• Normal males: projection of the anal fin called the gonopodium.
• Normal females: gravid spot and urogenital papilla.
• Papermill effluent-exposed females: elongation of the anal fin resembling the gonopodium of a maturing male (Bortone & Cody 1999), with length dependent on distance from effluent outfall (Noggle, unpublished data).
   
• Mosquitofish have been proposed as bioindicators of papermill effluent exposure (Bortone & Davis 1994, Cody & Bortone 1997).
   
• In order to use mosquitofish as bioindicator species, appropriate methods and potential biases must be established beforehand.
   
• Error may arise from preservation state, observer bias, and instrument bias, to name a few.
   
• In tandem with a field study of these fish (see Noggle et al. 2001 - SETAC poster #PM183), effects of such biases were addressed.
   

OBJECTIVE

• To determine the influence of preservation state, observer bias, and instrument bias on gender identification, anal fin length, and body measurements in Eastern mosquitofish exposed to papermill effluents.
   

MATERIALS & METHODS

• Location: Rice Creek, tributary of St. Johns River, Palatka, FL, USA.
• receives papermill effluent from Georgia-Pacific mill.
• survey sites:  three downstream, an upstream, and a reference.
• Collection: backpack electroshocking and dip nets, near vegetated banks.
• Processing: Table 1 presents measurements taken under four different conditions.
• USGS lab measured fish fresh (before preservation) and fixed (after preservation with formalin) to account for preservation state – all manually (using digital calipers & a dissecting scope with ocular micrometer).
• USGS and NCASI labs collaborated to determine observer bias, comparing hand measurements.
• NCASI lab measured fish by hand and by computer (SigmaScan Pro 5.0, Jandel Scientific) to examine instrument bias.
• Statistics: Pearson's product moment correlation coefficient (p< 0.05 significance).

DISCUSSION

• Preservation state did not alter measurements (Figure 1), although fresh values were consistently larger than fixed values (formalin dehydrating the fishes).
• In Figure 2, computer-aided versus manual measurements for standard length had a tight correlation, while anal fin measures showed more variation.
• Perhaps fixation influenced measurements.
• Computer measures are more specific – calibration is important.
• Strong correlation between labs for gender identification (Figure 3) supports the use of female urogenital papilla as a noninvasive, reliable sexing technique for this species.
• Consistently larger values in USGS lab versus NCASI lab for anal fin measurements could be due to time of preservation (since NCASI measured fish weeks after USGS) and/or observer bias – see Figure 4.
• Overall, these measurements were repeatable within and between labs using the same instrumentation.
• Fixation appears to decrease measurements.
• Focus on computer-aided measurements with future studies.
   

ACKNOWLEDGMENTS

• National Council for Air and Stream Improvement, Inc. (NCASI) provided funding, and the NCASI Southeastern Aquatic Biology Center participated in this study.
• Georgia-Pacific Corporation, Atlanta, GA, also contributed funding for this project.