Product Citations and Abstracts

Weathered Alaska North Slope crude oil (ANS 521) was stirred for 14 days at 15° C in 20 o/oo salinity sterilized seawater, amended with nutrients and a hydrocarbon degrading microorganism (EI2V) isolated from an oil-contaminated beach in Prince William Sound, Alaska. A total of 13.7 mg/L water soluble fraction (WSF) of neutral fraction hydrocarbons was recovered. Toxicity/tetratogenicity tests were conducted with WSF neutral fraction hydrocarbons recovered from the biodegradation system using embryonic and larval Pacific herring, Clupea pallasi. Exposures were begun at 4, 48 and 96 h post fertilization of herring eggs. Exposure concentrations were 1, 10 and 100% (w/v) of the recovered WSF fraction (redissolved in 20 o/oo salinity sterile seawater at 15° C). Sterile 20 o/oo salinity seawater without the addition of redissolved neutral fraction hydrocarbons was used for controls. Significant (p < or = 0.05) teratogenic responses or embryo mortality was observed at WSF concentrations of 10 and 100% (w/v). On days 5 through 8 of embryogenesis, counts of heart contraction rates were significantly lower (p < or = 0.05) at the 100% WSF concentration of hydrocarbons for embryos exposed beginning 4 and 48 h post fertilization. Grow-out of larvae from selected exposures was conducted. High mortality was noted in larvae that were exposed to the 10% WSF concentration beginning at 4 and 48 h post fertilization. Most of these larvae died 5 to 8 days after hatching when they elicited broken backs at a time concurrent with the onset of feeding behavior.

Middaugh, Douglas P. and Toru Takita. 1983. Tidal and Diurnal Spawning Cues in the Atlantic Silverside, Menidia menidia. EPA-600/J-82-015. Environ. Biol. Fishes. 8(2):97-104. (ERL,GB 150). (Avail. from NTIS, Springfield, VA: PB83-209809)

Field and laboratory observations revealed tidal and diurnal cues for spawning in the Atlantic silverside, Menidia menidia. In the field, spawning runs began approximately when daytime flood tide velocities ranged from 3 to 16 (x = 11) cm sec-1. Spawning runs ended at ebb tide velocities ranging from 5 to 22 (x = 17) cm sec-1. In the laboratory, M. menidia were reared from embryos to sexual maturity in 10 months (April 1979 to January 1980). During this time, approximately 50 fish were held in each of two, 1 m diameter tanks. A pump was used to maintain a constant current velocity of 8 cm sec-1 in the holding tanks. Water temperature ranged from 16° to 25°C, the salinity was 30 ± 2 o/oo. Fish were fed Tetramin flake food and Artemia nauplii each day. During January and February 1980, the seawater circulation pump was turned off twice daily for one hour, from 1200 to 1300 and 2400 to 0100. Current velocities decreased from 8.0 cm sec-1 to 0.0 cm sec-1 during these periods. M. menidia held under a 24 h light:0 h dark (24 L:0 D) photoperiod spawned from 1200 to 1300 and 2400 to 0100 in response to decreased current velocities. Modification of the photoperiod to 14 L:10 D (with the circulating pump turned off from 1200 to 1300 and 2400 to 0100) resulted in spawning between 0500 and 0600 in response to "lights-on" and between 1200 and 1300 in response to decreased current velocities. No spawning occurred in response to the decreased current velocities during darkness. The importance of timing of spawning runs to coincide with low current velocities was revealed in tests to determine the duration of viability of sperm and unfertilized eggs. Eggs remained highly viable for only 5 min, sperm for 10 min. Spawning when current velocities were low reduced dispersion of milt, thus increasing the likelihood of egg fertilization.

Middaugh, D.P., P.J. Chapman, M.E. Shelton, C.L. McKenney, Jr. and L.A. Courtney. 2002. Effects of Fractions from Biodegraded Alaska North Slope Crude Oil on Embryonic Inland Silversides, Menidia beryllina. Arch. Environ. Contam. Toxicol. 42(2):236-243. (ERL,GB 1136).

Embryonic inland silversides, Menidia beryllina, were exposed to neutral, water soluble fractions (WSFs) resulting from microbial degradation of artificially weathered Alaska North Slope (ANS) crude oil. Three individual microbes obtained from Prince William Sound, Alaska and designated Phe#6 (enriched on phenanthrene), Hexaco#2 (enriched on the straight-chain alkane, hexacosane) and E12V (grown by enrichment on Bushnell-Haas medium containing 0.2% pristane, a branched alkane) were used to individually biodegrade weathered ANS crude oil for 14 days in darkness in 20 L glass carboys containing nutrient enriched, sterilized 20 o/oo salinity seawater at 20 ± 1° C. Neutral WSFs resulting from biodegradation of ANS (lot 521) by each microbe were recovered and weighted. Neutral WSFs recovered were: 1.76 mg/L for Phe#6, 1.85 mg/L for Hexaco#2 and 13.02 mg/L for the E12V microbe. Embryo toxicity and teratogenicity tests revealed that exposure of embryos to the WSFs from the E12V incubation (with a total recovered neutral fraction approximately seven times greater than the Phe#6 and Hexaco#2 incubations) resulted in the most severe responses in craniofacial, cardiovascular, and skeletal organ systems. The total neutral WSFs recovered from the E12 V biodegradation of weathered ANS 521 were subfractionated into saturated (eluted with hexane), aromatic (eluted with CH2 Cl2), polar (eluted with ethyl ether), and recombined (saturated + aromatic + polar) fractions. Developing fish embryos were then exposed to each subfraction and the recombined subfractions. The polar subfraction and recombined subfractions proved to be the most embryo toxic and teratogenic. They resulted in statistically significant (p less than or equal to 0.05) responses (compared to controls) for craniofacial, cardiovascular, skeletal and total severity effects in one or both tests with these subfractions.

Middaugh, Douglas P. and Charles L. McKenney, Jr. 2003. Occurrence and Orientation of Flounders (Bothidae: Paralichthys) on an Intertidal Beach. J. North Carol. Acad. Sci. 119(4):157-171. (ERL,GB 1172).

The intertidal movement and burying pattern of paralichthid flounders was studied on DeVeaux Bank, a semi-permanent island at the mouth of the North Edisto River in South Carolina. A total of 1,366 flounders buried within the study site during the two yr study. The standard length of flounders burying ranged from 19 to 48 cm. The frequence of flounders digging into substrates in the upper intertidal zone to form "beds" was greater on nighttime high tides than during daytime high tides. Most flounders, 99.99%, buried in sand, the remainder in mud. There was no significant difference in the directional orientation of buried flounders and the direction of tidal currents. The orientation of flounder beds generally deviated from the orientation of tidal current flow by 10 degrees azimuth or less. Flounder beds were oriented into flood tide currents an average of 68% and 32% into ebb tide currents. Periodic washover events formed megaripples in the sand within the study site. There was a significant difference in the size distribution of flounder beds on megaripples and between megaripples. Smaller flounders were positioned on megaripples while larger individuals were found between the sand ripples. Tidal flow turbulence or water depth may have played a role in the spatial distribution of flounders in these two locations. There was a significant correlation between the standard length of flounder beds and the water depth where they occurred. Smaller flounders buried at shallower water depths than larger flounders. Avoidance of predators, including the bottlenose dolphin, could have influenced the location where flounders buried in the intertidal zone.

Middaugh, Douglas P., H.W. Kohl and L.E. Burnett. 1983. Concurrent Measurement of Intertidal Environmental Variables and Embryo Survival for the California Grunion, Leuresthes tenuis and Atlantic Silverside, Menidia menidia (Pisces: Atherinidae). EPA-600/J-81-177. Calif. Fish Game. 69(2):89-96. (ERL,GB 221).

Concurrent daily measurements of environmental variables and embryo survival were made for two atherinid fishes, the California grunion, Leuresthes tenuis, observed at Blacks Beach, La Jolla, California; and the Atlantic silverside, Menidia menidia, observed at the Point of Pines, Edisto Island, South Carolina. Measurements were made during April 1980. Both species spawned in the upper intertidal zone on high tide. L. tenuis eggs were deposited approximately 4 cm below the beach surface during nighttime. Subsequent sand deposition buried embryos to a depth of approximately 8 cm where they were protected from thermal and desiccation stresses. Daily survival of incubating embryos averaged 97%. M. menidia utilized three spawning substrates: 1) abandoned crab burrows, 2) detrital mats, and 3) the stems and primary leaves of cordgrass, Spartina alterniflora. These substrates provided embryos with varying degrees of protection from thermal and desiccation stresses. Daily survival of embryos located 15 cm deep in abandoned crab burrows averaged 88%. Survival was less, 76% at the entrance. Daily survival averaged 94% at the surface of detrital mats and at the axis of stems and primary leaves of cordgrass. Survival was lower at other locations on these substrates.

Middaugh, Douglas P., Wayne R. Davis and Ruth L. Yoakum. 1975. Response of Larval Fish, Leiostomus xanthurus, to Environmental Stress Following Sublethal Cadmium Exposure. Contrib. Mar. Sci. 19(1):13-19. (ERL,GB 223).

The toxicity of cadmium to larval fish, Leiostomus xanthurus, was studied. An incipient LC50 concentration of approximately 0.2-0.3 mg/l cadmium was first estimated. Subsequent short-term sublethal tests were conducted to determine the relationship of cadmium exposure and accumulated whole body residues of the metal on the response of larvae to thermal stress and low dissolved oxygen. Results of this study indicated a significant decrease (a=0.05, t-Test) in the critical thermal maximum (CTM) for larvae exposed to 0.5 and 0.8 mg/l cadmium for 96 hours at 20°C. Significant decreases (a=0.05, X2) in survival of larvae subjected to a dissolved oxygen (D0) level of 1.6 mg/l after exposure to 0.5 And 0.8 mg/l cadmium were also observed.

Middaugh, Douglas P. and John M. Dean. 1977. Comparative Sensitivity of Eggs, Larvae and Adults of the Estuarine Teleosts, Fundulus heteroclitus and Menidia menidia to Cadmium. Bull. Environ. Contam. Toxicol. 17(6):645-652. (ERL,GB 231).

The present study measured the sensitivity of two common species of estuarine fish, the mummichog, Fundulus heteroclitus, and the Atlantic silverside, Menidia menidia, to cadmium at specific stages in their life histories. Developing eggs were tested because of the proven sensitivity of this life stage to cadmium toxicity in marine fish. Three age groups of larvae, 1-, 7- and 14-days old were tested to determine if changes in sensitivity occur during the first few weeks after emergence. Bioassays were also conducted with adults for comparison with data from egg and larval bioassays. The duration of tests was limited to 48 hrs so that comparisons of sensitivity to cadmium could be made for specific developmental stages (ages) of each species.

Middaugh, D.P. and P.W. Lempesis. 1976. Laboratory Spawning and Rearing of a Marine Fish, the Silverside Menidia menidia. Mar. Biol. 35(4):295-300. (ERL,GB 252).

Adult silversides, Menidia menidia menidia (Linnaeus), were collected in early March, 1974 and maintained in 3 recirculating seawater tanks in the laboratory. Respective groups were fed Moore-Clark Fry Fine at 3, 7 and 10% of their body weight per day. The photoperiod (light intensity approximately 2000 lux) was increased in increments of 10 min/day from 12 h light to 14 h light. The water temperature was increased by 1°C/day from the ambient collection temperature, 14° C, to 22° C. Twenty-four days after beginning laboratory conditioning, fish in each tank were stripped. There was a significant increase (X2, a= 0.05) In the number of ripe males at all three feeding levels, compared to an initial field-collected group that was checked at the beginning of the conditioning period. Females also showed significant increases in ripeness at the 7 and 10% but not at the 3% feeding level. The gonadal indices (gonad weight expressed as percentage of body weight) of both sexes were significantly greater than those measured for the initial field-collected group, but did not differ from those of adults collected from the field at the time laboratory conditioning was terminated. Techniques for maintaining eggs from field-ripened adults in the laboratory have been developed, and the effect of salinity on the percentage emergence of larvae determined. The highest emergence rate of larvae was 61% when eggs were maintained at 30 o/oo S. Emergence was 56% at 20 o/oo S and 47% at 10 o/oo S. The effect of delayed feeding on survival and growth was best for larvae fed Artemia sp. nauplii immediately after emergence at 30 o/oo S.

Middaugh, Douglas P. and Genie Floyd. 1978. Effect of Prehatch and Posthatch Exposure to Cadmium on Salinity Tolerance of Larval Grass Shrimp, Palaemonetes pugio. EPA-600/J-78-079. Estuaries. 1(2):123-125. (ERL,GB 260). (Avail. from NTIS, Springfield, VA: PB-290 085)

Groups of embryonic grass shrimp, Palaemonetes pugio, were exposed to 0.1 and 0.3 mg/l cadmium at 30 ppt salinity and 25°C for the last 1, 4 or 8 days prior to hatching. Other groups of embryos were cultured in uncontaminated seawater. Prehatch exposure to cadmium was found to have no additive effect on the sensitivity of the larvae to cadmium exposure and salinity stress for 14 days after hatching. Only one group of larvae, exposed to 0.1 mg/l cadmium for 4 days before hatching, and transferred to 10 ppt salinity water containing 0.1 mg/l cadmium after hatching, showed a significant (X2, p < 0.05) decrease in survival, compared to control survival. No significant decreases in survival were observed for any larvae transferred to 15 and 30 ppt salinity at a pre- and posthatch cadmium concentration of 0.1 mg/l. At a pre- and posthatch cadmium concentration of 0.3 mg/l, significant decreases in survival were observed for all of the larvae transferred to 10 and 15 ppt salinity after hatching. Significant decreases in survival were observed for only 2 of the groups exposed before hatching and transferred to 30 ppt salinity and 0.3 mg/l cadmium after hatching.

Middaugh, Douglas P., John A. Couch and Allan M. Crane. 1977. Responses of Early Life History Stages of the Striped Bass, Morone saxatilis to Chlorination. Chesapeake Sci. 18(1):141-153. (ERL,GB 304). (Avail. from NTIS, Springfield, VA: PB-269 932)

The toxicity of total residual chlorination (TRC) to early life stages of the striped bass, Morone saxatilis, was determined using percent embryo hatchability, incipient LC50 bioassays, histopathology, and avoidance responses. Beginning 8 to 9 hours after fertilization, developing embryos were exposed continuously to TRC in flowing water at 1.0-3.0 ppt salinity and 18 ± 1 C. Fifty-six percent of the control group (no TRC exposure) hatched. None of the embryos exposed to a measured TRC concentration of 0.21 mg/l hatched. Only 3.5 percent of the embryos exposed to 0.07 mg/l TRC and 23 percent of those exposed to 0.01 mg/l TRC hatched. Incipient LC50 bioassays were used to determine the sensitivity of 2-, 12- and 30-day-old striped bass to concentrations of TRC in flowing water (1.0-3.0 ppt salinity at 18 ± 1 C). The estimated incipient LC50 was 0.04 mg/l TRC for 2-day-old prolarvae, 0.07 mg/l for 12-day-old larvae and 0.04 mg/l for 30-day-old juveniles. Histological examination of 30-day-old juveniles which survived exposure in the incipient LC50 bioassay indicated gill and pseudobranch damage for fish exposed to 0.21 to 2.36 mg/l TRC. Statistical analysis of avoidance tests conducted at 1.0-3.0 ppt salinity and 18 ± 1 C with 24-day-old larvae showed significant (X2, p < 0.05) and reproducible avoidance responses to measured TRC concentrations of 0.79-0.82 mg/l and 0.29-0.32 mg/l. No avoidance was indicated at TRC concentrations of 0.16-0.18 mg/l.

Middaugh, D.P., J.M. Dean, R.G. Domey and G. Floyd. 1978. Effect of Thermal Stress and Total Residual Chlorination on Early Life Stages of the Mummichog Fundulus heteroclitus. EPA-600/J-78-070. Mar. Biol. 46(1):1-8. (ERL,GB 308). (Avail. from NTIS, Springfield, VA: PB-291 003)

Effects of simultaneous short-term (7.5 to 60 min) thermal stress (24° to 34°C) and total residual chlorination (0.05 to 1.0 mg 1-1) on specific development stages of the mummichog Fundulus heteroclitus (Pisces: Cyprinodontidae), were investigated. For the embryonic stages, the total number of successfully hatched larvae was used as the criterion to measure effect. For the larval stages, survival 24 h after exposure was used. In the embryonic stages, temperature was the most important main variable. Only one embryonic stage (gastrula) was confounded by second-order interactions (temperature x duration of exposure x total residual chlorination). Both 0-day and 7-day-old larval stages showed significant higher-order interactions for all combinations of test parameters, suggesting the presence of synergistic effects of the three main experimental variables.

Middaugh, D.P. and W.P. Davis. 1976. Impact of Chlorination Processes on Marine Ecosystems. In: Water Quality Criteria Research of the U.S. Environmental Protection Agency. EPA-600/3-76-079. U.S. Environmental Protection Agency, Environmental Research Laboratory, Corvallis, OR. Pp. 46-62. (ERL,GB 326). (Avail. from NTIS, Springfield, VA: PB-257 091)

The use of chlorine as a disinfectant and antifouling agent is reviewed. Chemical reactions of chlorine in aquatic environments are discussed, with particular emphasis on the formation of halogenated organic constituents in freshwater and marine systems. Studies of the effect of chlorinated sewage effluents and cooling water from generating stations on marine organisms and ecosystems are summarized.

Middaugh, D.P., A.M. Crane and J.A. Couch. 1977. Toxicity of Chlorine to Juvenile Spot, Leiostomus xanthurus. EPA-600/J-78-080. Water Res. 11(12):1089-1096. (ERL,GB 364). (Avail. from NTIS, Springfield, VA: PB-290 976)

The sensitivity of juvenile spot, Leiostomus xanthurus, to total residual chlorine (TRC) in flowing sea-water was investigated. Incipient LC50 bioassays, histopathology, avoidance tests and the combined effect of thermal stress and TRC were used to assess sensitivity. Estimated incipient LC50 values were 0.12 mg1-1 TRC at 10 degrees C and 0.06 mg1-1 TRC at 15 degrees C. Histological examination of spot used in the incipient LC50 bioassay at 15 degrees C and sacrificed while alive indicated pseudobranch and gill damage occurred in individuals exposed to a measured TRC concentration of 1.57 mg1-1. Spot exposed to lower concentrations of TRC, 0.02-0.06 mg1-1 at 15 degrees C and sacrificed alive showed no consistent tissue damage.Spot demonstrated temperature dependent avoidance responses to TRC. At 10 degrees C, a concentration of 0.18 mg1-1 was required for significant (X2; P is less than 0.05) avoidance; at 15 and 20 degrees C, spot showed significant avoidance of TRC concentrations as low as 0.05 mg1-1. Simultaneous exposure of spot to thermal stress (5, 10 or 13 degrees C above the acclimation temperature of 15 degrees C) at measured TRC concentrations of 0.05-0.07 and 0.34-0.52 mg1-1 demonstrated a significant, (X2 with Yates correction, P is less than 0.05) increase in sensitivity to TRC with increased temperature and exposure times for some of the groups tested.

Middaugh, Douglas P., Louis E. Burnett and John A. Couch. 1980. Toxicological and Physiological Responses of the Fish, Leiostomus xanthurus, Exposed to Chlorine Produced Oxidants. Estuaries. 3(2):132-141. (ERL,GB 378). (Avail. from NTIS, Springfield, VA: PB81-126559)

The sublethal and lethal effects of chlorine produced oxidants (CPO) on juveniles of the estuarine teleost, Leiostomus xanthurus, were investigated in flowing water tests conducted at 30 ± 1°C and 26 to 31 ppt salinity. Short-term LT50 tests were conducted at two nominal concentrations of NaOCl, 1.0 and 1.4 mg/l (respective measured CPO concentrations 0.09 and 0.12 mg/l) which were sublethal in 2,880 minute exposures; and three nominal concentrations, 1.6, 1.8 and 3.2 mg/l NaOCl (respective measured CPO concentrations 0.13, 0,20 and 0.37 mg/l) which were acutely toxic. Opercular ventilation rates in exposed spot were much higher than in control fish, but returned to rates only slightly above those of controls during the latter portion of the 2,880 minute exposure to the two sublethal CPO concentrations. Opercular rates at the three acutely toxic CPO concentrations remained much higher than control rates until the exposed fish died. Blood pH after 2,880 minutes of exposure to the sublethal concentrations of CPO; or at the respective estimated LT50 for lethal concentrations, showed significant decreases (X as low as 6.84) compared to controls (X 7.35). No significant changes in the percent methemoglobin were observed. Oxygen uptake by spot was depressed at all of the measured concentrations of CPO tested. Histopathological examination showed that gill respiratory epithelial tissues sloughed away from the underlying pillar cells. Complete denudation of circulatory tissues and hemangiectic secondary lamellae were observed in gill tissues from fish exposed to the highest CPO concentration of 0.37 mg/l.

Middaugh, Douglas P. 1981. Reproductive Ecology and Spawning Periodicity of the Atlantic Silverside, Menidia menidia (Pisces: Atherinidae). EPA-600/J-81-103. Copeia.(4):766-776. (ERL,GB 400). (Avail. from NTIS, Springfield, VA: PB82-205808)

The reproduction ecology and spawning periodicity of the Atlantic silverside, Menidia menidia, was studied in the North Edisto River estuary, South Carolina, at Bears Bluff and the Point of Pines during the spring and summer, 1976-1978. Spawning runs occurred only in daytime and coincided precisely with the predicted time of high tide. Spawning by large numbers of fish in a small area at high tide, when tidal current and velocities were low, caused depletion of dissolved oxygen to more than l.0 mg/l. M. menidia spawned in the upper intertidal zone at elevations of 1.20 to 2.40 meters above mean low water. Observed maxima in spawning-run index values occurred near the time of new and full moons, suggesting that the spawning population is synchronized by a lunar cue. The coincidence of a high tide at the time of sunrise every two weeks (high tide-sunrise cue) also may have served as a synchronizer for the spawning population. Determination of the relative importance of each factor was made difficult by their approximate synchrony during the March-July spawning season. Many predators fed on spawning M. menidia. Several predators, including the ruddy turnstone, Arenaria interpres morinella, semipalmated sandpiper, Ereunetes pusillus, and blue crab, Callinectes sapidus, fed on developing M. menidia embryos. Larval emergence was limited to times of tidal inundation. More larvae hatched during high tides at night than during the day.

Middaugh, Douglas P., Geoffrey I. Scott and John M. Dean. 1981. Reproductive Behavior of the Atlantic Silverside, Menidia menidia (Pisces, Atherinidae). EPA-600/J-80-008. Environ. Biol. Fishes. 6(3/4):269-276. (ERL,GB 409). (Avail. from NTIS, Springfield, VA: PB82-236407)

The spawning behavior of the Atlantic silverside, Menidia menidia, was studied at two sites on the North Edisto River estuary in South Carolina. Prespawning schools moved back and forth along the shoreline as the time of high tide approached. Spawning runs took place in the upper intertidal zone at high tide. Spawning fish deposited # their eggs on three types of substrates: 1) the lower stems of cordgrass plants, Spartina alterniflora, 2) detrital mats, and 3) exposed cordgrass roots along erosional scraps. Spawning behavior during egg deposition and fertilization was similar for all three substrates. Females released eggs during a rapid fluttering motion of the posterior half of the body. A similar movement accompanied release of sperm by males. Behavior of fish just prior to spawning insured deposition of gametes at locations that provided protection from thermal and drying stress during development. Eggs were deposited at mean intertidal elevations of 1.8 and 1.5 meters above mean low water (MLW) at respective study sites. They were exposed to the atmosphere for approximately ten hours between successive high tides. During spawning runs in which eggs were deposited at the base of cordgrass plants, ambient dissolved oxygen concentrations of the water in the spawning area were sometimes reduced to <1.0 mg . 1-1. Spent fish, apparently incurring an oxygen debt while spawning, formed a nonschooling aggregation offshore from the spawning zone. The recurrent use of specific spawning substrates for egg deposition resulted in an uneven distribution of spawning runs along the shoreline at each study site.

Middaugh, Douglas P. and Michael J. Hemmer. 1984. Spawning of the Tidewater Silverside, Menidia peninsulae (Goode and Bean), in Response to Tidal and Lighting Schedules in the Laboratory. Estuaries. 7(2):139-148. (ERL,GB 441).

Tidewater silverside, Menidia peninsulae were maintained in 1.3 m diameter holding tanks in identical laboratory recirculating systems. During two weeks under constant conditions (a current velocity of 8 cm s-1 and continous illumination, 24 L:O D) there was a low relative frequency of arrhythmic spawning. In the subsequent two-week period, fish in one pair of tanks were maintained under the singular influence of twice daily decreases in current velocity from 8 to 0 cm s-1 at 0600-0700 and 1800-1900, under continous illumination. The relative frequency of spawning remained low and there was no evidence of a daily spawning rhythm. However, the mean number of eggs per spawn increased substantially. Fish in the second holding system were subjected to diel light cycle of 13 L:11 D with a constant current velocity of 8 cm s-1 for two weeks. The relative frequency of spawning remained low and there was no indication of spawning rhythmicity; moreover, there was only a slight increase in the mean number of eggs per spawn. During the third two-week period, fish in the first pair of tanks were provided a 13 L:11 D diel light cycle, in conjunction with preexisting twice daily decreases in current velocity; those in the second pair of tanks were provided twice daily decreases in current velocity in conjunction with the preexisting 13 L:11 D light cycle. Under the combined influence of decreases in current velocity and a diel light cycle, there was a marked increase in the relative frequency of spawning in both pairs of tanks. Fish manifested a discernible spawning periodicity, spawns typically occurred between 1800 and 2400; the mean number of eggs per spawn also increased. When fish were returned to constant conditions, current velocity 8 cm s-1 and 24 L:0 D for two weeks, the frequency of spawning decreased and there was no indication of a spawning periodicity. Results of another experiment (decreased current velocities at 1200-1300 and 2400-0100 with 13 L:11 D light cycle) indicated gradual expression of a tidal spawning rhythm during nighttime, 2000-0359. Our laboratory results indicate that M. peninsulae is predominantly a nocturnal spawner and that spawning coincides with decreased current velocities.

Middaugh, Douglas P., Richard G. Domey and Geoffrey I. Scott. 1984. Reproductive Rhythmicity of the Atlantic Silverside. EPA-600/J-84-191. Trans. Am. Fish. Soc. 113(4):472-478. (ERL,GB 491).

The reproductive periodicity of the Atlantic silverside Menidia menidia was studied at two locations on the North Edisto River estuary in South Carolina during March-July 1976-1978. Spawning runs occurred in the upper intertidal zone and coincided precisely with daytime high tides. Time-series analysis of daily changes in the intensity of spawning runs revealed a fortnightly reproductive periodicity and indicated that the observed reproductive rhythmicity in Atlantic silversides may be mediated by a high tide-sunrise cue that also occurs at fortnightly intervals. During the 1976 and 1977 reproductive seasons, there were highly significant correlations (P < 0.01) among the male gonadal index, the female gonadal index, and the occurrence of intermediate, maturing, and hydrated-egg stages of sexual development in females. The percentage of females with hydrated eggs was greatest on days when a high tide occurred within 1 hour after sunrise.

Middaugh, Douglas P., Michael J. Hemmer and Yara Lamadrid-Rose. 1986. Laboratory Spawning Cues in Menidia beryllina and M. peninsulae (Pisces, Atherinidae) with Notes on Survival and Growth of Larvae at Different Salinities. EPA/600/J-86/086. Environ. Biol. Fishes. 15(2):107-117. (ERL,GB 508). (Avail. from NTIS, Springfield, VA: PB86-208543)

Spawning patterns of inland silversides, Menidia beryllina, and tidewater silversides, Menidia peninsulae, were examined in the laboratory under several combinations of 'tidal' and diel light cycle cues. M. beryllina showed a high frequency of spawning throughout the day when held under constant conditions (24L: OD, current velocity 8 cm sec-1) and when 'tidal' and diel light cycles were presented singly or in combination. In contrast, M. peninsulae demonstrated a high frequency of spawning only when presented a combination of 'tidal' and diel light cycle cues and spawned predominantly at night. Menidia beryllina embryos were euryhaline. Hatching ranged from 73 to 78% at salinities of 5, 15 and 30 o/oo. M. peninsulae embryos showed an inverse relationship between the percentage hatch and the incubation salinity, 90% at 5 o/oo and only 65% at 30 o/oo. Survival and growth of larval M. beryllina from the day of hatching through 16 days old was optimal at 15 o/oo. Although survival of M. peninsulae larvae was optimal at 30 o/oo, no trend was apparent in growth of larvae held for 16 days at 5, 15, or 30 o/oo salinity.

Middaugh, D.P., P.G. Hester, M.V. Meisch and P.M. Stark. 1985. Preliminary Data on Use of the Inland Silverside, Menidia beryllina, to Control Mosquito Larvae. EPA/600/J-85/376. J. Am. Mosq. Control Assoc. 1(4):435-441. (ERL,GB 532). (Avail. from NTIS, Springfield, VA: PB86-187382)

A study of procedures for spawning and culture of the inland silverside, Menidia beryllina, was conducted. The efficacy of young Menidia, 20 to 22 and 31 to 33 days old, to control mosquito larvae was determined in the laboratory with first and second larval instars of the saltmarsh mosquito Aedes taeniorhynchus. Feeding trials were run at salinities of 1, 5, 15 and 25 o /oo. Field trials were also conducted to determine if Menidia would effectively control Culex quinquefasciatus in brackish water impoundments.

Middaugh, Douglas P., Michael J. Hemmer and Daniel E. Penttila. 1987. Embryo Ecology of the Pacific Surf Smelt, Hypomesus pretiosus (Pisces: Osmeridae). EPA/600/J-87/468. Pac. Sci. 41(1-4):44-53. (ERL,GB 557). (Avail. from NTIS, Springfield, VA: PB89-208623)

A study of the ecology of developing embryos of the Pacific surf smelt, Hypomesus pretiosus, was conducted. Embryos were maintained in the laboratory at 7.6, 12.1 and 17.6°C and the time to specific embryonic stages determined. Embryos held at 7.6° C developed to stage 24, 18 days after collection; those held at 12.1°C hatched after 13 days; at 17.6°C hatching occurred 8.5 days after collection. Embryos maintained at 15°C and salinities of 20, 25 and 30 o/oo averaged 84% survival. There was no significant difference in survival among groups (ANOVA, P=0.53). Field observations indicated that embryos are spawned in patches in the upper intertidal zone near the time of high tide. They are attached to gravel substrates by the zona radiata membrane which ruptures and quickly turns inside out at the time embryos are fertilized. After several days of development, stage 18 to 22 embryos detach from the original spawning substrates and are washed seaward and down into the gravel substrate in the intertidal zone. However, there was no significant difference (ANOVA, P >=0.09) in the number of eggs found at each of 4 depth strata in the upper, middle and lower intertidal zones.

Middaugh, Douglas P. and Michael J. Hemmer. 1987. Reproductive Ecology of the Tidewater Silverside, Menidia peninsulae (Pisces: Atherinidae) from Santa Rosa Island, Florida. Copeia. 1987(3):727-732. (ERL,GB 561). (Avail. from NTIS, Springfield, VA: PB88-171061)

The reproductive ecology of the tidewater silverside, Menidia peninsulae, was studied during 1982-1983 along the shoreline of Santa Rosa Island, Florida. Adult Menidia were observed at low tide spawning on a red alga, Ceramium byssoideum. Pinfish, Lagodon rhomboides, were noted preying upon newly spawned Menidia eggs. The annual reproductive cycle of M. peninsulae extended from Feb.-July with the highest spawning activity during March-June at water temperatures of 16.7 to 30.8° C. Several statistical analyses of tidal stages and gonadal indices failed to reveal significant differences between spring and neap tides and reproductive activity. However, on four occasions peaks in the percentage occurrence of mature eggs within ovaries coincided with the time that the moon was positioned over the equator. Moreover, analysis of young-of-the-year Menidia (6-28 mm SL) revealed distinct length classes, suggesting that spawning and subsequent hatching of larvae may have occurred in periodic pulses throughout the spring and early summer.

Middaugh, D.P. and M.J. Hemmer. 1987. Influence of Environmental Temperature on Sex-Ratios in the Tidewater Silverside, Menidia peninsulae (Pisces: Atherinidae). EPA/600/J-87/332. Copeia.(4):958-964. (ERL,GB 568). (Avail. from NTIS, Springfield, VA: PB88-199435)

The sex-ratios of Menidia peninsulae from Santa Rosa Island, Florida, were studied during a 13-month survey. Weekly samples revealed significant deviations from the expected sex-ratio of 1:1. During May-October, young-of-the-year (YOY) females comprised 70 to 94% of the individuals collected in the 32.5 to 62.4 mm standard length SL size class. These females were the presumptive progeny of reproduction during cold to cool fluctuating temperatures, (14.1-24.2 C), during February-April. In contrast, collections of YOY Menidia during November-April yielded 35-60% females. These individuals were the presumptive progeny of reproductive activity and sexual differentiation in May-August at warm fluctuating temperatures of 23.5-31.5 C. The pattern in sex-ratios of older Menidia (62.5 - 102.4 mm SL) paralleled that of YOY individuals. The annual (13 month) sex-ratio for collections of YOY were identical (69% females) to sex-ratios in older Menidia.

Middaugh, D.P., M.J. Hemmer and E.M. Lores. 1988. Teratological Effects of 2,4-Dinitrophenol, 'Produced Water' and Naphthalene on Embryos of the Inland Silverside Menidia beryllina. Dis. Aquat. Org. 4:53-65. (ERL,GB 613).

Embryos of the inland silverside Menidia beryllina were exposed to 3 teratogens: (1) 2,4-dinitrophenol, (2,4-DNP), (2) 'produced water' (PW), and (3) naphthalene (NPH). Tests were conducted by placing single embryos in glass tissue culture tubes containing 6 ml of saline exposure media. Twenty tubes were used for each exposure concentration and for controls. A severity-index based upon craniofacial, cardiovascular, and skeletal terata was used to rank response each day. The compounds tested caused teratogenic expressions in embryos and larvae exposed from the 2- to 4-cell and blastula stage through 7 to 8 d post-fertilization. However, combined survival in control embryos and larvae, and those exposed to respective teratogens, were not significantly different in 5 of 6 tests, except the 2- to 4-cell embryos exposed to 2,4-DNP. There was a marked increase in the relative frequency of terata with increasing nominal exposure concentrations of each compound. Post-hoc comparison of mean rank scores for severity of expression between control and exposed individuals revealed statistically significant (a= 0.05) levels of terata at 1.8 and 3.2 mg 2,4-DNP 1-1; 10 and 20% PW; and 5.6 and 10 mg NPH 1-1.

Middaugh, Douglas P. and Michael J. Hemmer. 1994. Fish Model as an Indicator for Teratogenic Substances. In: Biological Monitoring of the Environment: A Manual of Methods. EPA/600/A-94/202. J. Salanki, D. Jeffrey, and G.M. Hughes, Editors. CAB International, Wallingford, England. Pp. 116-120. (ERL,GB 621). (Avail. from NTIS, Springfield, VA: PB95-122966)

A fish model, suitable for use as an indicator for teratogenic substances, is described. Individual blastula stage embryos of the inland silverside, Menidia beryllina, are exposed to teratogens in sealed tissue culture tubes containing 6 ml of saline test media, 5 o/oo salinity and 25 +- 1°C. Individual embryos are examined daily and terata enumerated using a system that ranks craniofacial (CR), cardiovascular (CV) and skeletal (SK) responses. Procedures for statistical analysis of data are described.

Middaugh, Douglas P. and Jonathan M. Shenker. 1988. Salinity Tolerance of Young Topsmelt, Atherinops affinis, Cultured in the Laboratory. EPA/600/J-88/469. Calif. Fish Game. 74(4):232-235. (ERL,GB 626). (Avail. from NTIS, Springfield, VA: PB90-147745)

Topsmelt, Atherinops affinis, were cultured at 21 +- 1°C at salinities of 10 o /oo and 30 o/oo from the day of hatching to 24 days old. Thereafter, the salinity was lowered 2 o/oo per day for the group originally maintained at 10 o /oo and raised 2 o/oo per day for the group cultured at 30 o/oo salinity. Observations for mortality were made daily throughout the study. No fish died at 10 o/oo salinity during the first 24 days after hatching and no mortalities occurred as salinity was lowered from 10 o/oo to 2 o/oo, or during the 24-day period after hatching. As salinity was increased by 2 o/oo daily, no deaths occurred until day 40 at 60 o/oo salinity. Thereafter, cumulative mortality increased to 48 % at a salinity of 80 o/oo. At 82 o/oo salinity, mortality totalled 80%.

Middaugh, Douglas P., Michael J. Hemmer, Jonathan M. Shenker and Toru Takita. 1990. Laboratory Culture of Jacksmelt, Atherinopsis californiensis, and Topsmelt, Atherinops affinis (Pisces: Atherinidae), with a Description of Larvae. Calif. Fish Game. 76(1):4-13. (ERL,GB 646).

Embryonic and larval jacksmelt, Atherinopsis californiensis, and topsmelt, Atherinops affinis, were cultured in the laboratory. Larval A. californiensis were grown for 24 days at 10, 20 and 30 o/oo salinity. Survival, 80-91%, was highest at 10 o/oo salinity. Increases in standard length (SL) and wet weight were greatest for larvae cultured at 10 or 20 o/oo. Survival of larval A. affinis cultured at 10, 20 and 30 o/oo for 24 days ranged from 99-100%. Increases in SL and wet weight were greatest for larvae cultured at 20 or 30 o /oo salinity. Illustrations of day of hatch, 8-, and 24-day-old larvae are presented with morphometric descriptions for each species. Unique melanophore patterns provide a useful character for identification of these two closely related atherinid fishes which occur sympatrically in California bays and estuaries.

Middaugh, Douglas P., John W. Fournie and Michael J. Hemmer. 1990. Vertebral Abnormalities in Juvenile Inland Silversides Menidia beryllina Exposed to Terbufos During Embryogenesis. EPA/600/J-90/382. Dis. Aquat. Org. 9(2):109-116. (ERL,GB 695). (Avail. from NTIS, Springfield, VA: PB91-163956)

Embryos of the inland silverside, Menidia beryllina, were exposed to a nominal concentration of 50 µg terbufos 1-1 during the first five days of embryogenesis. Silversides were maintained in clean dilute seawater until 37 days after hatching. Radiographs revealed compressed and fused vertebrae and dorsal-ventral misalignment of pre- and post-zygapophyseal processes. Histopathological examination of individuals exposed to terbufos during hyperostoses to almost complete fusion of some vertebrae.

Middaugh, D.P., B.S. Anderson and M.J. Hemmer. 1992. Laboratory Spawning of Topsmelt, Atherinops affinis, with Notes on Culture and Growth of Larvae. EPA/600/J-92/218. Environ. Toxicol. Chem. 11(3):393-399. (ERL,GB 703). (Avail. from NTIS, Springfield, VA: PB92-195759)

Topsmelt, Atherinops affinis, were spawned repeatedly in the laboratory during May-July, 1989. A periodic "temperature spike" from the holding temperature of approximately 18°C, up to approximately 20.5°C, introduced at 7 to 9 day intervals, resulted in maximum production of viable embryos on the fourth morning after the spike. Examination of embryonic stages and comparison to known developmental rates for A. affinis embryos revealed that spawning was generally nocturnal, occurring between 1900 and 0500 hrs. Survival of embryonic and larval A. affinis cultured at 21 + or - 1°C and 20 + or - 2 o/oo salinity was excellent (>80%). A larval growth curve was developed for the first 24 days post-hatch.

Middaugh, Douglas P. and Michael J. Hemmer. 1992. Reproductive Ecology of the Inland Silverside, Menidia beryllina (Pisces: Atherinidae) from Blackwater Bay, Florida. EPA/600/J-92/220. Copeia. 1992(1):53-61. (ERL,GB 724). (Avail. from NTIS, Springfield, VA: PB92-195775)

The reproductive ecology of the inland silverside, Menidia beryllina, was studied during Feb. 1988--March 1989 at Robinson Point, Blackwater Bay, Florida. Environmental variables including pH, rainfall, salinity, water temperature, and dissolved oxygen were measured weekly or biweekly. Fish were sampled weekly with a seine designed to catch adult, juvenile, and young-of-the year (YOY) individuals. Most reproductive activity occurred during Feb.--April 1988. The maximum mean weekly female gonadosomatic index (GSI) of 12.5 occurred in April. Fecundity ranged from 63 to 419 hydrated eggs/female. The maximum mean weekly male GSI of 6.1 occurred in early March. Catches of YOY individuals 7.6-37.5 mm SL were greatest in May. Some of these YOY individuals matured in July-Sept. and spawned. This reproductive activity resulted in recruitment of a second group of YOY fish into the population during Aug.-Oct. Growth rates of YOY in May-July, calculated by regression methods from weekly frequency distributions of standard length, was 0.34 mm/day for females and 0.31 mm/day for males. The reproductive pattern of M. beryllina from Blackwater Bay, Florida indicates that qualitatively it is an r-strategist with rapid growth of YOY, sexual maturation at an early age, relatively high fecundity, and multiple spawning within the first reproductive period for YOY fish in July-Sept. and again as 1- to 1-plus-year-old individuals.

Middaugh, D.P., J.G. Mueller, R.L. Thomas, S.E. Lantz, M.J. Hemmer, G.T. Brooks and P.J. Chapman. 1991. Detoxification of Pentachlorophenol and Cresote Contaminated Groundwater by Physical Extraction: Chemical and Biological Assessment. EPA/600/J-91/336. Arch. Environ. Contam. Toxicol. 21(2):233-244. (ERL,GB 732). (Avail. from NTIS, Springfield, VA: PB92-129733)

Chemical analyses revealed that polycyclic aromatic hydrocarbons (PAH's) and other organic compounds were present in a perennial freshwater stream that flowed through the abandoned American Creosote Works, designated for Superfund cleanup by the U.S. Environmental Protection Agency. A study was conducted to determine efficacy of ultrafiltration for removal of these organics from groundwater at the Superfund site. Ultrafiltration reduced the concentration of total identified organics from 210.0 mg/L in groundwater to 1.5 mg/L in the post-filtration permeate. Tests for toxicity/teratogenicity in embryonic inland silversides, Menidia beryllina; and Microtox® were conducted with: streamwater, untreated groundwater, feedwater used in the ultrafiltration system and permeate water that passed through the ultrafiltration system. A concentration of 100% streamwater caused significant (a < or =0.05) teratogenic responses in fish embryos and larvae. Groundwater and feedwater caused significant embryo toxic or teratogenic responses at concentration of 100, 10 and 1%; Microtox® EC50's were 0.85 and 0.48%, respectively. In contrast, only 100% permeate water caused significant increases in terata, compared to the control response; at 10 and 1% concentrations >90% of hatched larvae appeared normal. The Microtox EC50 was 30% permeate water

Middaugh, D.P., S.M. Resnick, S.E. Lantz, C.S. Heard and J.G. Mueller. 1993. Toxicological Assessment of Biodegraded Pentachlorophenol: Microtox(R) and Fish Embryos. EPA/600/J-93/058. Arch. Environ. Contam. Toxicol. 24(2):165-172. (ERL,GB 777). (Avail. from NTIS, Springfield, VA: PB93-168920)

A Gram-negative bacterium, Pseudomonas vesicularis, (strain SR3) was isolated from soil at a former wood treatment plant in north central Florida. The ability of this bacterium to degrade pentachlorophenol (PCP) was confirmed by growing cells in a basic salt medium in which PCP was the only source of carbon and energy. Degradation from a measured concentration of 39-40 ug PCP/mL to 0.0006 ug PCP/mL was observed within 120 hrs of incubation in the presence of PCP induced cells of P. vesicularis. The initial cell density in these cultures was 6 x 10 to the sixth power cfu/mL. Microtox(R) 5 min EC50 toxicity tests revealed that aqueous solutions of PCP, measured concentrations 39-40 ug/mL were toxic but that final biodegraded samples, 0.0006 ug PCP/mL were nontoxic. However, bioassays with embryonic inland silversides, Menidia beryllina, showed that the biodegraded samples were embryotoxic or teratogenic. Water containing PCP at concentrations up to 30 times higher than measured in the final biodegraded samples were less toxic/teratogenic. These results indicate that while biodegradation of PCP was nearly complete, intermediate metabolites of the degradation process were toxic or teratogenic. Thus, the M. beryllina bioassay allows extremely sensitive assessment of toxicity associated with biodegraded environmental pollutants and may be a useful criterion for determining whether bioremediated water or soil is safe for discharge back into the environment.

Middaugh, D.P. and B.S. Anderson. 1993. Utilization of Topsmelt, Atherinops affinis, in Environmental Toxicology Studies Along the Pacific Coast of the United States. EPA/600/J-94/234. Rev. Environ. Toxicol. 5:1-49. (ERL,GB 835). (Avail. from NTIS, Springfield, VA: PB94-170065)

This review provides a synopsis of information available on the geographical distribution, identification, natural history and reproductive habits of the topsmelt, Atherinops affinis. We also review and report on procedures for laboratory spawning of adult topsmelt, culturing of the early-life stages including embryos and larvae, and conducting several different toxicity tests with early-life stages. The toxicological protocols were developed as part of a continuing effort by the U.S. Environmental Protection Agency and the California Water Resources Control Board to provide toxicity testing protocols for indigenous indicator fishes that can be used in pollution research and effluent monitoring programs in the Pacific coastal region of the United States.

Middaugh, D.P. and F.J. Genthner. 1994. Infectivity and Teratogenicity of Beauveria bassiana in Menidia beryllina Embryos. EPA/600/J-94/274. Arch. Environ. Contam. Toxicol. 27(1):95-102. (ERL,GB 863). (Avail. from NTIS, Springfield, VA: PB94-191004)

Developing embryos of the inland silverside fish, Menidia beryllina, were exposed to conidiospores of the insect pathogenic fungus, Beauveria bassiana, that possessed activity against the migratory grasshopper, Melanoplus sanguinipes. Various adverse effects were observed in Menidia beryllina embryos and larvae. They included rupture of the chorion, embryo death, developmental defects (vertebral abnormalities) in the embryo of hatched larvae, and fungal infections on the mandibles of larvae. Although there was little evidence of a definitive dose response trend based on densities of viable conidiospores, statistically significant (p < or = 0.01) responses were observed in tests in which conidiospore densities were as low as 7.1 x 103/ml and as high as 1.3 x 106/ml. Viable spores were required for adverse effects to occur; heat-killed spores failed to cause significant adverse effects.

Middaugh, D.P., R.L. Thomas, S.E. Lantz, C.S. Heard and J.G. Mueller. 1994. Field-Scale Testing of a Hyperfiltration Unit for Removal of Creosote and Pentachlorophenol from Ground Water: Chemical and Biological Assessment. EPA/600/J-94/280. Arch. Environ. Contam. Toxicol. 26(3):309-319. (ERL,GB 888). (Avail. from NTIS, Springfield, VA: PB94-191061)

Chemical analyses and biological response data were used to assess the efficacy of a field-scale hyperfiltration unit in the removal of polylcyclic aromatic hydrocarbons (PAHs) and other organic compounds from creosote- and pentachlorophenol (PCP)-contaminated ground water recovered from the former American Creosote Works in Escambia County, Pensacola, Florida. The hyperfiltration unit consisted of 4 modules containing porous stainless steel tubes which were coated with a formed-in-place zirconium hydrous oxide-polyacrylic acid (ZOPA) membrane. A 5-fold concentraion of the feedwater (80% volume reduction) with up to 97% removal of high molecular weight PAHs was achieved during pre-demonstration and field-demonstration runs of the hyperfiltration unit. Approximately 68% of PCP was removed by the unit. Performance for phenolics was less successful, averaging 27 and 36%, respectively for the two runs. Toxicological and teratogenic data for embryonic inland silversides, Menidia beryllina, indicated that 100, 10 and 1% solutions of the ground water sample used in the pre-demonstration run caused statistically significant (p < or = 0.05) biological responses when compared to controls. However, the 1% solution of permeate in the pre-demonstration run was nontoxic and not teratogenic. This 1% solution was also not very toxic in Microtox® tests. The 5 min EC50 was 56.44%. Acute toxicity tests with Ceriodaphnia dubia revealed a 48 h LC50 of greater than 1% permeate. Similar results were obtained in the field-demonstration run of the hyperfiltration unit. In this run, feedwater was toxic or teratogenic to Menidia beryllina embryos at 100, 10 and 1% concentrations. Permeate from the field-demonstration run was embryo toxic or teratogenic at 100 and 10% concentrations but not at 1%. The Microtox® 5 min EC50 was 58.50% at the 1% concentration and the Ceriodaphnia 48 h LC50 was 5.6% permeate in the field-demonstration run. The Escambia County Utilities Authority (ECUA) set a pre-discharge requirement of non-toxicity to Ceriodaphnia dubia for 1% permeate solutions. Permeates from both runs, diluted to 1%, met the pre-condition of non-toxic responses in 48 h tests with Ceriodaphnia dubia. Meeting this requirement allowed for discharge of diluted permeate into the county's sanitary sewerage collector system.

Middaugh, D.P., S.E. Lantz, C.S. Heard and J.G. Mueller. 1994. Field-Scale Testing of a Two-Stage Bioreactor for Removal of Creosote and Pentachlorophenol from Ground Water: Chemical and Biological Assessment. EPA/600/J-94/279. Arch. Environ. Contam. Toxicol. 26(3):320-328. (ERL,GB 889). (Avail. from NTIS, Springfield, VA: PB94-191053)

A two-stage, field-scale bioreactor system was used to determine the efficacy of bioremediation of creosote- and pentachlorophenol (PCP)-contaminated ground water at the abandoned American Creosote Works (ACW) site in Pensacola, Florida. In separate 15-day runs of the field-scale (454L) system, bioreactor performance in the presence of specially-selected microbial inoculants was compared to that observed using non-specific biomass. In the first run, Bioreactor #1 was amended with nutrients, surfactants and microorganisms (strains CRE 1-13) that were isolated from soil at ACW and selectively cultured for their ability to biodegrade monitored creosote constituents. After four days of organism acclimation and degradation of organic contaminants, the batch system was converted to a flow-through regime. Effluent was transferred to Bioreactor #2 where Pseudomonas paucimobilis strain EPA 505 and Pseudomonas sp. strain SR 3 were introduced. A second 15-day bioreactor run was conducted using indigenous bacteria and microorganisms from a waste water treatment facility that was designed to treat effluents containing PAHs and phenolics. Bioreactor performance was evaluated by chemical analysis of feed water initially pumped into Bioreactor #1 and clarified effluent from Bioreactor #2. These materials were also tested for toxic/teratogenic responses with developing embryonic Menidia beryllina, with Microtox® 5 min EC50 tests, and with Ceriodaphnia dubia 48 h LC50 tests. Results obtained with specialty organisms in the first run of the field-scale bioreactor showed that, on average, 70.6% of polycyclic aromatic hydrocarbons (PAHs) and heterocycles were degraded. Only 36.9% of the pentachlorophenol (PCP) present was biodegraded, apparently because of low cell counts and incomplete induction of Pseudomonas sp. strain SR 3. A concomitant reduction in toxicity/teratogenicity occurred. In the second 15-day run of the bioreactor, microorganisms from an industrial waste water treatment facility averaged 51.0% biodegradation of PAHs and heterocycles. Degradation of PCP was 81.0%, a value substantially higher than in the first run. Reductions in toxicity/teratogenicity were also observed for effluent from the second run of the field-scale bioreactor but the magnitude of toxicity reduction was less than in the first run.

Middaugh, D.P. and D.D. Whiting. 1995. Responses of Embryonic and Larval Inland Silversides, Menidia beryllina, to No. 2 Fuel Oil and Oil Dispersants in Seawater. Arch. Environ. Contam. Toxicol. 29(4):535-539. (ERL,GB 897).

Embryonic inland silversides, Menidia beryllina, in the early blastula stage were exposed to the water-soluble fraction (WSF) of No. 2 Fuel oil and the oil dispersants Corexit 7664® and 9527®, singly and in combination. An ordinal ranking system was used to score observed daily craniofacial, cardiovascular, and skeletal responses in control embryos and those exposed to 1%, 10%, and 100% concentrations of the WSF of No. 2 Fuel oil, the dispersants Corexit 7664® and 9527® applied at the recommended field application concentrations, and the combination of No. 2 Fuel oil and respective dispersants in seawater. The non-parametric Kruskal-Wallis analysis of variance (ANOVA) and post hoc analyses were used to identify stastically significant differences for control embryos and those exposed to No. 2 Fuel oil and dispersants. Embryos exposed to No. 2 Fuel oil in 20 o/oo salinity seawater showed significant (a less than or equal to 0.01) responses only at the 100% WSF concentration. Corexit 7664® tested singly elicited significant responses at 10% and 100% concentrations. When No. 2 Fuel oil and Corexit 7664® were combined at recommended field application concentrations of the dispersant, the oil and dispersant mixture resulted in significant (a less than or equal to 0.01) responses at 1%, 10%, and 100% exposure concentrations. In contrast, Corexit 9527® did not cause significant responses at the three test concentrations of 1%, 10%, and 100% of the recommended field application rate. However, when No. 2 Fuel oil and Corexit 9527® were combined in seawater, the 10% and 100% exposure concentrations resulted in statistically significant (a less than or equal to 0.01) embryonic responses, relative to controls. Chemical analyses indicated that both dispersants increased the total WSF of No. 2 Fuel oil in seawater.

Middaugh, D.P., P.J. Chapman and M.E. Shelton. 1996. Responses of Embryonic and Larval Inland Silversides, Menidia beryllina, to a Water-Soluble Fraction Formed During Biodegradation of Artificially Weathered Alaska North Slope Crude Oil. Arch. Environ. Contam. Toxicol. 31(3):410-419. (ERL,GB 934).

Weathered Alaska North Slope crude oil (ANS 521) was stirred for 2 and 14 days in 20o/oo salinity sterile seawater or for 14 days in seawater with nutrients and a group of three (GO3) microorganisms from Prince William Sound, Alaska, (PWS) that were capable of biodegrading hydrocarbons. A total of 0.65 and 0.69 mg/L water soluble fraction (WSF) of neutral fraction hydrocarbons were recovered from the 2 and 14 day stirred sterile systems, respectively. In contrast, a total of 7.5 mg/L WSF neutral fraction hydrocarbons was recovered from systems containing ANS 521 that was stirred and biodegraded by the GO3 microbes for 14 days. Toxicity/teratogenicity tests were conducted with neutral fraction hydrocarbons recovered from the sterile and biodegraded systems using embryonic inland silversides, Menidia beryllina. Hydrocarbons from the sterile systems did not cause statistically significant teratogenic responses at concentrations of 1%, 10% and 100% of recovered fractions (redissolved in 20o/oo sterile seawater). Counts of heart contraction rate were significantly lower (a< or = to 0.05) at the 100% WSF concentration of hydrocarbons on day 5 and 6 of embryogenesis. Recovered and redissolved neutral fraction hydrocarbons from ANS 521 that were biodegraded for 14 days caused statistically significant (a< or = to 0.05) teratogenic responses at the 10% and 100% WSF concentrations. Measurement of heart contraction rates showed statistically significant (a< or = to 0.05) reductions at the 100% WSF concentration on days 2 through 6 of embryogenesis, compared with controls.

Middaugh, D.P., N. Beckham, J.W. Fournie and T.L. Deardorff. 1997. Evaluation of Bleached Kraft Mill Process Water Using Microtox(R), Ceriodaphnia dubia, and Menidia beryllina Toxicity Tests. Arch. Environ. Contam. Toxicol. 32(4):367-375. (ERL,GB 953).

To determine whether a 7 to 10-day embryo toxicity/teratogenicity test with the inland silverside fish Menidia beryllina is a sensitive indicator for evaluation of bleached kraft mill effluents, we compared this test with the Microtox® 15-minute acute toxicity test and the Ceriodaphnia dubia 7-day chronic toxicity test. Water samples used in each test were collected from three areas in a bleached kraft pulp and paper mill using a 100% chlorine dioxide bleaching process: 1) river water prior to use in the mill; 2) the combined acid/base waste stream from the pulping process prior to biological treatment in the aerated stabilization basin (ASB); and 3) the effluent from the ASB with a retention time of approximately 11 days. Relative toxicity determined by the three tests for each water sampling location was compared. All three toxicity tests were predictive; however, the C. dubia and M. beryllina tests were the more similar and sensitive indicators of toxicity. Process water (ASB influent) prior to biological treatment in the ASB was toxic at all concentrations using the Microtox® and C. dubia tests. The fish embryo test showed no toxicity at 1% concentrations, slight toxicity at 10%, and acute toxicity at the 100% ASB influent concentration. Tests with biologically treated ASB effluent indicated a substantial reduction in observed toxicity to Microtox® bacteria, C. dubia, and M. beryllina. No toxic responses were observed in any test at a 1% ASB effluent concentration, the approximate effluent concentration in the receiving river following mixing. No relationship was found among any toxicological response and effluent levels of adsorbable organic halides, polychlorinated phenolic compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin,2,3,7,8-tetrachlorodibenzofuran, total suspended solids, color, chemical oxygen demand, or total organic carbon.

Middaugh, Douglas P., Michael J. Hemmer and Larry R. Goodman. 1987. Methods for Spawning, Culturing and Conducting Toxicity Tests with Early Life Stages of Atherinid Fishes. EPA/600/8-87/004. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 56 p. (Avail. from NTIS, Springfield, VA: PB87-174934)

Procedures are presented for spawning, culturing and conducting acute and chronic toxicity tests with four atherinid fishes: the inland silverside, Menidia beryllina, Atlantic silverside, M. menidia, tidewater silverside, M. peninsulae, and California grunion, Leuresthes tenuis. Guidelines also are provided for growing of food organisms (Chlorella sp., Brachionus plicatilis, and Artemia sp.) that are required for successful culture and testing of the atherinid fishes.

Middaugh, Douglas P. and Ruth L. Yoakum. 1974. Use of Chorionic Gonadotropin to Induce Laboratory Spawning of the Atlantic Croaker, Micropogon undulatus, with Notes on Subsequent Embryonic Development. Chesapeake Sci. 15(2):110-114. (ERL,GB X105).

We studied the feasibility of spawning and rearing the Atlantic croaker, Micropogon undulatus, in the laboratory. Adult croaker were collected during mid-October, 1972, placed in flowing seawater holding tanks, and administered injections of either 125 or 250 I.U. of chorionic gonadotropin three times per week for three weeks. An apparent period of latency (6-7 days) was observed between the initial hormone injections and successful ovulation. Fish dosed at the 125 I.U. level were successfully spawned and eggs and larval development monitored. Larvae were carried through to 4 days after hatching when mass mortalities occurred, apparently as a result of nutritional deficiencies.

Middaugh, D.P. and C.L. Rose. 1974. Retention of Two Mercuricals by Striped Mullet, Mugil cephalus. Water Res. 8(3):173-177. (ERL,GB X106).

The uptake and retention of phenyl mercuric nitrate (PMN) and mercuric chloride (HgCl(2)) were studied in Mugil cephalus, the common saltwater mullet. Fish were exposed to calculated doses of 10, 50, 100 and 500 parts per billion (ppb) mercury of each toxicant in seawater for three hours. Thereafter, they were held in tanks with flowing seawater for 216 h. Fish were sacrificed at 72-h intervals during the holding period to determine levels of mercury in gill, liver and muscle tissues. Higher mercury levels were found in mullet exposed to PMN than in those exposed to equivalent concentrations of HgCl(2). No deaths occurred among mullet dosed with HgCl(2) whereas 58 per cent of those exposed to the highest level of PMN died within 21 h of exposure. Mercury levels detected in tissues appeared to be related to the toxicant used, dose levels, and time of sampling after exposure.

Middaugh, Douglas. 1985. Distribution, Life Cycle, Taxonomy, and Culture Methods: 4. Silversides (Menidia). In: Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms (Third Edition). EPA/600/4-85/013. William H. Peltier and Cornelius I. Weber, Editors. U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH. Pp. 126-137. (ERL,GB X499).

Silversides occur in estuaries along the Atlantic and Gulf coasts. The Atlantic silverside, Menidia menidia, is a resident of estuaries from Maine to northern Florida. It occurs at intermediate to high salinities, typically of 12 to 30 parts per thousand (ppt), and remains in Atlantic estuaries throughout most of the year. Recent evidence indicates an offshore migration at northern latitudes in the fall and reappearance of adults in estuaries in late spring. This species is an important component in estuarine ecosystems, serving as forage fish for commercially and recreationally valued species such as striped bass, bluefish and spotted seatrout. Although culturing methods described in this section were written primarily for Menidia menidia, they are also suitable for the inland silverside, M. beryllina, and the tidewater silverside, M. peninsulae. The staff of the Environmental Research Laboratory, Gulf Breeze, Florida, is currently developing procedures for spawning, culturing, and testing of other fishes, including the California grunion, Leuresthes tenuis, and the Pacific surf smelt, Hypomesus pretiosus. The availability of these fishes as test organisms will permit the use of indigenous fish in toxicity tests of wastes discharged along the entire coast line of the contiguous United States and Alaska.

Discharge of chlorine into estuarine and coastal waters may result in undesirable toxic effects to many of the organisms residing in these habitats. There is a substantial volume of information on short-term chlorination toxicity in marine invertebrates and fishes; however, little is known about the long-term effects of chlorination on the American oyster, Crassostrea virginica (G). Galtsoff reports that adult C. virginica showed reduced pumping rates at concentrations of chlorine of less than 0.05 mg/l. At concentrations of 1.00 mg/l or greater, oysters closed their valves and ceased to pump; the longest period of exposure, however, was only 48 hr. Recent work of Bongers et al reports little mortality in adult C. virginica exposed to chlorine (0.35-0.85 mg/l) and bromine chloride (BrCl) (0.17-0.86 mg/l) for 15 days. Sublethal responses revealed that new shell growth was greater in controls than in chlorine and BrCl-exposed oysters, with the amount of shell deposition decreasing with increased toxicant concentrations. No significant difference in the reduction of new shell deposition occurred in tests with chlorinated and BrCl-treated effluents. A study of the acute toxicity of chlorination to molluscan larvae revealed that chlorination is very toxic to both oyster (C. virginica) and clam (Mercenaria mercenaria) larvae. The 48-hr median effective concentration (EC50) was estimated to be less than 0.005 mg/l for larval oysters and 0.006 mg/l for larval clams. The estimated 96-hr EC50 (by shell deposition) for juvenile oysters was 0.023 mg/l. Oyster larvae survival under intermittent chlorination was much higher than for continuous chlorination. Products formed from chlorination of natural waters are a function of physical and chemical parameters of the water, including but not limited to temperature, pH, ammonia, sunlight (UV) and salinity (or the amount of bromine available as a reaction component). Since sea water typically contains 60 mg/kg bromide, bromination rather than chlorination may predominate as salinity increases. Thus, the chlorine-produced oxidant (CPO)12 levels measured during this study may include varying proportions of hypochlororus acid and hypochlorite ion, as well as hypobromous acid and hypobromite ion, depending upon the ambient salinity. In addition, photolysis may influence the level of bromate produced during chlorination of saline waters. The objective of this study was to examine the effects (lethal and sublethal) of chlorination to adult oysters, Crassostrea virginica, during chronic exposures on a seasonal basis.

Scott, Geoffrey I., Douglas P. Middaugh, Allan M. Crane, Nancy H. McGlothlin and Normitsu Watabe. 1980. Physiological Effects of Chlorine-Produced Oxidants and Uptake of Chlorination By-Products in the American Oyster, Crassostrea virginica (Gmelin). In: Water Chlorination: Environmental Impact and Health Effect, Vol. 3. Robert L. Jolley, William A. Brungs, and Robert B. Cumming, Editors. Ann Arbor Science Publishers, Ann Arbor, MI. Pp. 501-516. (ERL,GB 412).

Previous chlorination studies with the American oyster, Crassostrea virginica, documented the toxic effects of chlorine-produced oxidants (CPO) during the fall, winter, and spring. However, little is known about the toxicological or sublethal physiological effects of chronic CPO exposure during the summer months. The objectives of this study were to (1) determine the lethal effects of chronic CPO exposure during the summer; and (2) examine the sublethal effects of seasonal chronic CPO exposure, including the potential uptake of CPO-produced bromoform (CHBr3) in adult American oysters.

Crabtree, Roy E. and Douglas P. Middaugh. 1982. Oyster Shell Size and the Selection of Spawning Sites by Chasmodes bosquianus, Hypleurochilus geminatus, Hypsoblennius ionthas (Pisces, Blenniidae) and Gobiosoma bosci (Pisces, Gobiidae) in Two South Carolina Estuaries. Estuaries. 5(2):150-155. (ERL,GB 417).

Utilization of empty oyster shells as a habitat and spawning substate by the naked goby, Gobiosoma bosci, the striped blenny, Chasmodes bosquianus, crested blenny, Hypleurochilus geminatus, and freckled blenny, Hypsoblennius ionthas, was studied in the North Inlet and North Edisto River estuaries of South Carolina. Gape and length of shells occupied by fishes were measured. Fish inhabited and spawned in only a small portion of the size range of shells available. G. bosci, the smallest species studied, spawned in shells with narrower gapes than did C. bosquianus or H. geminatus and in shorter shells than did C. bosquianus. Thus, G. bosci may escape competition for spawning sites. Overall, there were a positive correlation between fish length and both gape and length of shells occupied by egg-guarding males.

Scott, Geoffrey I., Douglas P. Middaugh and Scott Klingensmith. 1983. Bioconcentration of Bromoform by American Oyster, Crassostrea virginica (G.) Exposed to Chlorinated and Dechlorinated Seawater, with Notes on Survival and Feeding. In: Water Chlorination: Environmental and Health Effects, Vol. 4. EPA-600/D-83-019. Robert L. Jolley, Editor. Ann Arbor Science Publishers, Ann Arbor, MI. Pp. 1029-1037. (ERL,GB 429).

Chlorine added to seawater reacts with organic precursors to form numerous oxidation products including bromoform. Although several studies have examined the toxicological, behavioral and physiological responses of bivalve molluscs to continuous chlorination, in most instances, only active oxidants were measured; the potential effects of exposure to, or bioconcentration of, bromoform were not considered. Notable exceptions include studies by Gibson et al., in which bioconcentration of bromoform by several marine organisms was examined, and Scott et al., in which bromoform, generated during chlorination of seawater, was bioconcentrated by American oyster, Crassostrea virginica. Recently, Roberts determined the response of American oysters to chlorinated seawater and seawater dechlorinated with sodium thiosulfate. Chlorine-produced oxidants (CPO) were apparently detoxified by dechlorination; however, the potential for bioconcentration of bromoform was not determined. Objectives of our study were to (1) determine the rate of bromoform production in chlorinated seawater; (2) assess the survival and response of oysters exposed to chlorinated seawater, containing active oxidants, bromoform, and other chlorinated organics, and to dechlorinated seawater, containing bromoform plus other chlorinated organics; and (3) determine the potential for bioconcentration of bromoform in oysters exposed to chlorinated and dechlorinated seawater.

Takita, Toru, Douglas P. Middaugh and John M. Dean. 1984. Predation of a Spawning Atherinid Fish, Menidia menidia, by Avian and Aquatic Predators. EPA/600/J-84/266. Jpn. J. Ecol. 34(4):431-437. (ERL,GB 465).

Observations were made of predation on Atlantic silversides, Menidia menidia, during spawning runs in the intertidal zone of the North Edisto River estuary, South Carolina. Several fishes and avian predators captured M. menidia. Snowy egrets, Egretta thula, and Great egrets, Casmerodius albus, were the most dominant avian predators. We found specific differences in each mode and actual predatory pressure for the two wading birds. Before the onset of spawning runs, Snowy egrets often made hovering catches which were inefficient, while Great egrets always made standing catches, striking at M. menidia from a standing position. Once a spawning run began, Snowy egrets ceased hovering and both Great and Snowy egrets frequently struck at fish from a standing position. Because of inefficiency in catching prey prior to a spawning run, Snowy egrets always remained unsatiated after a run had ended. A Great egret was satiated after consuming 114 fish (42% of its body weight) during a run. Intraspecific disturbances were often observed in both Snowy and Great egrets. Interspecific interaction did not present a serious problem to the subordinate species, Snowy egret.

Goodman, Larry R., Douglas P. Middaugh, David J. Hansen, Peggy K. Higdon and Geraldine M. Cripe. 1983. Early Life-Stage Toxicity Test with Tidewater Silversides (Menidia peninsulae) and Chlorine-Produced Oxidants. EPA-600/J-83-158. Environ. Toxicol. Chem. 2(3):337-342. (ERL,GB 466). (Avail. from NTIS, Springfield, VA: PB84-170877)

Early life-stage toxicity tests (continuous exposure from embryonic stage to 3 weeks or more into the exogenous feeding stage) with North American marine fishes have been conducted almost exclusively with cyprinodontids. In this report, we present methods for testing a representative of another family, Atherinidae. Embryos of the tidewater silverside Menidia peninsulae were obtained by a laboratory spawning procedure that required lighting and tidal (current) stimuli. A 28-d toxicity test with chlorine-produced oxidants (CPO) began with stage 21 and 22 embryos (approximately 36 h old). Average measured CPO concentrations in exposure water were nondetectable (<0.01 mg/L) in the control and in the two lowest exposure concentrations, and 0.01, 0.04 and 0.21 mg/L. Survival of embryos to hatching averaged 99%, with no significant difference among treatments. Although no fish survived exposure to 0.21 mg CPO/L, survival was >or= 88% in the control and the four other CPO treatments. Average wet weights of individual fish ranged from 11.7 mg in 0.04 mg/L to 13.2 mg in 0.01 mg/L, with no significant difference among treatments.

McMullen, Dennis M. and Douglas P. Middaugh. 1985. Effect of Temperature and Food Density on Survival and Growth of Menidia peninsulae Larvae (Pisces: Atherinidae). EPA/600/J-85/052. Estuaries. 8(1):39-47. (ERL,GB 489). (Avail. from NTIS, Springfield, VA: PB85-210102)

Day of hatch tidewater silversides, Menidia peninsulae, were stocked at 5 fish per liter in 3 l of seawater at 30o/oo and raised for 16 days at 20°, 25° and 30° C. Food organisms (Brachionus sp. or Artemia nauplii) were maintained at 500, 1,000, 5,000 or 10,000 organisms per l. The influence of food density on growth of larval M. peninsulae was temperature dependent. At 20° C, there was no difference in final size of fish based on food densities. But at 25° and 30° C there was an increase in final body size as food density increased. There were no significant differences in survival among food densities in tests at 20°, 25° or 30° C. However, for any given temperature and food density, the number of survivors in a replicate affected the final size attained. Optimal culture condition for larval M. peninsulae, considering both survival and growth was determined to be 5,000 food organisms per l at 25° C.

Borthwick, Patrick W., James M. Patrick, Jr. and Douglas P. Middaugh. 1985. Comparative Acute Sensitivities of Early Life Stages of Atherinid Fishes to Chlorpyrifos and Thiobencarb. EPA/600/J-85/122. Arch. Environ. Contam. Toxicol. 14(4):465-473. (ERL,GB 517). (Avail. from NTIS, Springfield, VA: PB86-100278/AS)

Sensitivity, expressed as the 96-hr LC50 derived from acute lethality tests, was compared for four ages (day-of-hatch, 7-day, 14-day, and 28-day) of three atherinid fishes: Leuresthes tenuis (California grunion), Menidia menidia (Atlantic silverside), and Menidia peninsulae (tidewater silverside). Responses of each age-species combination exposed to the organophosphate insecticide chlorpyrifos and the carbamate herbicide thiobencarb were compared in both static and flowing seawater toxicity tests. Chlorpyrifos was highly toxic to all atherinids (96-hr LC50's ranged from 0.4 to 6.7 µg/L); toxicity of thiobencarb was approximately two orders of magnitude lower (LC50 values from 199 to 1,405 µg/L). Responses to each pesticide were similar among the three species. Sensitivity was generally highest for 7-day and 14-day age groups, and flowing water tests were more sensitive measures of toxicity than static tests, especially for chlorpyrifos. Comparisons of three computational methods indicate that probit and moving average methods calculate comparable LC50 estimates with the binomial method being the least uniform point estimator.

Clark, James R., James M. Patrick, Jr., Douglas P. Middaugh and James C. Moore. 1985. Relative Sensitivity of Six Estuarine Fishes to Carbophenothion, Chlorpyrifos, and Fenvalerate. EPA/600/J-85/336. Ecotoxicol. Environ. Saf. 10(3):382-390. (ERL,GB 541). (Avail. from NTIS, Springfield, VA: PB86-171634)

The acute toxicity (96-hr LC50) of carbophenothion, chlorpyrifos, and fenvalerate to six estuarine fishes was determined in flow-through laboratory tests. The atherinid fishes (Menidia menidia, M. peninsulae, M. beryllina, and Leuresthes tenuis) consistently were among the most sensitive species tested and were similar to each other in their sensitivity to pesticides. The sensitivity of sheepshead minnows (Cyprinodon variegatus) to carbophenothion was the same as that of the atherinids. For fenvalerate, the sheepshead minnow LC50 was an order of magnitude greater than that of the most sensitive atherinid, whereas the LC50 for chlorpyrifos and sheepshead minnows was two orders of magnitude greater. Gulf toadfish (Opsanus beta) were the least sensitive fish tested with carbophenothion and chlorpyrifos and their 96-hr LC50 for fenvalerate ranked between the LC50 for sheepshead minnows and atherinids. Test results were compared to acute toxicity data for other estuarine fishes and invertebrates.

Scott, Geoffrey I., Douglas P. Middaugh and Tommy I. Sammons. 1985. Interactions of Chlorine-Produced Oxidants (CPO) and Salinity in Affecting Lethal and Sublethal Effects in the Eastern or American Oyster, C. virginica (Gmelin), Infected with the Protistan Parasite, P. marinus. In: Marine Pollution and Physiology: Recent Advances. EPA/600/D-85/196. F. John Vernberg, Editor. University of South Carolina Press, Columbia, SC. Pp. 351-375. (ERL,GB 553). (Avail. from NTIS, Springfield, VA: PB85-243129)

Exposure of oysters, infected with the protistan parasite, Perkinsus marinus, to high salinity and high salinity, chlorine-produced oxidants (CPO) conditions was quite toxic. The combination of high salinity and CPO exposure generally increased toxicity above levels measured in high salinity controls and was generally correlated with parasitism by the protistan parasite, P. marinus. Low salinity and the combination of low salinity and CPO exposure resulted in a significant reduction in P. marinus related oyster mortalities. Low salinity, CPO exposure reduced oyster mortalities from P. marinus infections by as much as 56%. The observed lower mortality rates in oysters exposed to low salinity, glycogen and free amino acid levels, which may have limited the intensification of the disease to lethal hypnospore infection levels.

Scott, Geoffrey I., Edward O. Oswald, Tommy I. Sammons, Douglas S. Baughman and Douglas P. Middaugh. 1985. Interactions of Chlorine-Produced Oxidants, Salinity, and a Protistan Parasite in Affecting Lethal and Sublethal Physiological Effects in the Eastern or American Oyster. In: Water Chlorination: Chemistry, Environmental Impact and Health Effects, Vol. 5. EPA/600/D-86/037. Robert L. Jolley, Editor. Lewis Publishers, Chelsea, MI. Pp. 463-480. (ERL,GB 564). (Avail. from NTIS, Springfield, VA: PB86-166931)

Results from this study have generally indicated the significant interactive effects of salinity and CPO exposure in affecting oyster mortalities resulting from P. marinus infection. Comparisons of control oysters in high- and low-salinity exposures generally indicated that low-salinity conditions resulted in exposure of oysters to water which was reduced in both salt content (major ions) and phytoplankton density (as food availability was reduced by 66%). Bio-deposition rates (or feeding rates) were significantly reduced in low-salinity-exposed oysters, probably related to less food availability.

Hemmer, Michael J., Douglas P. Middaugh and James C. Moore. 1990. Effects of Temperature and Salinity on Menidia beryllina Embryos Exposed to Terbufos. EPA/600/J-90/375. Dis. Aquat. Org. 8:127-136. (ERL,GB 667). (Avail. from NTIS, Springfield, VA: PB91-163881)

Embryos of the inland silverside, Menidia beryllina, were exposed to the organophosphorus pesticide terbufos at nine combinations of temperature (20°, 25° and 30° C) and salinity (5, 12.5 and 20 o/oo). Nominal exposure concentrations were 12.5, 25, 50 and 100 µg terbufos l-1 with an acetone and seawater control for each temperature/salinity combination. Test durations were temperature dependent and ranged from 5 to 14 days. Endpoints were embryo survival, hatching and percentage of larvae with normal vertebrae. Embryo survival was significantly (a= 0.05) lower in tests conducted at 20° C for all salinities. Salinity affected survival only at combinations of 20 o/oo and 100 µg terbufos l-1. Both temperature and salinity affected the percentage hatch, with the lowest hatching occuring in 20° C tests, and in tests conducted at 20 o/oo. The percentage of larvae with normal vertebrae was significantly (a=0.05) reduced from controls at terbufos concentrations of 25 (7 to 32 %), 50 (44 to 62 %) and 100 µg l-1 (58 to 73 %) for the 3 temperatures tested, whereas salinity showed no significant effect. Anomalies in the development occurred across all temperature and salinity combinations, and were observed at concentrations as low as 12.5 µg terbufos l-1.

Winstead, James T., Douglas P. Middaugh and Lee A. Courtney. 1991. Ovarian Mycosis in the Topsmelt, Atherinops affinis (Ayres). EPA/600/J-91/332. Dis. Aquat. Org. 10:221-223. (ERL,GB 702). (Avail. from NTIS, Springfield, VA: PB92-129691)

An ovarian mycosis in a topsmelt, Atherinops affinis (Ayres) collected from Elkhorn Slough, Monterey County, California, is reported. The fungus caused a considerable host response resulting in numerous granulomas, fibrosis and egg compression. Based on morphologic characteristics and the elimination of other fungal pathogens, the fungus is probably a member of the class Oomycetes. This is the first report of an invasive ovarian mycosis in a marine fish.

Hemmer, Michael J., Douglas P. Middaugh and Valerie Comparetta. 1992. Comparative Acute Sensitivity of Larval Topsmelt, Atherinops affinis, and Inland Silversides, Menidia beryllina to 11 Chemicals. EPA/600/J-92/209. Environ. Toxicol. Chem. 11(3):401-408. (ERL,GB 718). (Avail. from NTIS, Springfield, VA: PB92-195668)

Larval topsmelt (Atherinops affinis) and inland silversides (Menidia beryllina) were exposed in 96-hr static acute toxicity tests to eleven chemicals to determine the relative sensitivity to the two atherinid species. High to low LC50 ratios for endosulfan, methoxychlor, carbophenothion, chlorpyrifos, terbufos, fenvalerate, permethrin, 4-nitrophenol, and sodium lauryl sulfate were within a factor of < 2 for the two species. A. affinis was more sensitive to both azinphos-methyl and 2,4-dinitrophenol by factors of 6.7 and 4.4, respectively. Comparison of the relative sensitivity of A. affinis with three freshwater fish species (Lepomis macrochirus, Oncorhynchus mykiss, Pimephales promelas) and one estuarine fish species (Cyprinodon variegatus) are also presented. Sensitivities were similar between A. affinis and the two most sensitive freshwater species, L. macrochirus and O. mykiss. A. affinis is easily transported, cultured and maintained in the laboratory, and readily adaptable for use in toxicological studies.

Goodman, Larry R., Michael J. Hemmer, Douglas P. Middaugh and James C. Moore. 1992. Effects of Fenvalerate on the Early Life Stages of Topsmelt (Atherinops affinis). EPA/600/J-92/217. Environ. Toxicol. Chem. 11(3):409-414. (ERL,GB 719). (Avail. from NTIS, Springfield, VA: PB92-195742)

Flow-through acute and early life-stage (ELS) toxicity tests were conducted with topsmelt (Atherinops affinis), a Pacific Coast saltwater fish, and fenvalerate, a synthetic pyrethroid insecticide. The 96-h LC50 for juvenile fish was 0.66 µg/L. In the 30-d ELS test with laboratory-spawned embryos, average measured fenvalerate concentrations were nondetectable (< 0.075 µg/L) in two control treatments, 0.14, 0.34, 0.82, 1.5, and 3.2 µg/L. Survival of embryos to hatching ranged from 94% to 100%, with no statistically significant difference among treatments. No fry survived exposure to fenvalerate concentrations > 0.82 µg/L; overall survival in lower concentrations and control treatments ranged from 86% to 97%. There were no consistent concentration-dependent differences in weight between fish in the carrier-control treatment and fish exposed to fenvalerate. Mean wet weights of surviving fish ranged from 16.9 mg in 0.34 µg/L to 20.3 mg in 0.14 µg/L. The average bioconcentration factor for fish exposed to 0.14 and 0.34 µg fenvalerate/L was 315.

Mueller, James G., Douglas P. Middaugh, Suzanne E. Lantz and Peter J. Chapman. 1991. Biodegradation of Creosote and Pentachlorophenol in Contaminated Groundwater: Chemical and Biological Assessment. EPA/600/J-91/328. Appl. Environ. Microbiol. 57(5):1277-1285. (ERL,GB 728). (Avail. from NTIS, Springfield, VA: PB92-129659)

Shake flask studies examined the rate and extent of biodegradation of pentachlorophenol (PCP) and 42 components of coal-tar creosote present in contaminated groundwater recovered from the American Creosote Works (ACW) Superfund site, Pensacola, Florida. The ability of indigenous soil microorganisms to remove these contaminants from aqueous solutions was determined by gas chromatographic analysis of organic extracts of biotreated groundwater. Changes in potential environmental and human health hazards associated with the biodegradation of this material were determined at intervals by Microtox® assays and fish toxicity and teratogenicity tests. After 14 days incubation at 30°C, indigenous microorganisms effectively removed 100, 99, 94, 88 and 87% of measured phenolic, lower-molecular-weight polycyclic aromatic hydrocarbons (PAH), S-heterocycle, N-heterocycle and O-heterocycle constituents of creosote, respectively. However, only 53% of the higher-molecular-weight PAHs were degraded; PCP was not removed. Despite the removal of a majority of the organic contaminants through biotreatment, only a slight decrease in toxicity and teratogenicity of biotreated groundwater was observed. These data suggest that toxicity and teratogenicity are associated with compounds difficult to treat biologically, and that one may not necessarily rely on indigenous microorganisms to effectively remove these compounds in a reasonable time span; to this end, alternative or supplemental approaches may be necessary. Similar measures of toxicity and teratogenicity of treated material may offer a simple, yet important, guide to bioremediation effectiveness.

Sherrill, Marie T. and Douglas P. Middaugh. 1993. Spawning Periodicity of the Inland Silverside, Menidia beryllina, (Pisces: Atherinidae) in the Laboratory: Relationship to Lunar Cycles. EPA/600/J-93/391. Copeia. 1993(2):522-528. (ERL,GB 773). (Avail. from NTIS, Springfield, VA: PB93-236297)

Our study was conducted to determine if laboratory populations of M. beryllina exhibited a spawning periodicity and to determine if the reproductive activity could be related to geophysical forces such as the lunar declinational cycle (period 13.66 days between successive passages of the moon over the equator, thus producing equatorial tides) or the semilunar cycle (period 14.77 days between successive new and full moons, thus producing spring tides). M. beryllina from the Blackwater Bay, Florida demonstrated a semilunar periodicity in the laboratory by examination of daily egg production over a 85 day period. The daily timing of spawning by M. beryllina in the laboratory and in nature seems to be linked to daytime high tides that occur between 0852 and 1354 hrs in Blackwater Bay. A similar diel reproductive periodicity has been observed for M. beryllina in Lake Texoma, Oklahoma (Hubbs, 1976) and for the Atlantic silverside, Menidia menidia, from a South Carolina estuary (Middaugh, 1981).

Genthner, Fred J. and Douglas P. Middaugh. 1992. Effects of Beauveria bassiana on Embryos of the Inland Silverside Fish (Menidia beryllina). EPA/600/J-92/406. Appl. Environ. Microbiol. 58(9):2840-2845. (ERL,GB 774). (Avail. from NTIS, Springfield, VA: PB93-131811)

A chemical toxicity/teratogenicity test was adapted to assess potential adverse effect of a microbial pest control agent on a nontarget fish. Developing embryos of inland silverside, Menidia beryllina, were exposed to conidiospores of the insect-pathogenic fungus, Beauveria bassiana. Adherence of spores to the chorion followed by germination and penetration by the germ tube caused the embryos to rupture, sometimes resulting in death. Conidiospores treated with a biological detergent showed significantly less binding (p < or = to 0.01) to embryos than did untreated spores. Both detergent-treated and heat-killed spores failed to cause significant pathogenic responses.

Genthner, Fred J., Douglas P. Middaugh and Steven S. Foss. 1992. Development of Test Methods to Determine Fate of Microbial Pest Control Agents and Their Effects on Nontarget Aquatic Animals. In: Proceedings of the 2nd International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms, May 11-14, 1992, Goslar, Germany. R. Casper and J. Landsmann, Editors. Biologische Bundesanstalt fur Land- und Forstwirtschaft, Braunschweig, Germany. Pp. 107-115. (ERL,GB 789).

Two test methods are described. The first involved the design and field validation of a fully contained test system. This system was used to assess potential adverse effects of microbial pest control agents (MPCAs) on a nontarget invertebrate and determine fates of the MPCAs in the animal. American oysters, Crassostrea virginica, were exposed to various MPCA types represented by the vegetative bacterium, Pseudomonas fluorescens, gram-positive bacterial spores of Bacillus sphaericus, and fungal spores of Colletotrichum gloeosporioides. Each MPCA cleared from the oysters at a different rate. None of the microbes caused infection, pathogenesis or toxicity. Spore germination was not observed in oyster tissue. In the second method, developing embryos of the inland silverside fish, Menidia beryllina, were exposed to spores of the insect pathogenic fungus, Beauveria bassiana. Fungal growth on the chorion surface as well as embryo rupture and death were observed. Spores treated with a dispersant (biological detergent, Triton X-100) showed significantly less binding (p less than or equal to 0.01) to embryos than untreated spores. Neither detergent-treated or heat-killed spores caused significant adverse effects.

Mueller, James G., Suzanne E. Lantz, Derek Ross, Richard J. Colvin, Douglas P. Middaugh and Parmely H. Pritchard. 1993. Strategy Using Bioreactors and Specially Selected Microorganisms for Bioremediation of Groundwater Contaminated with Creosote and Pentachlorophenol. EPA/600/J-93/217. Environ. Sci. Technol. 27(4):691-698. (ERL,GB 822). (Avail. from NTIS, Springfield, VA: PB93-205003)

A two-stage, sequential inoculation bioreactor strategy for the bioremediation of groundwater contaminated with creosote and pentachlorophenol (PCP) was evaluated at bench scale (1.2 L) and pilot scale (454 L). Bioreactor performance using specially selected microorganisms was assessed according to chemical analyses of system influent, effluent, and bioreactor residues, a chemical mass balance evaluation, and comparative biological toxicity and teratogenicity measurements. During pilot-scale operations, the concentration of creosote constituents was reduced from ca. 1000 ppm in the groundwater feed (flow rate 114 L/day) to <9 ppm in the system effluent (total removal efficiency of >99%). Notably, the cumulative concentration of carcinogenic polycyclic aromatic hydrocarbons was reduced from 368 ppm in the feed to 5.2 ppm in the system effluent. Moreover, the toxicity and teratogenicity of the bioreactor effluent were significantly reduced. In general, field data correlated well with those obtained from bench-scale studies.

Genthner, Fred J. and Douglas P. Middaugh. 1995. Nontarget Testing of an Insect Control Fungus: Effects of Metarhizium anisopliae on Developing Embryos of the Inland Silverside Fish Menidia beryllina. EPA/600/J-95/460. Dis. Aquat. Org. 22(3):163-171. (ERL,GB 898).

Developing embryos of the inland silverside fish, Menidia beryllina, were exposed to conidiospores of the entomopathogenic fungus, Metarhizium anisopliae. Several adverse effects were observed in both embryos and newly hatched larvae. These included transitory effects on the heart resulting in decreased cardiac output or circulation velocity, rupture of the chorion, fungal growth on the mandibles of larvae, focal vetebral abnormalities in larvae and teratogenic expressions in embryos and larvae. An ordinal ranking system was used to enumerate responses to conidiospores. This ranking system allowed significance to be determined by nonparametric analysis of variance. Responses were highly variable with significant (p< or = to 0.05) adverse effects observed in five of the six experiments conducted. Heat-killed spores failed to cause significant adverse effects, indicating that viable spores were required for the adverse effects.

Mueller, James G., Suzanne E. Lantz, Beat O. Blattmann, Douglas P. Middaugh and Peter J. Chapman. 1990. Alternative Biological Treatment Processes for Remediation of Creosote-Contaminated Materials: Bench-Scale Treatability Studies. EPA/600/9-90/049. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 74 p. (Avail. from NTIS, Springfield, VA: PB91-179085)

Bench-scale biotreatability studies were performed to determine the most effective of two bioremediation application strategies to ameliorate creosote- and pentachlorophenol (PCP)-contaminated soils present at the American Creosote Works Superfund site, Pensacola, Florida: solid-phase bioremediation or slurry-phase bioremediation. When indigenous microorganisms were employed as biocatalysts, solid-phase bioremediation was slow and ineffective (8-12 weeks required to biodegrade greater than 50% of resident organics). Biodegradation was limited to lower-molecular-weight constituents rather than the more hazardous, higher-molecular-weight (HMW) compounds); PCP and HWM polycyclic aromatic hydrocarbons (PAHs) containing 4 or more fused rings resisted biological attach. Moreover, supplementation with aqueous solution of inorganic nutrients had little effect on the overall effectiveness of the treatment strategy. Alternatively, slurry-phase bioremediation was much more effective: greater that 50% of targeted organics were biodegraded in 14 days. Again, however, more persistent contaminants, such as PCP and HMW PAHs, were not extensively degraded when subjected to the action of indigenous microorganisms.

Davis, William P. and Douglas P. Middaugh. 1978. Revised Review of the Impact of Chlorination Processes upon Marine Ecosystems: Update 1977. In: Water Chlorination: Environmental Impact and Health Effects, Vol. 1. Robert L. Jolley, Editor. Ann Arbor Science Publishers, Ann Arbor, MI. Pp. 283-310. (ERL,GB X057).

For over 175 years chlorine gas has been used in industrial, biocidal and disinfection applications. The chemistry of chlorine in fresh water is relatively well known, but long-range effects on the organisms and the ecological communities of marine waters have barely been studied. Until recently, the so-called 'chlorine demand' of treated or receiving waters has been considered a desirable feature which assured degradation of actively oxidizing states of chlorine to a nontoxic state. With continuing and increased use of chlorine as an antifouling biocide in powerplants, and as a disinfectant of municipal wastes, concern has arisen that resulting by-products, such as induced halogenated hydrocarbons, could potentially reach environmentally harmful levels. For example, in the state of Maryland the quantity of chlorine used, which subsequently reaches the Chesapeake Bay, would have sterilized that body of water were not chemical/biological degradation processes in effect. But, what are the limits of natural degradation systems? What, for one example, are the known environmental costs of our present rates of chlorine applications on renewable fishery resources? What kind and at what rate are persistent halogenated compounds being produced? Where do these go in natural systems? From partial or complete answers to these questions will come meaningful environmental management criteria. This paper presents a theoretical degradation model of chlorine added to marine waters. Additionally it summarizes literature reporting laboratory or ecological effects of chlorinations. It is revised to attempt incorporation of pertinent literature through 1977.

Davis, William P. and Douglas P. Middaugh. 1976. Review of the Impact of Chlorination Processes upon Marine Ecosystems. In: Environmental Impact of Water Chlorination: Proceedings of the Conference on the Environmental Impact of Water Chlorination, Oak Ridge National Laboratory, Oak Ridge, Tennessee, October 22-24, 1975. Robert L. Jolley, Editor. Oak Ridge National Laboratory, Oak Ridge, TN. Pp. 299-325. (ERL,GB X155). (Avail. from NTIS, Springfield, VA: CONF-751096)

For over 175 years chlorine gas has been used in industrial, biocidal and disinfection applications. The chemistry of chlorine in freshwater is relatively well known, but long-range effects on the organisms and the ecological communities of marine waters has barely been studied. Until recently, the so-called 'chlorine demand' of treated or receiving waters has been considered as desirable feature which assured degradation of actively oxidizing states of chlorine to a non-toxic state. With continuing and increased use of chlorine as an antifouling biocide in powerplants, and as a disinfectant of municipal wastes, concern has arisen that resulting byproducts, such as induced halogenated hydrocarbons, could potentially reach environmentally harmful levels. For example in the State of Maryland the quantity of chlorine used, which subsequently reaches the Chesapeake Bay, would have sterilized that body of water were not chemical/biological degradation processes in effect. But, what are the limits of natural degradation systems? What, for one example, are the known environmental costs of our present rates of chlorine applications on renewable fishery resources? What kind and at what rate are persistent halogenated compounds being produced? Where do these go in natural systems? From partial or complete answers to these questions will come meaningful environmental management criteria. This paper presents a theoretical degradation model of chlorine added to marine waters. Additionally it summarizes literature reporting laboratory or ecological effects of chlorine.

Scott, Geoffrey I., Tommy I. Sammons, Douglas P. Middaugh and Michael J. Hemmer. 1982. Impacts of Water Chlorination and Coliform Bacteria on the American Oyster, Crassostrea virginica (Gmelin). In: Physiological Mechanisms of Marine Pollutant Toxicity. W.B. Vernberg, A. Calabrese, and F.P. Thurberg, Editors. Academic Press, Inc., New York, NY. Pp. 505-529. (ERL,GB X232).

In estuaries such as Murrells Inlet, South Carolina, public health officials have chlorinated nonpoint source run-off so that bacterial water quality can be maintained and oyster resources made harvestable. There is a trade-off as the more immediate risk of bacterial pollution is reduced to an acceptable level, but in its place chlorination by-products such as bromoform, a potent carcinogen, are introduced into estuarine waters where they may be bioconcentrated by oysters and may ultimately affect human consumers. Throughout this attempt to control bacterial pollution, the primary concern has been focused on protecting human health without giving consideration to the potential physiological effects of the disinfection process on oysters. The purpose of this paper was to review and contrast the potential physiological effects of coliform bacteria and chlorine on the American oyster, Crassostrea virginica (Gmelin) and to evaluate the potential risks and benefits of each pollutant type in an effort to gain insight into the proper management of shellfish resources.

Brungs, William A. and Douglas P. Middaugh. 1985. Ambient Aquatic Life Water Quality Criteria for Chlorine. EPA-440/5-84-030. U.S. Environmental Protection Agency, Office of Water Regulations and Standards, Criteria and Standards Division, Washington, DC. 57 p. (ERL,GB X504).

The criteria presented herein supersede previous aquatic life water quality criteria for chlorine (U.S. EPA, 1976) because these new criteria were derived using improved procedures and additional information. Whenever adequately justified, a national criterion may be replaced by a site-specific criterion (U.S. EPA, 1983b), which may include not only site-specific criterion concentrations (U.S. EPA, 1983c), but also site-specific durations of averaging periods and site-specific frequencies of allowed exceedences (U.S. EPA, 1985). The latest literature search for information for this document was conducted in May, 1984; some newer information was also included.

Echelle, Anthony A., Alice F. Echelle and Douglas P. Middaugh. 1989. Evolutionary Biology of the Menidia clarkhubbsi Complex of Unisexual Fishes (Atherinidae); Origins, Clonal Diversity, and Mode of Reproduction. In: Evolution and Ecology of Unisexual Vertebrates, Bull. N.Y. State Mus. 466. Robert M. Dawley and James P. Bogart, Editors. New York State Museum, Albany, NY. Pp. 144-152. (ERL,GB X659).

Experimental crosses with four allozymically-defined clones of the Menidia clarkhubbsi complex of unisexual fishes are consistent with expectations for gynogenetic reproduction. Successful reproduction occurred after exposure of eggs to milt from either of the bisexual species, M. beryllina or M. peninsulae, while eggs not exposed to milt failed to initiate embryogenesis. Progeny assayed allozymically were identical to the mother, and all of those assessed for sex were females. Rare occurrences of wild-caught triploids suggest occasional amphimixis between eggs from the M. clarkhubbsi complex and haploid sperm from the bisexual species. The evolutionary origin of the unisexual complex is reviewed against the background of new data on geographic surveys and clonal diversity. The various clones appear to have arisen polyphyletically through multiple hybridizations between M. beryllina and a missing ancestral population similar to M. peninsulae.

Genthner, Fred J. and Douglas P. Middaugh. 1995. Nontarget Testing of an Insect Control Fungus: Effects of Metarhizium anisopliae on Developing Embryos of the Inland Silverside Fish Menidia beryllina. In: Biotechnology Risk Assessment: Proceedings of the Biotechnology Risk Assessment Symposium, June 22-24, 1994, College Park, Maryland. Morris Levin, Chris Grim, and J. Scott Angle, Editors. University of Maryland Biotechnology Institute, College Park, Maryland. Pp. 250-268. (ERL,GB X853).

Developing embryos of the inland silverside fish Menidia beryllina, were exposed to conidiospores of the entomopathogenic fungus, Metarhizium anisopliae. Several adverse effects were observed in both embryos and newly hatched larvae. These included transitory effects on the heart resulting in decreased cardiac output or circulation velocity, rupture of the chorion, fungal growth on the mandibles of larvae, focal vertebral abnormalities in larvae and teratogenic expressions in embryos and larvae. An ordinal ranking system was used to enumerate responses to conidiospores. This ranking system allowed significance to be determined by nonparametric analysis of variance. Responses were highly variable with significant (p less than or equal to 0.05) adverse effects observed in five of the six experiments conducted. Heat-killed spores failed to cause significant adverse effects, indicating that viable spores were required for adverse effects to occur.