The mini mobile environmental monitoring unit: a novel bio-assessment tool

Abstract

This paper introduces a new bio-assessment tool, the mini-mobile environmental monitoring unit (MMU). The MMU is a portable, lightweight, energy-efficient, miniaturized laboratory that provides a low-flow system for on-site exposure of aquatic animals to local receiving waters in a protected, controllable environment. Prototypes of the MMU were tested twice in week-long studies conducted during the summers of 2008 and 2009, and in a 12-day study in 2010. In 2008, fathead minnows and polar organic chemical integrative samplers (POCIS) were deployed downstream from the Hastings, Nebraska wastewater treatment plant (WWTP), a waterway known to contain estrogenic contaminants in biologically active concentrations. In 2009, minnows and POCIS were deployed downstream, upstream and within the Grand Island, Nebraska WWTP, a site where the estrogenic contaminants had been detected, but were found at levels below those necessary to directly impact fish. In 2010, an advanced prototype was tested at the Sauk Center, Minnesota WWTP to compare its performance with that of traditional fish exposure methods including caged fish and static-renewal laboratory testing of effluent. Results from the prototype illustrate the capabilities of the MMU and offer an inexpensive monitoring tool to integrate the effects of pollutant sources with temporally varying composition and concentration.

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Environmental impact

This paper introduces a novel piece of bio-assessment equipment, the mini-mobile environmental monitoring unit. Current methods used to evaluate the toxicity of contaminants in surface waters often employ caged fish. Low or high flow conditions, low or fluctuating water levels, and the presence of apex predators (e.g., alligators) can all preclude the successful use of cages deployed in the field. The proposed technology allows fish or other aquatic organisms to be exposed to field conditions on-site in real-time, but also allows the animals to remain in a controlled and secure environment.

1. Introduction

A number of studies have revealed that biologically active compounds, such as steroids, human and veterinary pharmaceuticals, plasticizers, cleaning solutions, and personal care products are widespread in aqueous environments.^1,2 These compounds are usually found in complex mixtures of variable composition and concentration.^3,4 As a consequence, integration of the variable nature of these pollutant sources is a crucial component in assessing any environmental detriment associated with their presence.

One approach commonly used to evaluate the biological effect of complex mixtures is to study the effects that environmental effluents have on sentinel organisms. Fish used in this capacity have been captured from polluted sites,^5–7 have been placed in cages and deployed at polluted sites,^8,9 and have been exposed to natural waters in mid-sized mobile laboratories, often built within the confines of an recreational vehicle (RV).^3,10 Effluent samples have also been transported to traditional laboratories for static renewal fish exposures.¹¹ All of these methodologies have strengths and limitations. For many field environments, the exposure history of wild fish cannot be ascertained, limiting their utility as sentinel organisms. Mobile laboratories are expensive, complex to operate, and cumbersome to move between sites, thereby constraining the design of experiments and limiting spatial geography as a variable. Static renewal laboratory exposures provide logistical challenges and naturally limit the ability to model the variability of treated wastewater effluent.

Most often the method of choice for exposing fish to surface waters is direct caging in receiving streams for set periods of time, but this also has limitations. Caged fish are subject to vagaries within the environment (flash-floods, drawdowns), are susceptible to predation, and cannot be used in conditions in which the receiving stream is too shallow, the water temperature too cold or too warm, or the river velocity too fast or too stagnant. Furthermore, caged fish have limited foraging capabilities and are likely to be ration-restricted. All of these eventualities can introduce adverse biologic factors into the exposure study that could mask underlying effects due to effluent exposure.

The objective of this paper is to present field data from a prototype of a new bio-assessment tool, the mini-mobile environmental monitoring unit (MMU). The MMU is a small, energy efficient exposure laboratory in which fish or other aquatic organisms can be exposed to field water in real-time while maintaining the animals in a controlled environment.

2. Materials and methods

The MMU is a terrestrial, streamside mobile laboratory that allows for precise control of the abiotic environment, while simultaneously exposing fish to flowing stream water. The exposure unit is approximately 0.45 m³MMU (Fig. 1). Water is pumped from the local waterway or effluent source into the MMU, where it is allowed to gravity feed back into the stream. The water from the receiving stream is diverted into two streams: a high volume stream that flows into an outer insulation jacket, and a low volume stream that flows into an inner 10-L stainless steel aquarium (Fig. 1). Flow can be regulated so that fish contained in the inner tank do not need to maintain position against the current but high enough to ensure two water turnovers within the tank per day. The high flow of circulating water outer jacket of the unit acts to provide thermal insulation for the inner unit. The unit includes an air pump, supplying aeration for the fish in the inner exposure chamber.

Fig. 1 (A) A diagrammatic illustration of a battery powered MMU. The smaller box (1) contains two 12 V, 120 amp-hour marine dry cell batteries. The batteries are connected (solid lines), and supply power, to air (2) and water pumps (3). Water is pumped (dashed lines) from the receiving stream into the MMU where valves (4) split the water into two streams, a high volume stream that irrigates the outer insulating tank, and a low volume stream that irrigates the inner exposure tank (5). The battery powered air pump (2) aerates the water within the exposure tank. (B) An MMU deployed in the field.

The MMU was designed so that there could be simultaneous exposure of fish and integrative chemical samplers (in this case, polar organic chemical integrative samplers, POCIS) with the exposure chamber. The use of POCIS, alongside the fish, was tested during 2008 and 2009. The prototypic MMUs were designed with one attachment site for the POCIS within the inner exposure chamber, which prevented statistical interpretation of the POCIS data. While POCIS results from the 2008 and 2009 field trials were only qualitative, additional attachment sites could be added to the MMU to allow for quantitative, statistical treatment of the POCIS data.

2.1 2008 and 2009 deployments

Three deployments of MMU prototypes were conducted during the summers of 2008, 2009 and 2010 (Table 1). The first (2008) deployment occurred downstream from the Hastings, Nebraska wastewater treatment plant (WWTP) from September 13 to 20, 2008. Effluent released from the Hastings WWTP has been shown to contain estrogenic compounds at concentrations high enough to induce the hepatic gene expression of vitellogenin in male fathead minnows in 7 days.⁹

Table 1 Experimental variables for three deployments of the MMU prototypes

Experimental variables	Deployments
	2008	2009	2010
Location	Hastings, NE	Grand Isle, NE	Sauk Centre, MN
Source water	Treated effluent receiving river	Final treated effluent	Upstream river water; final effluent; downstream river water
Exposure duration	7 days	7 days	12 days
POCIS	YES	YES	NO
Data loggers	Temperature	Temperature	Temperature
MMU location	River bank	In wastewater treatment plant	River bank
Power source	12V dry cell batteries	12 V dry cell batteries	125V power grid
Exposure organisms	10 male fathead minnows	8 male fathead minnows	10 male fathead minnows (each site)
Comparative exposures	10 caged male fathead minnows downstream	15 caged male fathead minnows each upstream and downstream	10 caged male fathead minnows each upstream, final effluent or downstream river water. 10 static renewal male fathead minnows in mobile laboratory receiving upstream, treated effluent, or downstream river water
Endpoints	Body mass	Body mass	BCF
	HSI	LSI	LSI
	GSI	GSI	GSI
	Hepatic vitellogenin mRNA	Heptic vitellogenin mRNA	Secondary sex characteristics
			Plasma vitellogenin

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The Hastings WWTP represents the headwaters of the N. Fork of the Big Blue River, therefore fish could only be deployed downstream from the WWTP (Table 1). During the 7-day deployment, 10 adult male fathead minnows were deployed in minnow cages directly downstream from the Norfolk WWTP. In addition, polar organic chemical integrated samplers (POCIS, Environmental Sampling Technologies, Inc, St. Joseph, MO) and a temperature logger (Onset, Bourne, MA) were also deployed in the river. The MMU was deployed on the riverbank approximately 1 m above the river's surface and 3 m away from the bank. Water was pumped from the river through the MMU, then returned back to the river by means of a 12 V submersible pump. Inside the MMU, 10 adult male fathead minnows and a single POCIS were maintained in a 10-L stainless steel aquarium. Air and water temperature within the MMU were also collected during deployment.

The second (2009) deployment occurred downstream from the Grand Island, Nebraska wastewater treatment plant (WWTP) from September 25 to October 3 2009 (Table 1). The effluent from the Grand Island WWTP enters a fairly large, channelized stream where the effluent becomes diluted with upstream water. Downstream from the WWTP the stream water has been found to contain estrogenic compounds, however the concentration of these compounds was not sufficient enough to induce the hepatic gene expression of vitellogenin in male fathead minnows.⁹ It is possible that the dilution of the effluent with river water has effectively reduced the estrogenic waste stream to the point that is it no longer biologically active, therefore part of the rationale for this study was to determine if undiluted WWTP effluent, sampled within the plant, was sufficiently estrogenic to feminize male fathead minnows.

To test this hypothesis, the MMU was deployed within the plant, after the water had traveled through the last battery of treatment, UV disinfection. The water released from the disinfectant building traveled to the nearby receiving stream through a large cement conduit buried approximately 4 m below ground. The MMU was deployed near the disinfectant building, and water was continuously pumped from the conduit through the MMU.

During the 2009 deployment, 15 adult male fathead minnows were deployed in minnow cages directly upstream and 15 minnows downstream of the Grand Island WWTP outflow. In addition, polar organic chemical integrated samplers (POCIS, Environmental Sampling Technologies, Inc, St. Joseph, MO) and a temperature logger (Onset, Bourne, MA) were also deployed upstream and downstream of the outflow. Eight adult males fathead minnows were deployed within the MMU, alongside a POCIS and temperature logger. In addition a POCIS and temperature logger were attached to the pump head 4 m below the MMU.

Following deployments, fathead minnows were measured for body mass and liver mass. Hepatosomatic (HSI) and gonadosomatic (GSI) indices were generated by dividing the mass of the liver and gonad tissues, respectively, into the body mass of the fish, then multiplying by 100. Immediately upon dissection, livers were flash frozen in liquid nitrogen for subsequent gene expression analysis.

2.1.1 POCIS analysis for estrogenic compounds. The POCIS from the field deployments and laboratory exposures were analyzed for 11 different steroids or presumptive estrogenic compounds and the wastewater indicator, caffeine (Table 2) by the University of Nebraska at Lincoln Water Science Laboratory. In addition, the extracts were also analyzed for caffeine, as an indirect indicator of the presence of treatment plant wastewater. For the steroids, ¹³C₃-estradiol and D5-testosterone were used as internal standards, while α-methyltestosterone was used as a surrogate. A single laboratory fortified blank containing each of the analytes measured was utilized to determine recovery values for each analysis. The reported amounts of steroids and pesticides in POCIS, water samples and sediment samples were not corrected for recovery values.

Table 2 Mass (ng) of 11 steroids/estrogenic compounds and caffeine extracted from POCIS during the 2008 and 2009 field deployments at the Hastings and Grand Island wastewater treatment plants. A dash indicates a nondetect of the compound or a spurious detection below the detection limit of 0.5 ng. n = 1 for each sample (see text)

	Hastings - 2008		Grand Island – 2009
	MMU	Downstream	MMU	Conduit	Upstream	Downstream
17b-estradiol	13.2	36.4	—	—	—	—
Estriol	14.8	6.0	—	—	—	—
Estrone	46.0	110.7	2.6	—	—	—
17a-estradiol	—	—	—	—	—	—
Testosterone	0.8	3.1	16.9	17.3	12.8	12.1
4-Androstenedione	9.9	16.5	—	—	—	—
Androsterone	27.9	20.1	—	—	—	—
Progesterone	0.6	0.6	—	—	—	—
a-Zearalanol	—	—	—	—	—	—
a-Zearalenol	145.7	25.4	—	—	—	—
b-Zearalenol	129.6	22.6	—	—	—	—
Caffeine	3042.7	1623.8	5.2	—	25.3	78.9

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Processing and extraction of the POCISs for steroid hormone and pesticide analysis was conducted according to previously published protocols.^12,8 Purified POCIS extracts from each of the POCIS deployed at each site were analyzed for steroid hormones using LC/MS/MS. Average (±SD) recovery of steroid hormones from POCIS was determined to be 65 ± 11% and the detection limit was estimated to be 0.5 ng.

2.1.2 Gene expression analysis. Hepatic mRNA expression was evaluated for six male fish from each treatment group (Table 3). Fish were chosen for analysis based on their body mass, GSI and HSI. Hepatic vitellogenin 1 (vtg) mRNA expression analysis was conducted using previously published protocols,⁸ and was normalized relative to that of ribosomal L8 expression, a constituently expressed “housekeeping” gene. Hepatic mRNA expression was determined using a Bio-Rad MyiQ Real-Time Polymerase Chain Reaction Detection System managed by Optical System Software version 1.0. Data were quantified by the standard curve method using series diluted cDNA samples as a standard.

Table 3 Results from the fish deployed at the Hastings wastewater treatment plant in cages and in the MMU. An asterisk designates a statistically difference between the value for the fish in the MMU and the fish caged in the waterway

	MMU	Downstream
Body mass	2.32 ± 0.26	2.05 ± 0.23
LSI	1.69 ± 0.14	2.62 ± 0.31^*
GSI	1.44 ± 0.57	1.42 ± 0.52
Vtg/L8	0.085 ± 0.13	0.18 ± 0.26

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2.2 2010 Deployment

The third deployment occurred at the Sauk Centre, MN WWTP between May 18 and May 30, 2010 (Table 1). The 12-day deployment of male fathead minnows was designed to compare the performance of the MMU with that of two other common fish exposure techniques: static renewal laboratory exposures and caged fish. Together these three techniques span the range of exposures from highly controlled laboratory environments lacking flow but providing controlled water temperature and ad libitum fed, viaMMU units with fluctuating water temperatures but ad libitum fed and real time pollutant exposure, to caged fish exposed to flow, water temperature fluctuations and ration-limited. This experimental design tested the hypotheses that similar exposures to wastewater will result in differential effects as a consequence of abiotic and biotic experimental factors. Groups of 10 male fathead minnows were caged upstream, in WWTP effluent and in the mixing zone just downstream of the effluent in the Sauk River. Each caged group was paired with a group of 10 male fathead minnows exposed on shore in an MMU. Finally, groups of 10 male fathead minnows were exposed to 50% daily static renewal waters from the upstream, downstream and 100% effluent sites. Following exposures, we assessed body condition factor (BCF), secondary sex characteristics, GSI, HSI and plasma vitellogenin concentrations using methods described previously.^7,13,14 A concurrent study analyzed water samples from nearby upstream, effluent and downstream locations for a suite of 72 suggested endocrine active compounds.

2.2.2 Statistical analysis. Differences among groups of fish were determined using student's T-tests and one-way analysis of variance (ANOVA, JMP, SAS) followed by Newman-Keuls multiple comparison tests. Alpha was set at 0.05.

3. Results

3.1 2008 Prototype - Hastings, Nebraska WWTP

3.1.1 Temperature and POCIS. The MMU deployment occurred during late summer, and as such the internal air temperature within the MMU varied dramatically over the day. The warmest air temperature within the MMU was 44 °C whereas the coolest temperature was 10.3 °C. In contrast, the water temperature within the N. Fork of the Big Blue River was much less variable, with a maximum water temperature of 23.8 °C, and a minimum water temperature of 16 °C. The thermal insulation system used within the MMU was effective, as the maximum water temperature recorded within the MMU was 27.5 °C, and the minimum was 14.8 °C. The mean air, field water and MMU water temperatures were 21.3 (±8.9) °C, 21.2 (±1.4) °C and 21.3 (±2.4) °C, respectively.

The POCIS deployed in the waterway as well as in the MMU collected contaminants. Androsterone, 4-androstenedione, 17b-estradiol, estriol, estrone, progesterone, testosterone, a-zeralenol, b-zearalenol and caffeine were detected in both the waterway, as well as within the POCIS. There was no discernable trend regarding whether the POCIS in the MMU collected more compound than did the POCIS in the waterway, as five compounds were found in higher concentrations in the MMU, whereas four were in higher levels within the creek (Table 2).

3.1.2 Body mass, organ indices and hepatic vtg induction. After the 7-day deployment, the body mass of the males deployed in the downstream from the Hastings were not significantly different from those deployed in the MMU (Table 3, P > 0.05). LSI for the fish in the MMU was significantly less than that of the fish from the stream, however GSI was not (Table 3). Hepatic vtg expression was induced by exposure to the wastewater in both the stream and the MMU. The expression, however was not significantly different between the two groups of fish (Table 3, P > 0.05).

3.2 2009 Prototype - Grand Island, Nebraska WWTP

3.2.1. Temperature and POCIS. The MMU deployment occurred during late summer and early fall, and the air temperature both outside and within the MMU varied dramatically throughout the day. The warmest air temperature during the exposure period was 30.0 °C whereas the coolest temperature was a chilly 3.4 °C. Within the MMU, the air temperature ranged from 30 °C to a minimum of 9.8 °C. Overall, the average air temperatures were: external air temperature, 18.9 (±4.9) °C; air within the MMU, 20.2 (±3.0) °C. Variations in daily water temperatures were less than the air temperatures. Temperatures within the stream ranged from a maximum of 24.9 °C to a minimum of 21.0 °C; within the WWTP conduit, water temperatures ranged from a maximum of 24.7 °C to a minimum of 21.8 °C, and within the MMU water temperatures ranged from a maximum of 25.1 °C to a minimum of 12.0 °C. Average water temperatures were: stream temperature, 22.8 (±0.9) °C; WWTP conduit, 23.3 (±0.8) °C; MMU 20.2 (±3.0) °C.

The POCIS deployed upstream and downstream of the WWTP outfall, the WWTP conduit and the MMU collected similar contaminants (Table 2). Testosterone was detected at all four sites, whereas caffeine was detected at three of the fours sites. In addition, estrone was detected within the MMU but not at any of the other three sites.

3.2.2 Body mass, organ indices and hepatic vtg induction. After the 7-day deployment, there were no significant differences in body mass among the male minnows deployed upstream and downstream from the WWTP outfall or within the MMU (Table 4, P > 0.05). Average male GSI was similar among the three groups of fish, with no significant differences among any of the groups. There were significant differences (ANOVA P < 0.05) in LSI among the three groups of fish. Post hoc mean comparisons revealed that the LSI for the downstream fish was significantly greater then the upstream fish and than the fish from within the MMU (Tukey-Kramer, post hoc analysis, P < 0.05). Relative vitellogenin gene expression in the male minnows was uniformly low regardless of location (Table 4). There were no significant differences in male vtg expression among the fish regardless of location.

Table 4 Results from the fish deployed at the Grand Island wastewater treatment plant in cages upstream and downstream from the discharge and in the MMU. Different subscript letters designates statistical differences among the mean values for the fish in the MMU and the fish caged in the waterway

	MMU	Downstream	Upstream
Body mass	2.6 ± 0.5	2.8 ± 0.6	2.8 g ± 0.4
LSI	1.03 ± 0.25^b	1.51 ± 0.39^a	1.06 ± 0.28^b
GSI	0.63 ± 0.29	0.79 ± 0.31	0.84 ± 0.23
Vtg/L8	0.0017 ± 0.001	0.0018 ± 0.001	0.0013 ± 0.001

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3.3. 2010 - Sauk Center, Minnesota WWTP – comparing exposure methods

3.3.1 Temperature. The MMU deployment occurred during early summer 2010 with river temperatures exhibiting daily temperature fluctuations of 3–4 °C as is reflected in the upstream data logger data from the caged fish site (Table 5). Effluent temperatures varied similarly, but were generally 4–5 °C lower than river water temperatures. Static renewal laboratory exposures were conducted at laboratory room temperatures of 23 °C for all three treatments.

Table 5 Comparison of fathead minnow endpoints expressed in fish either caged in the river or deployed in MMUs (Sauk Center), or exposed in a daily 50% static renewal laboratory exposure system. Absolute fish numbers vary due to logistical considerations. Bold numbers indicate results that differed significantly between deployment methods at the same site (ANOVA with Tukey post-test, p < 0.05). Small letters indicate differences between deployment methods. VTG; BCF; GSI; LSI; SUM SSC = sum of secondary sex characteristics (tubercles, dorsal pad, color, each scored on a scale of 0 to 3)

Fish placement		Cage	Upstream			Effluent			Downstream
			MMU	Lab	Cage	MMU	Lab	Cage	MMU	Lab
T/°C	Mean (S.E.)	21 (0.1)	22 (0.1)	23 (0.1)	15 (0.1)	18 (0.2)	23 (0.1)	20 (0.1)	21 (0.1)	23 (0.1)
	Median	21	22	23	15	17	23	20	20	23
Survival (%)		80	100	100	80	100	88	100	100	63
n for analysis		10	15	12	10	16	7	10	15	5
SUM SSC	Mean (S.E.)	7.8 (0.6)	6.7 (0.5)	5.5 (0.6)	7.4 (0.5)	6.3 (0.4)	6.4 (0.7)	8.5 (0.2)	7.6 (0.4)	6.9 (0.6)
BCF	Mean (S.E.)	13 ^a (0.8)	13 ^ab (0.4)	11 ^b (0.4)	14 ^a (0.3)	13 ^ab (0.5)	11 ^b (0.9)	14 (0.7)	14 (0.5)	13 (0.7)
GSI	Mean (S.E.)	1.6 (0.3)	1.3 (0.1)	1.8 (0.9)	1.3 (0.2)	1.4 (0.2)	1.0 (0.3)	2.0 (0.2)	1.4 (0.1)	1.8 (0.5)
LSI	Mean (S.E.)	2.4 ^ab (0.3)	2.5 ^a (0.3)	1.7 ^b (0.6)	3.5 (0.4)	2.8 (0.2)	2.2 (0.5)	2.6 (0.3)	2.4 (0.3)	2.0 (0.3)
Plasma Vtg (ug/ml)	Mean (S.E.)	113 ^a (25)	176 ^a (48)	2028 ^b (540)	352 ^a (100)	336 ^a (43)	1307 ^b (430)	392 ^a (69)	176 ^a (38)	1665 ^b (687)

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In a concurrent study,¹⁵water samples were taken upstream, from the effluent and downstream of the Sauk Center WWTP in the vicinity of the fish deployment sites. Analysis indicated 8 of 72 tested suggested endocrine active compounds in the upstream river water including high alkylphenol concentrations. The effluent was found to contain 38 of 72 in the treated wastewater effluent and 18 of the compounds were detected in the mixing zone below the WWTP.

3.3.2 Body mass and organ indices. After the 12 day deployment, there were significant differences among the male minnows deployed upstream of the WWTP outfall with caged and MMU fathead minnows having higher BCFs than laboratory exposed minnows (ANOVA with Tukey post-test; P < 0.05)(Table 5). The same pattern was found in fish exposed to 100% effluent but was not retained downstream where the exposure method had no effect on fish BCF. There were significant differences (ANOVA P < 0.05) in LSI among the three groups of fish exposed to upstream waters with fish exposed in the laboratory having significantly smaller livers than fish exposed either in cages or MMUs. No differences were observed among secondary sex characteristics of fish exposed using either exposure method at any of the sites.

3.3.3 Vitellogenin induction. Plasma vitellogenin concentrations differed strongly between exposure methods (Table 5). At all three locations results for caged and MMU deployed fish were similar and differed significantly (ANOVA with Tukey post test, P < 0.05) from laboratory exposed fish, which were much higher. Interestingly, while plasma vitellogenin concentrations in caged and MMU deployed fish roughly doubled from upstream sites to the effluent exposures, laboratory exposed fathead minnows exhibited a reverse trend with highest vitellogenin concentrations found in the plasma of fish exposed upstream of the effluent outfall and a 30% drop in plasma vitellogenin concentration in effluent exposed fish.

4. Discussion

The objective of this paper is to present field data from a prototype MMU, a novel bio-assessment tool. The three deployments outlined above are interesting in that they each show the utility, and potentially some drawbacks of the MMUs.

4.1 2008 and 2009 deployments

The 2008 deployment was conducted as a proof-of-concept for the MMU. Outflow from the Hastings WWTP has been shown historically to contain estrogenic compounds in concentrations that feminize male fathead minnows. In the current study, estrogenic compounds were found in the POCIS extracts downstream of the Hastings WWTP (Table 1) at levels that led to elevated levels of hepatic vtg expression. Furthermore, POCIS and male fish within the MMU also indicated the presence of estrogenic compounds in the water at levels that would feminize male fish.

The 2009 deployment illustrated one of the utilities of the MMU that being the ability to expose fish to water that is difficult to access. A previous study⁹ found elevated levels of estrogenic compounds downstream of the Grand Island wastewater treatment plant. Fish were caged in this effluent, however the males were not feminized. Clearly, the effluent stream was not of sufficient concentration to elicit feminization in the male fish, but was it due to dilution?

To address that question water was pumped from an underwater conduit that led from the UV disinfectant facility to the discharge structure. The conduit was approximately 4 m subterranean, and the water velocity within the conduit exceeded 3m s⁻¹. Maintaining live fish within the conduit was not possible. As such, the water was pumped from the conduit to the surface, where it cycled through the MMU. As can be seen from the POCIS data, the chemistry of the water within the MMU was consistent with the water in the conduit and the water downstream from the discharge structure (Table 1). Furthermore, none of the male fish used in the study, in cages or in the MMU, exhibited any signs hepatic vtg gene expression or of inappropriate feminization. There was no evidence that the water from within the treatment plant contained bioavailable estrogenic compounds.

4.2 2010 Deployment

This study was designed to build on the 2008 and 2009 deployments and specifically test the utility of the MMU against two well established fish exposure methods: caged fish and on-site exposure laboratories using static-renewal exposure systems. Several noteworthy differences in biomarker expression emerged among the three treatments, and interestingly all of the differences were between the laboratory held fish relative to either the caged fish, the fish in the MMU or both groups of fish. In no case were the fish caged in the river significantly different than those maintained in the MMU.

While not the primary objective of this portion of the study, it is interesting to explore the differences in the laboratory fish relative to the fish held in either cages or the MMU. First, mean BCF, an indication of the overall nutritional state of the fish in a treatment was significantly lower for laboratory-exposed fish relevant to the fish held in the cages upstream of, or within, the effluent. Second, LSI for fish held in upstream cages and in the upstream MMU was significantly greater than that for animals held in the lab. These findings appear to be counter-intuitive, as laboratory fish would have been fed ad libitum, would not have to content with river current or other environmental stressors, and should have elevated, not reduced levels of LSI and BCF.

Plasma vitellogenin concentrations may provide a clue towards understanding the LSI and BCF data. Plasma vitelligenin concentrations for laboratory treatments were also orders of magnitude greater than for male fish housed in cages in the river or in MMUs on the bank of the river. The energetic cost of producing large quantities of vitellogenin may have resulted in reduced BCF and LSI for laboratory exposed fathead minnows, despite their ad libitum feed. The difference in plasma vitellogenin concentrations is also noteworthy when comparing the three exposure methods as results for the more environmentally relevant exposures of fish in cages or MMUs tracked closely with each other for each treatment, while the laboratory exposure were quiet different.

In three studies conducted over three field seasons and two states, the MMU provided a reliable, inexpensive and easy to use field exposure system. Fish exposures in the MMUs were equal or superior to caged fish and allowed for a flow-through, real time exposure assessment not possible in laboratory settings and not as vulnerable to changing environmental conditions as the use of caged fish. The MMU will provide a new tool in the arsenal of aquatic toxicologists that may assist in bridging the knowledge gap between laboratory and field exposure to suggested endocrine active compounds.

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