The Safety of SLICE (0.2% Emamectin Benzoate) Administered in Feed to Fingerling Rainbow Trout James D

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North American Journal of Aquaculture Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/unaj20 The Safety of SLICE (0.2% Emamectin Benzoate) Administered in Feed to Fingerling Rainbow Trout James D. Bowker a , Dan Carty a & Molly P. Bowman a a U.S. Fish and Wildlife Service, Aquatic Animal Drug Approval Partnership Program , 4050 Bridger Canyon Road, Bozeman , Montana , 59715 , USA Published online: 31 Jul 2013.

To cite this article: James D. Bowker , Dan Carty & Molly P. Bowman (2013) The Safety of SLICE (0.2% Emamectin Benzoate) Administered in Feed to Fingerling Rainbow Trout, North American Journal of Aquaculture, 75:4, 455-462, DOI: 10.1080/15222055.2013.806383 To link to this article: http://dx.doi.org/10.1080/15222055.2013.806383

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The Safety of SLICE (0.2% Emamectin Benzoate) Administered in Feed to Fingerling Rainbow Trout

James D. Bowker,* Dan Carty, and Molly P. Bowman U.S. Fish and Wildlife Service, Aquatic Animal Drug Approval Partnership Program, 4050 Bridger Canyon Road, Bozeman, Montana 59715, USA

Abstract SLICE (0.2% emamectin benzoate [EB]) is an in-feed treatment that has been shown to be effective and safe for controlling infestations of several ectoparasitic crustacean copepods and branchiurans in a variety of seawater- and freshwater-reared fishes. Although the safety of EB (in a pre-SLICE formulation) for use with seawater-reared Rainbow Trout Oncorhynchus mykiss has been demonstrated, the safety of SLICE for freshwater-reared Rainbow Trout has not. Consequently, we conducted a trial to evaluate the safety of SLICE for freshwater-reared Rainbow Trout when administered in feed at a dose of 0 (0×), 50 (1 × the maximum proposed therapeutic dose [1×]), 100 (2×), or 150 (3×) µgofEB·kg of fish body weight (BW)−1·d−1 for 14 d (2 × the proposed 7-d treatment duration). Medicated feed was prepared by top-coating commercially available feed with SLICE. Rainbow Trout fingerlings (mean TL ± SD = 7.4 ± 0.7 cm; mean weight ± SD = 4.4 ± 1.2 g) were stocked into 57-L flow-through tanks at 20 fish/tank. Diets were randomly assigned to four replicate tanks per treatment; fish in four additional, nontrial tanks were fed control diets and were weighed weekly to calculate the proper feeding quantities. Throughout the trial, water quality was maintained within ranges suitable for Rainbow Trout culture, fish were fed the assigned feeds at 4% BW/d divided equally between three feedings, and fish behavior was characterized as normal. Fish in the 0× ,1× ,and2× exposure groups consumed all of the offered feed at least 92% of the time, whereas fish in the 3× exposure group consumed all of the offered feed 75% of the time. No fish died, and gross and microscopic fish health evaluations revealed no chronic toxicity patterns. Based on these results, we conclude that there is an adequate margin of safety associated with administering SLICE-medicated feed to fingerling Rainbow Trout at the proposed therapeutic treatment regimen of 50 µgEB·kg fish BW−1·d−1 for 7 d.

SLICE (0.2% emamectin benzoate [EB]) is an in-feed treat- Vertebrates are generally unaffected by avermectins because

Downloaded by [Department Of Fisheries] at 20:46 19 August 2013 ment that was originally developed to control infestations of of the absence of glutamate-gated chloride channels, reduced sea lice (e.g., Lepeophtheirus salmonis, Caligus elongatus, receptor affinities, and protection of GABA receptors by the and Caligus rogercresseyi) in seawater-reared salmon and trout blood–brain barrier; however, arthropods, platyhelminths, and (MSD Animal Health 2012). The active component of SLICE is nematodes do not possess these protective features, rendering EB. Emamectin is an avermectin (Lasota and Dybas 1991) that these and other invertebrates vulnerable to the effects of aver- was developed initially as an insecticide for use with food crops; mectins, including EB (Lasota and Dybas 1991; Jansson et al. EB is derived synthetically from avermectins produced by fer- 1997). When EB is fed to fish, it is absorbed from the gut and mentation of the soil bacterium Streptomyces avermitilis (MSD distributed throughout the body of the fish via the circulatory Animal Health 2012). Like other avermectins, emamectin inter- system (Sevatdal et al. 2005). When sea lice or other parasitic feres with neural transmissions by binding or blocking gamma crustaceans feed on the tissues or fluids of the host fish, EB aminobutyric acid (GABA) receptors and glutamate-gated chlo- is absorbed by the parasites, resulting in their paralysis and ride channels, causing neuromuscular paralysis (CAHS 2007). death (CAHS 2007). Emamectin benzoate has been shown to

*Corresponding author: jim [email protected] Received March 4, 2013; accepted May 11, 2013 455 456 BOWKER ET AL.

be an effective means for controlling and preventing sea louse at 50 (1× the maximum proposed therapeutic dose [1×]), 100 infestations in Atlantic Salmon Salmo salar (e.g., Stone et al. (2×), or 150 (3×) µgEB·kg fish BW−1·d−1 for14d(2× the 1999, 2000b, 2000c, 2002; Armstrong et al. 2000). Further, fish proposed 7-d treatment duration). slowly metabolize EB, resulting in an extended period of protection from parasites long after treatment has been completed (Stone et al. 2000a). Protection can extend for up to 10 weeks METHODS posttreatment (Horsberg 2012), thus making SLICE a viable Experimental design and procedures.—Test fish used in the candidate for long-term parasite control in cultured fish. Cur- trial were Rainbow Trout (∼4–5 months in age) that were rently, SLICE is approved for the control of sea lice in salmonid spawned from domestic broodstock held at the U.S. Fish species within the United Kingdom, Ireland, Finland, Spain, and Wildlife Service (USFWS) Ennis National Fish Hatchery Portugal, Iceland, Faroe Islands, Norway, Chile, and Canada. (Ennis, Montana); the fish were reared from the eyed egg Recently, SLICE has been shown to be effective for control- stage at the USFWS Bozeman Fish Technology Center (BFTC; ling infestations of freshwater ectoparasitic crustacean copepods Bozeman, Montana). After hatching, the resultant fry were and branchiurans in several freshwater-reared fishes. Examples reared by BFTC staff via standard hatchery procedures. Sex of include the control of Salmincola edwardsii in Brook Trout the fish was neither determined nor considered; however, it was Salvelinus fontinalis (Duston and Cusack 2002), Salmincola assumed that males and females were present in roughly equal californiensis in Rainbow Trout Oncorhynchus mykiss (Bowker proportions and were sexually immature. Fish were ultimately et al. 2012; Gunn et al. 2012), Argulus coregoni in Rainbow held in two fiberglass circular tanks (86 cm in diameter × 58 cm Trout (Hakalahti et al. 2004), Argulus foliaceus in Common high; ∼200 fish/tank) with a water depth of 51 cm (water vol- Carp Cyprinus carpio domestica (Braun et al. 2008), and Ar- ume = 297 L/tank). Water inflow to each tank was 15 L/min, gulus sp. in Koi (ornamental variant of Common Carp) and which produced a water exchange rate of 3.0 exchanges/h. As Goldfish Carassius auratus (Hanson et al. 2011). The observed per our FDA-approved research protocol (Bowker and Erdahl efficacy of SLICE against these freshwater parasites included, 2010), 30 fish were randomly collected from the reference pop- in most cases, the “extended period of protection” that was pre- ulation tanks (15 fish/tank) 1 week before the fish were moved viously documented for the use of SLICE against sea lice (Stone to test tanks to begin the acclimation period. The mean ± SD et al. 2000a, 2002; Skilbrei at al. 2008; Horsberg 2012). More- weight of the initial sample of fish was 4.4 ± 1.2 g; there was over, SLICE has been tested for controlling infections of the no significant difference in mean weight between fish from the endoparasitic nematode Anguillicoloides crassus in freshwater- two reference tanks (t-test: t = 0.248, df = 28, P = 0.806). held American Eels Anguilla rostrata (Larrat et al. 2012), and Mean weight was determined so that feed amounts could be EB has been tested for controlling infestations of two ectopar- accurately administered. During the holding period, fish were asitic crustacean copepods (Lernanthropus kroyeri in seawater- fed nonmedicated Silver Cup 1.0-mm pellets (Skretting/Nelson reared European Bass Morone [Dicentrarchus] labrax: Toks¸en and Sons, Inc., Murray, Utah) at 4% BW/d delivered with a belt et al. 2006; Caligus curtus in seawater-reared Atlantic Cod feeder (Ziegler Brothers, Inc., Gardners, Pennsylvania). Gadus morhua: Hamre et al. 2011) and for its in vitro effect The trial was masked, and only one nonmasked trial partici- against the endoparasitic monogenean Acolpenteron ureteroe- pant knew the treatment assignments of test tanks. Completely cetes collected from freshwater-reared Largemouth Bass Mi- randomized design procedures were used to assign each of the cropterus salmoides (Reimschuessel et al. 2011). four EB exposure treatments (0, 50, 100, and 150 µgEB·kg fish To obtain Food and Drug Administration (FDA) approval of BW−1·d−1) to 4 of the 16 test tanks and to stock 20 fish into SLICE for use on fish in the United States, data must be gener- each test tank (i.e., 10 fish from each reference population tank). Downloaded by [Department Of Fisheries] at 20:46 19 August 2013 ated to demonstrate that the standard treatment regimen (50 µg Test tanks were circular and made of fiberglass (46 cm in diame- of EB·kg of fish body weight [BW]−1·d−1 administered for 7 ter × 46 cm deep), with a water depth of 35 cm containing 57 L consecutive days) is not only effective but also safe for appli- of flow-through well water. Four additional, nontrial tanks were cation to the representative target animals. Several studies have similarly stocked for the purpose of determining proper feeding demonstrated the safety of SLICE for seawater-reared Atlantic rates as described below. Water inflow to each test tank was set Salmon, Rainbow Trout, and Brown Trout Salmo trutta (Roy to approximately 3.8 L/min, which produced a water exchange et. al. 2000; Stone et al. 2000a, 2002); one study has evaluated rate of 3.7 exchanges/h. Fish were allowed to acclimate to the the safety of SLICE for use with freshwater-reared Common test tanks for 6 d. Carp (Braun et al. 2008), and a preliminary study has investi- Fish were observed daily for mortality, general behavior, and gated the safety of SLICE for freshwater-held American Eels feeding behavior. Criteria for assessing general fish behavior in- (Larrat et al. 2012). Nevertheless, additional data are needed to cluded position in water column (e.g., crowding near the inlet or demonstrate the safety of SLICE for use with other freshwater- outlet pipe; at the water surface), gasping for air, flashing, hyper- reared fishes and to establish an associated margin of safety. Ac- activity or lethargy, loss of equilibrium, abnormal pigmentation, cordingly, we conducted a trial to evaluate the safety of SLICE and any other unusual behaviors or signs. During the acclima- when administered in feed to freshwater-reared Rainbow Trout tion and exposure periods, fish were fed three times daily at SAFETY OF SLICE FOR RAINBOW TROUT 457

4% BW/d divided equally between the three feedings. Feeding fish culture and handling procedures. A gross necropsy was behavior was assessed once daily during the acclimation period performed on all 30 fish. Ten of the 30 fish were randomly and at each of the three daily feeding events during the expo- selected, fixed in Davidson’s fixative solution, and subsequently sure period. Feeding behavior was scored by using a five-point processed for histological evaluation. scale as follows: 0 = approximately no feed was consumed, and At the end of the trial (1 d posttreatment), the 20 fish in each the fish showed no interest in feeding; 1 = approximately 25% of test tank (320 fish total) were collected, measured for TL and the feed was consumed, and the fish showed little interest in feed- weight, and euthanized by spinal severance. To assess general ing; 2 = approximately 50% of the feed was consumed, and the fish health, standard necropsy procedures were used to examine fish showed a moderate interest in feeding; 3 = approximately external and internal tissues for gross pathologies of all fish. 75% of the feed was consumed, and the fish showed moderate Each fish health evaluation comprised visual examination of interest in feeding; and 4 = approximately 100% of the feed (1) skin for discoloration and the presence and severity of dermal was consumed, and the fish fed aggressively. Daily rations for lesions, (2) gills for pallor, and (3) internal tissues (liver, spleen, fish in each test tank were weighed no more than 2 d in advance and kidney) for signs of gross lesions or abnormalities. While into separate containers and were stored at ambient temperature. fish were being necropsied, 10 fish from each test tank (total = Fish in the nontrial tanks were weighed on treatment days 1 and 160 fish) were randomly selected, fixed in Davidson’s fixative 8 before feed was offered, and the amount of feed used for all solution, and stored in a 70% solution of ethanol for subsequent treated tanks was determined based on these weights. histological examination. Water temperature (mean = 15.0◦C; range = 14.9–15.2◦C) Selected fixed tissues were dissected and processed in Fisher and dissolved oxygen (DO) concentration (mean = 7.0 mg/L; Omnisette Tissue Cassettes (Fisher Scientific, Fair Lawn, New range = 6.5–8.0 mg/L) were measured once daily in each tank Jersey). The tissues were infiltrated with paraffin in a Leica ASP with a YSI Model 550A Dissolved Oxygen and Temperature 300 Advanced Smart Processor (Leica Microsystems, Nussloch, Meter (YSI Environmental, Yellow Springs, Ohio). Water hard- Germany), and the paraffin-infiltrated tissue samples were em- ness (240 ± 20 mg/L as CaCO3 [mean ± SD]) and alkalinity bedded in paraffin blocks with a Leica EG 1160 Tissue Embed- (160 ± 7 mg/L as CaCO3) were measured with Hach reagents ding System. and equipment (Hach, Loveland, Colorado), and pH (8.0 ± 0.4) Tissues in paraffin blocks were sectioned on a Leica RM2255 was measured with a YSI EcoSense pH Pen three times during Rotary Microtome. The 5-µm tissue sections were mounted on the trial (once during the acclimation period and twice during the glass microscope slides and stained with hematoxylin and eosin exposure period). Lighting consisted of overhead lights turned by using a Leica AutoStainer XL; slides were evaluated mi- on for approximately 9–10 h daily (i.e., 0700–1700 hours). croscopically. A histologist documented lesions (e.g., necrosis Medicated and control feeds.—The SLICE premix was pro- or degeneration), whether lesions were scattered or focal, and vided by Merck Animal Health Corporation (Summit, New whether any lesions were artifacts of tissue dissection, preser- Jersey). Control and medicated feeds were prepared at the BFTC vation, or processing. As per our FDA-approved research study in a Marion Mixer (Model SPS-1244; Marion Mixers, Inc., protocol (Bowker and Erdahl 2010), 2 of the 10 fish that were Marion, Iowa). Medicated feeds were prepared by top-coating randomly selected from each tank for histology were used for commercial feed with appropriate amounts of SLICE and men- an evaluation of gill, liver, anterior kidney, posterior kidney, haden fish oil (0.5% weight per weight) to administer target brain, heart, muscle, skin, spleen, pyloric intestine, and rectal doses of 0, 50, 100, and 150 µgEB·kg fish BW−1·d−1 when fed at intestine tissues. Histological evaluations of the remaining 8 4% BW/d. Control feed was top-dressed with menhaden fish oil fish/tank were only focused on the gill, liver, anterior kidney, only. After each batch of medicated feed was prepared, samples and posterior kidney. Downloaded by [Department Of Fisheries] at 20:46 19 August 2013 of feed (n = 5) were collected to confirm the EB concentration as The tissues were evaluated histologically for evidence of follows: (1) three samples were collected from each batch imme- SLICE-induced toxicity. Tissues were scored under a six-point diately after medicated feed preparation (samples collected from ordinal severity scale: 0 = no change; 1 = normal (<5% of top, middle, and bottom of the feed container); (2) one sample the tissue was affected); 2 = mild (5–15% of the tissue was was collected from each batch (at the top) on the first day of the affected); 3 = moderate (15–25% of the tissue was affected); treatment period; and (3) one sample was collected from each 4 = marked (25–50% of the tissue was affected); or 5 = batch (at the top) on the last day of the treatment period. Control severe (>50% of the tissue was affected). Only scores of 4 feed samples (n = 3) were also collected (from the top, middle, or 5 were considered severe enough to adversely affect fish and bottom of the container) to verify that control feed was not health. Before analysis, lesions with a score of 0, 1, 2, or 3 contaminated with EB. Concentrations of EB in feed samples were coded as 0 (not biologically important), and lesions with a were determined via high-performance liquid chromatography score of 4 or 5 were coded as 1 (biologically important). As per by Eurofins AvTech Laboratories (Portage, Michigan). FDA guidance, to minimize the number of histological images Fish health and histology.—Before the trial started, reference that had to be scored, images from the pooled 0- and 150-µg fish (n = 30) were randomly collected and used to characterize EB·kg fish BW−1·d−1 treatment groups were evaluated first. baseline fish health and histopathology associated with routine If significant differences were not detected between these two 458 BOWKER ET AL.

TABLE 1. Relative frequency (%) of Rainbow Trout fingerlings with normal to moderate histology findings for tissue samples from the target animal safety study on administration of SLICE-medicated feed. Histology was not performed on fish in the 50- and 100-µg emamectin benzoate (EB)·kg fish body weight (BW)−1·d−1 treatment groups.

Treatment (µgEB·kg fish BW−1·d−1) Tissue and histological feature Number of samplesa Reference 0 150 Spleen: melanomacrophage centers 2, 7, 8 100 100 75 Heart: inflammation 2, 8, 8 0 13 0 Liver: degeneration 10, 40, 40 10 20 20 Liver: necrosis 10, 40, 40 0 20 18 Liver: vacuolation 10, 40, 40 40 52.5 37.5 Liver: inflammation 10, 40, 40 30 23 7.5 Gill: hypertrophy 10, 40, 40 30 2.5 5 Gill: proliferation 10, 40, 40 70 98 98 Gill: fusion 10, 40, 40 60 98 100 Posterior kidney: melanomacrophage centers 10, 40, 40 100 95 95 Posterior kidney: proliferation 10, 40, 40 90 98 98 Posterior kidney: degeneration of tubule epithelium 10, 40, 40 60 75 50 Posterior kidney: necrosis of tubule epithelium 10, 40, 40 60 63 40 Posterior kidney: regenerating tubules 10, 40, 40 90 75 83 Posterior kidney: calculi 10, 40, 40 30 23 23 Posterior kidney: hyaline droplet degeneration 10, 40, 40 0 0 2.5

aNumber of samples from the reference population, the 0-µgEB·kg ﬁsh BW−1·d−1 exposure group, and the 150-µgEB·kg ﬁsh BW−1·d−1 exposure group, respectively.

groups, we were not required to test for differences between icance level enhanced our ability to identify potentially subtle the 0- and 50-µgEB·kg fish BW−1·d−1 treatment groups or deleterious side effects associated with the use of EB. For qual- between the 0- and 100-µgEB·kg fish BW−1·d−1 treatment itative purposes (i.e., Tables 1, 2), fish were considered the groups (Bowker and Erdahl 2010). experimental unit (Gaikowski et al. 2009). Mean weight and TL Statistical analysis.—Binomial variables (histopathology of fish at the end of the trial were analyzed by one-way ANOVA data) were analyzed with a PROC GLIMMIX-based model (SYSTAT 2012), and the significance level (two-sided) for these (logit link; Wolfinger and O’Connell 1993) in the Statistical comparisons was the conventional α of 0.05. Feeding behavior Analysis System version 9.2 (SAS 2008) by using fish (the ob- was summarized by adding the feeding scores across replicates servation unit) nested within tanks (the experimental unit). As in each exposure group for each feeding event (e.g., day 1, first per Bowker and Erdahl (2010), histology results were tested feeding) and graphing the results in a mosaic plot (Microsoft at an α of 0.10 (two-sided). Choosing this conservative signif- Excel 2010).

Downloaded by [Department Of Fisheries] at 20:46 19 August 2013 TABLE 2. Relative frequency (%) of Rainbow Trout fingerlings with marked or severe histology findings for tissue samples from the target animal safety study on the administration of SLICE-medicated feed. Histology was not performed on fish in the 50- and 100-µg emamectin benzoate (EB)·kg fish body weight (BW)−1·d−1 treatment groups.

Treatment (µgEB·kg ﬁsh BW−1·d−1) Tissue and histological feature Number of samplesa Reference 0 150 Pb Liver: degeneration 10, 40, 40 0 7.5 5 0.7379 Liver: vacuolation 10, 40, 40 60 47.5 62.5 0.2902 Gill: hypertrophy 10, 40, 40 0 0 2.5 0.9734 Gill: proliferation 10, 40, 40 0 0 2.5 0.9734 Posterior kidney: degeneration of 10, 40, 40 0 2.5 0 0.9734 tubule epithelium Posterior kidney: regenerating tubules 10, 40, 40 0 18 15 0.8693

aNumber of samples from the reference population, the 0-µgEB·kg fish BW−1·d−1 exposure group, and the 150-µgEB·kg BW−1·d−1 exposure group, respectively. bThe P-value represents significance of differences between the 0- and 150-µgEB·kg fish BW−1·d−1 exposure groups. SAFETY OF SLICE FOR RAINBOW TROUT 459

TABLE 3. Sum of feeding scores (based on a five-point ordinal scale) for fingerling Rainbow Trout across the four replicate tanks per exposure group during the first, second, and third feeding periods on each study day (exposure groups: 0 [0×], 50 [1×], 100 [2×], and 150 [3×] µg of emamectin benzoate·kg of fish body weight−1·d−1; the nontrial group was used to determine feeding rates). White indicates that fish in each of the four replicate tanks within an exposure group appeared to consume approximately 100% of the feed offered. Sequentially darker shades of gray indicate that less feed was consumed. The five sequentially darker shades of gray indicate scores of 15 to 11, respectively, indicating that successively less feed was consumed.

0× 1× 2× 3× Nontrial Study day 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1 2 3 4 5 6 7 8 9 10 11 12 13 14

RESULTS The measured mean EB doses ± SD for the 50-, 100-, and − − No fish died during the trial, and general fish behavior was 150-µgEB·kg fish BW 1·d 1 treatment groups based on feed characterized as normal throughout the trial. At each feeding samples collected to confirm EB concentration were 43.0 ± 2.9 event, fish consumed all of the feed that they were going to (–14% from the target dose), 100.7 ± 12.5 (1% from target), − − consume within 20–30 s of it being offered. There was an and 155.9 ± 48.3 (12% from target) µgEB·kg fish BW 1·d 1, apparent trend of decreasing feed consumption with increasing respectively. No EB was detected in the control feed. EB concentration in the feed (Table 3). The minimum total feeding score observed in the 0× and 2× exposure groups Fish Health and Histology during a single feeding event was 15, indicating that fish in three Reference population.—External and internal tissues ap- replicate tanks appeared to consume approximately 100% of the peared grossly normal in the 30 reference fish that were necrop- feed (3 tanks × feeding score of 4 for each replicate) and fish sied for fish health; however, shortened opercula were noted on in one replicate tank consumed approximately 75% of the feed three of these fish. For the 10 reference fish that were subjected (1 tank × feeding score of 3). For the 1× exposure group, there to histological examination, no lesions were observed in the were two instances in which the minimum total feeding score heart, liver, skin, muscle, spleen, and pyloric intestine tissues.

Downloaded by [Department Of Fisheries] at 20:46 19 August 2013 was 14. Feeding scores for the 3× exposure group ranged from Lesions observed in other tissues included (1) mild prolifera- 11 to 16, and scores for the nontrial group ranged from 12 to tion of epithelium and fusion of lamellae primarily at filament 16. With two exceptions, fish in all tanks appeared to consume tips in gill tissue, with foci of cartilage and epithelial hyper- at least approximately 75% of the feed offered. On study day 9, plasia denoting previous areas of gill injury; (2) moderate to fish in two 3× tanks appeared to consume only 50% of the feed marked glycogen vacuolation of hepatocytes; (3) mild numbers offered. Lower total feeding scores were documented more of melanomacrophages in anterior kidney and the interstitial frequently at the first daily feeding event than at any other time. tissue of posterior kidney; and (4) mild to moderate prolifera- During the trial, test fish grew an average of 2.8 cm in length tion of hematopoietic cells and numbers of regenerating tubules and 8.7 g in weight. Mean TL and weight of fish in test tanks (Tables 1, 2). Early stages of nephrocalcinosis were observed (n = 320 fish in 16 test tanks) at the end of the trial were in three fish. The observed lesions did not appear to adversely 10.2 cm (range = 7.0–12.5 cm) and 13.1 g (range = 4.0–21.3 g), affect the health of the fish. respectively. No significant differences were detected in mean The 0× group.—All 40 of the 0× fish (0-µgEB·kg fish TL (P = 0.941) or mean weight (P = 0.920) among the four BW−1·d−1 group) appeared to be healthy at the end of the trial. exposure groups. Water temperature, DO concentration, water Observations during necropsy indicated that 1 fish had a fatty hardness, alkalinity, and pH were within acceptable ranges for liver, 10 fish had cream-colored livers, and 4 fish possessed Rainbow Trout culture (Piper et al. 1982). shortened opercula. Histological examinations showed that 460 BOWKER ET AL.

FIGURE 1. Gill tissue of a Rainbow Trout fingerling that was sampled from the reference population before the start of the study (left panel) and gill tissue of a fish that was sampled after treatment with 150 µg emamectin benzoate·kg fish body weight−1·d−1 in feed for 14 d (right panel).

changes observed in the 0× fish (Tables 1, 2) were similar to The 3× group.—All 40 of the 3× fish (150-µgEB·kg fish those described for fish from the reference population. Obser- BW−1·d−1 group) were healthy in appearance, and all external vations included increased severity of glycogen vacuolation of and internal tissues that were examined appeared to be nor- hepatocytes in the liver and changes related to nephrocalcinosis mal. During necropsy, three fish were observed to have fatty in the kidney. In addition, two fish from one of the 0× tanks livers, seven fish had cream-colored livers, and three fish had showed marked degeneration and necrosis of liver hepatocytes. shortened opercula. Histological examinations showed that the No histopathologies observed in other tissues were considered changes observed in the 3× fish (Tables 1, 2) were similar to severe enough to affect the health of these fish. those described for reference fish and 0× fish (Figures 1, 2). Ob- The 1× group.—All 40 of the 1× fish (50-µgEB·kg fish servations included increased severity of glycogen vacuolation BW−1·d−1 group) were healthy in appearance, and all external of hepatocytes in the liver and changes related to nephrocal- and internal tissues that were examined appeared to be normal. cinosis in kidney tissue. In addition, one fish showed marked Observations during necropsy indicated that three fish had fatty degeneration and necrosis of liver hepatocytes. livers, nine fish had cream-colored livers, and two fish had short- Lesions observed that were marked or severe (Table 2) in ened opercula. No tissues from fish in the 1× exposure group both the 0× and 3× exposure groups included (1) liver degen- were examined histologically. eration, (2) liver vacuolation, and (3) posterior kidney regener- The 2× group.—All 40 of the 2× fish (100-µgEB·kg fish ating tubules. Marked gill hypertrophy and proliferation were BW−1·d−1 group) appeared to be healthy, and all of the external observed in one fish from the 3× group. Marked posterior kid- and internal tissues examined were normal in appearance. Ob- ney degeneration was observed in one fish from the 0× group. servations during necropsy showed 13 fish with cream-colored Differences in the prevalence of marked or severe lesions be- livers and 4 fish with shortened opercula. No tissues from fish tween the 0× and 3× exposure groups were not significant in the 2× treatment were examined histologically. (Table 2). Downloaded by [Department Of Fisheries] at 20:46 19 August 2013

FIGURE 2. Kidney tissue of a Rainbow Trout fingerling that was sampled from the reference population before the start of the study (left panel) and kidney tissue of a fish that was sampled after treatment with 150 µg emamectin benzoate·kg fish body weight –1·d−1 in feed for 14 d (right panel). In both panels, nephrocalcinosis is indicated by the darkly pigmented areas. SAFETY OF SLICE FOR RAINBOW TROUT 461

DISCUSSION findings were minimal. In the experiment with Atlantic Salmon Based on (1) survival results, (2) the fact that fish feed- (Roy et al. 2000), one fish in the high-dose exposure group ing behavior was characterized as mostly aggressive regardless died at 2 d postexposure. Pathological findings included focal of the EB concentration in the offered feed, and (3) the fact necrosis, ceroid accumulation in the spleen, and melanin accu- that fish behavior was considered normal, with no dose-related mulation in the kidney, all of which are signs consistent with differences detected, we conclude that administering SLICE- prolonged anorexia. In the experiment with Rainbow Trout, one medicated feed to fingerling Rainbow Trout at a target dosage fish in the low-dose exposure group died on exposure day 1. of 150 µgEB·kg fish BW−1·d−1 for 14 d is safe. Although there When that fish was examined histologically, findings included was an apparent trend indicating a decrease in feeding with (1) scattered melanin granules in the spleen, suggesting toxic ef- increasing EB concentration in feed, the fish still appeared to fects like those seen with bacterial toxins; and (2) high levels of consume most of the feed offered. One concern with admin- melanin in the kidney, indicating that the fish had been anorexic istering top-coated, medicated feed to fish is the potential for for some time. Stone et al. (2002) conducted a study in which the drug to leach into the water. We know of no reports of EB Atlantic Salmon smolts were exposed to nominal EB doses of − − leaching from top-coated feed; however, Yanong et al. (2005) 0, 50, and 250 µgEB·kg fish BW 1·d 1 for 7 d in freshwater reported that a considerable amount of florfenicol can be lost and then were moved to seawater and observed for an additional from feed that is top-coated with Aquaflor if the feed remains in 14 d. Stone et al. (2002) reported that among smolts receiving the water for several minutes. In our study, fish oil was applied the high dose, approximately 50% exhibited darker coloration to feed immediately after it was top-coated with SLICE, and and one fish (1%) exhibited uncoordinated swimming behav- fish appeared to consume the feed within 20–30 s after it was ior. There was no mortality, and no histopathological changes offered. Regardless, it is possible that some EB leached from indicative of EB toxicosis were found. In the studies by Roy the feed before it was consumed; thus, the intended dose might et al. (2000) and Stone et al. (2002), dark coloration, abnor- not have been received by every fish. Furthermore, the fact that mal swimming behavior, and inappetence were reported among fish were fed within 30 min of the lights being turned on in the fish that were exposed to EB doses much higher than those ad- study building might have resulted in decreased feed consump- ministered in our trial. Our results were similar to their results tion during the morning feeding period. Despite the evidence with respect to a lack of mortality, lesions and cellular changes, of apparent decreased feed consumption as EB concentration and inappetence at higher doses of EB administered in feed. increased from 0 to 100 µg·kg fish BW−1·d−1, the fish in the Although a decrease in feed consumption was observed during treatment groups appeared to consume more feed than fish in the an intermediate period in our study, it appeared that the fish re- nontrial tanks. However, it was clear that fish in the 3× exposure sumed consumption of feed and thereafter consumed 100% of group appeared to consume less feed than fish in the other ex- the offered feed for a majority of the time until the study ended. posure groups and that the greatest degree of decreased feeding We conclude that there is a substantial margin of safety asso- occurred during study days 6–10. It has been speculated that ciated with treating Rainbow Trout with the standard treatment − − diminished feed consumption is the first sign of intoxication to regimen of 50 µgEB·kg fish BW 1·d 1 for 7 d to control in- avermectins, which limits further toxic effects (Richard Endris, festations of susceptible freshwater copepods and branchiurans. Merck Animal Health, personal communication). Furthermore, we hypothesize that other freshwater salmonids No fish health or histopathological lesions were detected to can be safely treated with the prescribed therapeutic dosage indicate that the administration of SLICE at 150 µgEB·kg fish when administered to control infestations of susceptible fresh- BW−1·d−1 for 14 d was not safe for fingerling Rainbow Trout. water copepods and branchiurans. Such findings are consistent with those of similarly conducted Downloaded by [Department Of Fisheries] at 20:46 19 August 2013 studies evaluating the safety of SLICE administered in feed to Rainbow Trout and Atlantic Salmon. Roy et al. (2000) con- ACKNOWLEDGMENTS ducted a study in which Atlantic Salmon and Rainbow Trout Merck Animal Health supplied the SLICE for the trial and were exposed to nominal EB doses of 0, 100, 250, or 500 µg paid for analysis of EB in feed samples. Niccole Wandelear − − EB·kg fish BW 1·d 1 for 7 d in seawater. After a 7-d postex- (Aquatic Animal Drug Approval Partnership Program) prepared posure period (also in seawater), no mortalities were reported the medicated feeds and helped to collect data. Beth Mac- and no pathognomonic signs of EB toxicity were observed dur- Connell (Headwaters Fish Pathology, LLC) evaluated the his- ing gross necropsy or histopathological examination. Roy et al. tology samples. Jennifer Royston was the independent Quality (2000) reported that unequivocal signs of toxicity were only ob- Assurance Officer. Jesse Trushenski (Southern Illinois Univer- served at the highest exposure dose (10× the standard dose), and sity) and Richard Endris (Merck Animal Health) reviewed the these signs included lethargy, dark coloration, and inappetence. manuscript. Mention of trade names or commercial products Further, Roy et al. (2000) reported that mortalities occurring dur- in this article is solely for the purpose of providing specific ing their study were not treatment related, that fish behavior was information and does not imply recommendation or endorse- characterized as normal in all exposure tanks, and that pathology ment by the USFWS. 462 BOWKER ET AL.

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North American Journal of Aquaculture Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/unaj20 Effects of Temperature on Inorganic Nitrogen Dynamics in Sequencing Batch Reactors using Biofloc Technology to Treat Aquaculture Sludge Chen Yao a , Hong-Xin Tan a , Guo-Zhi Luo a & Li Li a a College of Fisheries and Life Science , Shanghai Ocean University , Post Office Box 181, Huchenghuan Road 888, Shanghai , 201306 , China Published online: 29 Jul 2013.

To cite this article: Chen Yao , Hong-Xin Tan , Guo-Zhi Luo & Li Li (2013) Effects of Temperature on Inorganic Nitrogen Dynamics in Sequencing Batch Reactors using Biofloc Technology to Treat Aquaculture Sludge, North American Journal of Aquaculture, 75:4, 463-467, DOI: 10.1080/15222055.2013.808297 To link to this article: http://dx.doi.org/10.1080/15222055.2013.808297

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COMMUNICATION

Effects of Temperature on Inorganic Nitrogen Dynamics in Sequencing Batch Reactors using Bioﬂoc Technology to Treat Aquaculture Sludge

Chen Yao, Hong-xin Tan, Guo-zhi Luo,* and Li Li College of Fisheries and Life Science, Shanghai Ocean University, Post Ofﬁce Box 181, Huchenghuan Road 888, Shanghai 201306, China

tem as ammonia and organic nitrogen (Azim et al. 2003). High Abstract levels of waste production in the water are harmful to cultured As a new aquaculture technology, biofloc technology (BFT) species and should be minimized (Neori et al. 2004; Chen et al. makes it possible to minimize water exchange and convert the nitro- 2006). gen in aquacultural waste into biofloc, which can be a natural fish food within the culture system. In the current study, biofloc bacte- Biofloc technology (BFT; also called “activated suspension ria were employed to treat sludge from a recirculating aquaculture technique”) was first used during the early 1980s to solve water system under four different temperatures (15, 20, 25, and 30◦C) in quality issues in aquaculture (Serfling 2006). By maintaining three sequencing batch reactors (SBRs) per temperature. Ammo- a high carbon : nitrogen (C:N) ratio, toxic inorganic nitro- nium reduction was seen on days 19, 17, 13, and 17 for the 15, 20, gen species can be converted into microbial biomass, which 25, and 30◦C groups, respectively, indicating that the biofloc bacteria started to convert ammonium to microbial biomass on that day. in turn can be made available as food for cultured animals The nitrite concentration remained below 6.5 mg/L for each group (Avnimelech et al. 1994). Applications of BFT in aquaculture except the 30◦C group, in which the value reached 13.88 mg/L. The have received much attention in recent years. Recent studies nitrate concentration remained below 4.5 mg/L for each group. have focused on the effects of aquaculture wastewater treatment An ammonia nitrogen removal study revealed that all SBRs could and the availability of biofloc protein through biofloc bacteria remove a total ammonia nitrogen (TAN) concentration of 10 mg/L in 6 h without increasing the nitrite and nitrate concentrations; that are co-cultured with shrimp and fish. However, less attention the 25◦C group needed only 3 h to remove the TAN. The protein has been given to nutrients from the recycling of feces and feed content of the biofloc at the end of the study was 27.81 ± 1.99% residue (Avnimelech 2007; De Schryver et al. 2008; Lu et al. ◦ ◦ (mean ± SD) for the 15 C group, 28.6 ± 3.14% for the 20 C 2012). In a BFT system, variation in dissolved oxygen (DO), ± ◦ ± group, 36.80 3.31% for the 25 C group, and 35.06 3.94% for pH, and turbidity may have negative effects on sensitive fish the 30◦C group, indicating that the bacteria could be harvested as biological protein for feeding the cultured species. species. This culture model is only suitable for finfish cultures

Downloaded by [Department Of Fisheries] at 20:47 19 August 2013 to a lesser extent (Avnimelech 2006; Azim et al. 2008). With BFT, sequencing batch reactors (SBRs) can be used With an average annual growth rate of 8.9% since 1970, to treat wastewater from fish culture ponds without having a aquaculture continues to be the fastest-growing global farming negative impact on the cultured fish. The start-up of SBRs system in the world (FAO 2009). This increase in aquaculture requires growing and accumulating a high concentration of production is being driven by the increasing demand for seafood. active heterotrophic bacteria (De Schryver and Verstraete However, most seafood is produced by land-based aquaculture 2009). Environmental conditions, such as temperature, pH, operations that are far from the seas and oceans (Diana 2009). organic loading rate, and shear stress, can effectively influence Because water and land are scarce resources, most farmers at- biofloc bacterial growth in BFT systems (Avnimelech 1999; De tempt to improve production by increasing pond productivity Schryver et al. 2008). Temperature is known to influence the ac- per unit of land area and water, which demands a greater use of tivity, growth rate, biomass composition, nutrient requirements, feeds. In intensive systems, only 20–25% of the fed protein can nature of metabolism, and metabolic reaction rate of biofloc be assimilated by fish, with the remainder being lost in the sys- bacteria (Novak 1974; Pirt 1975; Esener et al. 1981). It has

*Corresponding author: [email protected] Received December 19, 2012; accepted May 15, 2013 463 464 YAO ET AL.

been reported that biofloc is deflocculated at a low temperature were spectrophotometrically analyzed once every 2 d according (4◦C) and bulked (sludge volume index ≥ 500 mL/g) at to standard methods (APHA 2005). Soluble TOC was measured 30–35◦C (Krishna and Van Loosdrecht 1999; Wilen´ et al. using a TOC analyzer (TOC-V.CPH; Shimadzu Seisakusho, 2000). Biofloc bacteria do not have the ability to regulate their Kyoto, Japan). internal temperature; this may in turn influence the growth of Crude protein content of the biofloc.—At the end of the exper- biofloc bacteria in the SBRs and their removal of nitrogen from iments, all biofloc was collected from each SBR after a settling the solid waste. The main objective of this experiment was phase of 30 min, and the biofloc was filtered through a glass- to investigate and determine the effects of temperature on the fiber filter. The biofloc was dried at 105◦C to a constant weight. efficiency of nitrogen removal by biofloc bacteria. Approximately 0.5–1.0 g of dried biofloc was analyzed by the Kjeldahl method (APHA 2005) to determinate the crude pro- METHODS tein content of each sample from each SBR. Three samples were Reactor design and operation.—The experiments were car- analyzed from the biofloc collected from each SBR. ried out in 12 SBRs situated at the Recirculating Aquaculture Statistical analysis.—All of the statistical analyses in the Engineering and Technology Laboratory, Shanghai Ocean Uni- present study were performed using SPSS version 17.0 for versity, Shanghai, China. All SBRs had an internal diameter of Windows. Differences were considered significant at P-values 15 cm and a liquid-filled height of 100 cm (equivalent to 10 L less than 0.05. of working volume). Each SBR was filled with 10 L of tap water. Dried fish waste containing 21.52 ± 1.5% (mean ± SD) RESULTS AND DISCUSSION crude protein was obtained from the sand filter of a recirculating aquaculture system (RAS; at the Recirculating Aquaculture En- Removal of Dissolved Inorganic Nitrogen gineering and Technology Laboratory) stocked with Jade Perch Concentrations of dissolved inorganic nitrogen (TAN, − − Scortum barcoo, and 30 g of the dried waste were added to each NO2 N, and NO3 N) for the experimental period are shown SBR (Lu et al. 2012). in Figure 1. Each temperature group’s TAN had accumulated at ◦ Four different temperatures (15, 20, 25, and 30 C) were the beginning of the experiment due to the organic matter de- maintained by the water bath (water temperature was controlled composition by micro-organisms. The TAN concentration for by a heating rod). Each temperature group consisted of three the 25◦C group then decreased to 1.09 mg/L on day 13, and the replicates (i.e., each SBR was a replicate) in a randomized block TAN concentration for the 20◦C group decreased to 1.73 mg/L design. Sufficient mixing intensity and saturated DO were en- on day 17. Concentrations of TAN for the 15◦C and 30◦C groups sured in each reactor by use of a bubbling diffusive air stone, fluctuated more often than those observed for the 20◦C and 25◦C which was connected to a 135-W air pump (Model ACO-008; groups. The nitrite concentration remained below 6.5 mg/L for SenSen Co., Ltd., Zhejiang, China). Glucose was added to ob- all groups except the 30◦C group, in which nitrite reached a tain a C:N ratio greater than 20 after each water sample was concentration of 13.88 mg/L. The nitrate concentrations for all collected and analyzed. The SBRs were operated from Decem- groups remained below 4.5 mg/L. ber 8, 2011, to January 7, 2012. Nitrogen removal efficiency.—To study the effects of Ammonia Nitrogen Removal Study temperature on biofloc growth in SBRs for treating aquaculture The effects of different temperatures on TAN removal were wastewater, a laboratory study was conducted on day 31 of studied after all of the SBRs had stabilized. We found that almost SBR operation. An ammonium chloride (NH4Cl) solution was all of the ammonium added to the SBRs was removed over a added to each SBR to obtain a total ammonia nitrogen (TAN) period of 6 h (Figure 2a), and the 25◦C group required only Downloaded by [Department Of Fisheries] at 20:47 19 August 2013 concentration of 10 mg/L, and glucose was added as a carbon 3 h to remove the ammonium. According to one-way ANOVA, source at a concentration 20 times greater than that of TAN. The the concentration of TAN at hour 3 was lower for the 25◦C initial concentrations of soluble total organic carbon (TOC), group than for the other groups (P = 0.001). In additional tests, − − − − TAN, nitrite-nitrogen (NO2 N), and nitrate-nitrogen (NO3 N) there was no increase in NO2 NorNO3 N concentrations for − were determined. Samples for analyses of TAN, NO2 N, and any of the temperature groups. This indicated that the ammonia − NO3 N were taken every hour for 8 h. Sampling was performed nitrogen was directly converted into microbial biomass rather by allowing the flasks to settle for 5 min before the actual than being nitrified by the heterotrophic bacteria. sampling time and then filtering 15 mL of the supernatant over a 0.45-µm filter (Q/IEFJ01-1977; Shanghai Xingya Factory, Crude Protein Content of the Biofloc Shanghai, China). The samples were stored at 4◦C for further The measured protein content of the biofloc was 27.81 ± analysis (De Schryver and Verstraete 2009). 1.99% (mean ± SD) for the 15◦C group, 28.6 ± 3.14% for Water quality analyses.—Water temperature, DO concentra- the 20◦C group, 36.80 ± 3.31% for the 25◦C group, and 35.06 tion, and pH were monitored daily by using a YSI 556 multipa- ± 3.94% for the 30◦C group. The crude protein content of the rameter water quality instrument (YSI, Yellow Springs, Ohio). biofloc was higher in the 25◦C and 30◦C treatments than in Total alkalinity was measured by acid titration on a weekly basis the other two groups (P < 0.05), possibly because 25–30◦C − − (Stirling 1985). Total ammonia nitrogen, NO2 N, and NO3 N temperatures were more suitable for the metabolism and COMMUNICATION 465

− − FIGURE 1. Dissolved inorganic nitrogen (total ammonia nitrogen [TAN], nitrite [NO2 N], and nitrate [NO3 N]) in the four temperature groups throughout the experimental period: (a) 15◦C, (b) 20◦C, (c) 25◦C, and (d) 30◦C. Each value is the mean (±SD) of three replicates per sampling date for each treatment.

activity of the bacteria, which could transfer inorganic nitrogen The SBR used in our study was designed to be an indepen- to organic nitrogen. De Schryver and Verstraete (2009) found dent unit and is very easy to operate. The wastewater treatment that when BFT was applied to nitrogen removal, the protein process occurred within the SBR and was separated from the content of the biofloc increased to 61%. culture system to avoid the heterotrophic growth that may affect Water from aquaculture can be treated in the SBRs and then the cultured species. returned directly to the culture system. This procedure allows The daily nitrogen waste from an aquaculture system with water loss to be avoided. The biofloc bacteria in the SBR can di- a fish or shrimp culture density of 22–23 kg/m3 could be rep- rectly convert ammonia nitrogen into microbial biomass, which resented by an influent TAN concentration of 10–14 mg/L (De Downloaded by [Department Of Fisheries] at 20:47 19 August 2013 can be harvested as microbial protein for meeting the pro- Schryver et al. 2008). The biofloc bacteria have a 10-fold higher tein requirements of the cultured species (Avnimelech 2007; growth rate than nitrifying bacteria (Hargreaves 2006). Ma- Crab et al. 2007; Asaduzzaman et al. 2008; Kuhn et al. 2009). nipulation of the temperature in the SBR promotes the direct A previous study showed that nitrogen was nitrified into ni- conversion (i.e., by the biofloc bacteria) of ammonia nitrogen to trate, which tended to accumulate in RASs (Gutierrez-Wing and microbial biomass in 3 h, and this process produces no nitrite Malone 2006). This process cannot improve water quality or and nitrate, which could only be completely removed by further promote effective reuse of the nitrogen. In BFT systems, inten- denitrification into nitrogen gas or discharge into surrounding sive heterotrophic growth in the culture water may change the water bodies. As one of the most important environmental vari- environmental characteristics, including DO, pH, and alkalin- ables, temperature influences the activity and growth rate of ity. De Schryver and Verstraete (2009) reported that increased heterotrophic bacteria (Novak 1974; Pirt 1975). Similar results microbial metabolism resulting from carbon dosing may induce were previously demonstrated by De Schryver and Verstraete variation in DO levels. Azim et al. (2008) observed that in an (2009). Through BFT, nitrogen can be recycled back into the in-situ BFT system, the pH fluctuated between 4.61 and 8.02 culture system to feed the fish. The application of BFT by use and the alkalinity remained within the range of 5–57 mg/L, of SBRs in intensive aquaculture is an attractive alternative to which may have a negative influence on the cultured species. the various methods that are currently used in aquaculture. 466 YAO ET AL.

Conclusions The results of this study demonstrate that biofloc bacteria can improve the treatment effects on sludge at 25◦C. At this temperature, a significant amount of ammonia nitrogen removal was achieved in 3 h without increasing the nitrite and nitrate concentrations in the SBRs. The process was more efficient in 25◦C group than in the other three temperature groups. More- over, because the SBR is an independent unit separated from the culture system, it will not affect the cultured species, and the bacteria could be harvested from the SBR as biological protein for feeding the cultured species.

ACKNOWLEDGMENTS We are grateful to all members of the Recirculating Aqua- culture Engineering and Technology Laboratory for technical advice and helpful discussions. This study was funded by the National Natural Science Foundation of China (31202033).

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North American Journal of Aquaculture Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/unaj20 In Vitro Screening of Chinese Medicinal Plants for Antifungal Activity against Saprolegnia sp. and Achlya klebsiana Hu Xue-Gang a , Liu Lei a , Chi Cheng a , Hu Kun b , Yang Xian-Le b & Wang Gao-Xue a a College of Animal Science and Technology , Northwest A&F University , Xinong Road 22nd, Yangling , Shaanxi , 712100 , China b National Pathogen Collection Center for Aquatic Animals, College of Fisheries and Life Science , Shanghai Ocean University , 999 Hucheng Huan Road, Shanghai , 201306 , China Published online: 01 Aug 2013.

To cite this article: Hu Xue-Gang , Liu Lei , Chi Cheng , Hu Kun , Yang Xian-Le & Wang Gao-Xue (2013) In Vitro Screening of Chinese Medicinal Plants for Antifungal Activity against Saprolegnia sp. and Achlya klebsiana , North American Journal of Aquaculture, 75:4, 468-473, DOI: 10.1080/15222055.2013.808298 To link to this article: http://dx.doi.org/10.1080/15222055.2013.808298

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In Vitro Screening of Chinese Medicinal Plants for Antifungal Activity against Saprolegnia sp. and Achlya klebsiana

Hu Xue-Gang, Liu Lei, and Chi Cheng College of Animal Science and Technology, Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China Hu Kun and Yang Xian-Le National Pathogen Collection Center for Aquatic Animals, College of Fisheries and Life Science, Shanghai Ocean University, 999 Hucheng Huan Road, Shanghai 201306, China Wang Gao-Xue* College of Animal Science and Technology, Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China

In the aquaculture industry, fungal infection is one of the Abstract main factors responsible for mortality of cultured species and Saprolegniasis is a common fungal disease in aquaculture, caus- economic losses (Meyer 1991). Water molds (oomycetes) are ing severe damage to cultured fishes. To find natural agents for widespread in freshwater and brackish water (Bangyeekhun controlling and treating saprolegniasis, we investigated methanol extracts of 40 traditional Chinese medicinal plants. Saprolegnia et al. 2001; Hussein et al. 2001) and represent the most im- sp. strain JL and Achlya klebsiana wereusedtoevaluatethean- portant fungal group affecting cultured fish and their eggs (Ke tifungal activity of the plants. Cnidium monnieri, Magnolia offici- et al. 2009). Saprolegniasis causes severe damage in commer- nalis,andAucklandia lappa at a concentration of 62.5 mg/mL ex- cial aquaculture, including cultured salmon, trout, and anguillid hibited antifungal activity on Saprolegnia and Achlya klebsiana eels, and in noncommercial aquariums. Fungal pathogens infect mycelium and were selected for further evaluation. The three plant species were extracted with four solvents (petroleum ether the surface of the fish’s body after invading epidermal tissues, [PE], ethyl acetate, methanol, and water), and the extracts were thus causing epidermal damage and cellular necrosis (Takada evaluated with an in vitro bioassay using a rapeseed (Brassica et al. 2010). The organic dye malachite green is extremely effec- napus) microplate method. Among the extracts tested, the PE tive in the control and treatment of saprolegniasis and has been Downloaded by [Department Of Fisheries] at 20:47 19 August 2013 extracts of the three plants exhibited the highest efficacy. The widely used as a therapeutic agent in aquaculture (Willoughby PE extract of A. lappa exhibited the best anti-Saprolegnia and anti-Achlya activities (50% effective concentrations = 11.3 and and Roberts 1992). However, malachite green has been banned 26.1 mg/L, respectively), followed by C. monnieri and M. offic- for several years because it is a potential carcinogen and ter- inalis. Furthermore, the minimum fungicidal concentrations of atogen (Alderman 1985). Therefore, there is an urgent need to PE extracts from the three herbs were identified as 25, 12.5, and develop highly effective and environmentally safe fungicides 25 mg/L, respectively, against Saprolegnia spores and 25, 25, and for controlling saprolegniasis. 12.5 mg/L, respectively, against Achlya klebsiana spores. These findings demonstrate that the three traditional Chinese medicinal People are becoming increasingly interested in traditional plants—A. lappa, C. monnieri,andM. officinalis—have the poten- Chinese medicines because of their low toxicity and good ther- tial for use in developing a novel therapy to control saprolegniasis in apeutic performance (Li and Chen 2005). Screening and proper aquaculture. evaluation of medicinal plants could reveal possible alternatives that may be both sustainable and environmentally acceptable

*Corresponding author: [email protected] Received February 27, 2013; accepted May 16, 2013

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(Eguale et al. 2007). However, there is little available informa- that had the highest antifungal activities were selected from tion on the use of medicinal plants for the treatment of saproleg- among the 40 plants initially evaluated. Material from each plant niasis in fish. We conducted the present study to find possible (50.0 g) was extracted with petroleum ether (PE), ethyl acetate, alternatives for protecting cultured fish. Our preliminary inspec- methanol, and water for 2 h (complete extraction), and the pro- tion of the literature indicated that 40 plant species had potential cess was repeated three times. The ratio of sample to solvent antifungal activity, and those plants were selected for use in an- was 1:10 (mass per volume). All of the extracts were filtered, tifungal activity testing. We obtained the methanol extracts of combined, and evaporated under reduced pressure in a vacuum the 40 medicinal plants and evaluated their antifungal activity rotary evaporator. The extracts of the three plant species were against Saprolegnia sp. and Achlya klebsiana;wealsoexam- dissolved in DMSO to obtain the 200-mg/mL (sample/solvent) ined the in vitro bioactivity of different solvent extracts from stock solutions that were used in the assay. three plant species: Cnidium monnieri, Magnolia officinalis, Preliminary screening.—An agar dilution assay was used to and Aucklandia lappa. test antifungal activity, and a modified fungal growth inhibition assay (with some modifications) was also performed (Stueland et al. 2005). To obtain effective drugs, a final test concentration METHODS of 500 mg/L for each drug was produced by dilution in PDA Fungal cultures.—Pure strains of Saprolegnia sp. (strain JL; cooled at 60◦C in a six-well, flat-bottom tissue culture plate, and GenBank accession number HM637287; Cao et al. 2012) and each concentration was carried out in triplicate. Three indepen- Achlya klebsiana were obtained from the National Pathogen dent biological replicates were used. A DMSO control (2.5%) Collection Center for Aquatic Animals at Shanghai Ocean Uni- was included in the experiment. versity. The strains were cultured on potato dextrose agar (PDA) Rapeseeds were used in this study, and Saprolegnia was used slants and were stored at 4◦C until use. To obtain long-term in the preliminary screening via the rapeseed microplate method. growth cultures, the water mold was subcultured on 7-cm- Whole rapeseeds were autoclaved at 121◦C for 20 min and diameter PDA plates. A new subculture with fresh PDA medium cooled. The Saprolegnia strain was inoculated in the middle was started 24 h before inoculating the rapeseed Brassica napus of a 7-cm-diameter Petri dish and incubated at 25◦C for 48 h. cultures. The sterilized rapeseeds were then placed in a circle around To obtain the spore suspension, 20 fungal-colonized rape- the Saprolegnia colony, and the agar plates were incubated at seeds were placed with sterile forceps into a sterile, 300-mL 25◦C for another 48 h. Subsequently, the Saprolegnia-colonized Erlenmeyer flask (not shaken) and were incubated at 25◦C until rapeseeds were transferred with sterile forceps to the surface abundant zoospores were produced. The spore suspension was of the cooled medium in the wells of a six-well, flat-bottom filtered through carbasus, counted with a hemocytometer, and tissue culture plate. The plate was incubated at 25◦C for 48 h. diluted with sterile water to an approximate concentration of The results were recorded visually by determining mycelium 1 × 104 CFU/mL. presence as positive (“+”) or negative (“−”). Plant materials.—Forty plant species (Table 1) that are tradi- Minimum inhibitory concentration evaluation.—The medic- tionally used in Chinese folk medicine were collected in Decem- inal plants for which mycelium presence was recorded as ber 2011 and identified by X. P. Song at Northwest A&F Univer- negative for at the concentration of 500 mg/L were tested for sity (Shaanxi, China). The voucher specimens were deposited at minimum inhibitory concentration (MIC) values ranging from the Herbarium of the College of Life Science at Northwest A&F 15.6 to 500 mg/L using the above method. Control plates were University. The plants were washed, cut into small pieces, and treated with DMSO (2.5‰) alone. The MIC was read visually then placed in an oven at 45◦C until completely dried. The dried at 48 h and was defined as the concentration of extract that Downloaded by [Department Of Fisheries] at 20:47 19 August 2013 plants were separately crushed and reduced to a fine powder inhibited growth by at least 80% or more relative to the control. by using a strainer (30–40 mesh). The powdered samples were The MIC values of the four crude extracts from C. monnieri, freeze dried at −20◦C to ensure complete desiccation. M. officinalis, and A. lappa were tested at a different series Extraction of screened plants.—Dried powder (50.0 g) from of concentrations based on the initial tests, and the negative each of the 40 plant species (Table 1) was separately extracted control groups containing no plant extract were set up under the with methanol (500 mL, three times) for 2 h. The extracts were same conditions as the test groups. A DMSO control was also then filtered, combined, and evaporated under reduced pressure included. in a vacuum rotary evaporator (R-201; Shanghai Shen Sheng Mycelial growth inhibition test.—In vitro antifungal activ- Biotech Co., Shanghai, China) at 50◦C. The resulting extracts ities of PE extracts from the three plants that had lower MIC from each of the different plants were dissolved in dimethyl values were assessed for mycelial growth inhibition rate. Each sulfoxide (DMSO) to obtain stock solutions of 200 mg/mL crude extract was added to assay flasks containing heat-sterilized (sample/solvent), which were used to prepare the desired con- PDA, and final concentrations were adjusted to the correspond- centrations for the antifungal efficacy assay. ing concentrations based on the initial tests. After mixing with a Extraction of antifungal plants.—The three plant species vortex mixer, aliquots (10 mL) of treated medium were poured (Cnidium monnieri, Magnolia officinalis, and Aucklandia lappa) into 7-cm-diameter Petri dishes. A DMSO control was included. 470 HU ET AL.

TABLE 1. Species names, medicinal names, antifungal activity of extracts at a test concentration of 500 mg/L, and the minimum inhibitory concentration (MIC) values of 40 plant species (+=no inhibition of the fungus; −=inhibition) as well as the chemicals malachite green and DMSO.

MIC (mg/L)

Activity Saprolegnia Achlya No. Species or chemical Medical name Family Part used at 500 mg/L sp. klebsiana 1 Scutellaria barbata Herba scutellaria barbatae Lamiaceae Herb + 2 Curculigo orchioides Rhizoma curculiginis Amaryllidaceae Rhizome + 3 Angelica pubescens f. Radix angelicae Umbelliferae Roots + biserrata pubescentis 4 Cnidium monnieri Fructus cnidii Umbelliferae Fruits − 62.5 62.5 5 Areca catechu Semen arecae Palmaceae Seeds + 6 Melia toosendan Cortex meliae Meliaceae Bark + 7 Crataegus pinnatifida Fructus crataegi Rosaceae Fruits + 8 Scutellaria baicalensis Radix scutellariae Labiatae Roots − 125 250 9 Mentha haplocalyx Herba menthae Labiatae Herb − 125 250 10 Notopterygium forbesii Rhizoma et radix Umbelliferae Rhizome and roots + notopterygii 11 Phellodendron chinense Cortex phellodendri Rutaceae Bark + 12 Lycium barbarum Fructus lycii Solanaceae Fruits + 13 Acorus tatarinowii Rhizoma acori tatarinowii Araceae Rhizome − 500 500 14 Polygonum cuspidatum Rhizoma polygoni cuspidati Polygonaceae Rhizome + 15 Acanthopanax Radix et caulis Araliaceae Roots + (Eleutherococcus) acanthopanacis santicosi senticosus 16 Momordica cochinchinensis Semen momordicae Cucurbitaceae Seeds + 17 Cistanche deserticola Herba vistanches Orobanchaceae Herb + 18 Sabina vulgaris Sabina vulgaris Cupressaceae Stems − 125 125 19 Houttuynia cordata Herba houttuyniae Saururaceae Herb + 20 Prunus mume Fructus mume Rosaceae Fruits + 21 Zingiber officinale Rhizoma zingiberis Zingiberaceae Rhizome + 22 Magnolia officinalis Cortex magnoliae officinalis Magnoliaceae Bark − 62.5 62.5 23 Rheum officinale Radix et rhizoma rhei Polygonaceae Rhizome and roots + 24 Dictamnus dasycarpus Cortex dictamni Rutaceae Roots and bark + 25 Asparagus cochinchinensis Radix asparagi Liliaceae Roots + 26 Lilium lancifolium Bulbus lilii Liliaceae Herb + 27 Glycyrrhiza uralensis Radix glycyrrhizae Leguminosae Roots + 28 Foeniculum vulgare Fructus foeniculi Umbelliferae Fruits + 29 Tripterygium wilfordii Radix et rhizoma tripterygii Celastraceae Herb − 125 250 30 Bupleurum chinense Radix bupleuri Apiaceae Roots + Downloaded by [Department Of Fisheries] at 20:47 19 August 2013 31 Aconitum carmichaelii Radix aconiti lateralis Ranunculaceae Roots + preparata 32 Alpinia officinarum Rhizoma alpiniae Zingiberaceae Rhizome + officinarum 33 Salvia miltiorrhiza Radix salviae miltiorrhizae Labiatae Roots − 250 500 34 Quisqualis indica Fructus quisqualis Combretaceae Fruits + 35 Ophiopogon bodinieri Radix ophiopogonis Liliaceae Roots + 36 Citrus reticulata Semen citri reticulatae Rutaceae Seeds + 37 Lonicera japonica Flos lonicerae Caprifoliaceae Flower + 38 Aucklandia lappa Radix aucklandiae Compositae Roots − 62.5 31.3 39 Pseudolarix kaempferi Cortex pseudolaricis Pinaceae Roots and bark − 500 500 40 Cinnamomum cassia Cortex cinnamomi cassiae Lauraceae Bark + Malachite green 1.0 1.0 DMSO 35,000 35,000 COMMUNICATION 471

TABLE 2. Minimum inhibitory concentration (mg/L) values of four crude extracts from three plant species against Saprolegnia sp. and Achlya klebsiana mycelium at 48 h, and the minimum fungicidal concentration (mg/L) against spores at 72 h (−=an MIC was not detected).

Petroleum ether Ethyl acetate Methanol Water Species Against Saprolegnia Achlya Saprolegnia Achlya Saprolegnia Achlya Saprolegnia Achlya Cnidium monnieri Mycelium 62.5 62.5 250 250 >500 >500 >500 >500 Spores 25 25 −−−−−− Magnolia ofﬁcinalis Mycelium 62.5 62.5 250 250 500 >500 >500 >500 Spores 12.5 25 −−−−−− Aucklandia lappa Mycelium 62.5 31.3 250 125 >500 500 >500 >500 Spores 25 12.5 −−−−−−

The fungal-colonized rapeseeds were inoculated in the center promising antifungal activities, as each had an MIC value of of the PDA in Petri dishes, and three replicates of each concen- 62.5 mg/L. tration were used. The mycelial growth diameter was measured Table 2 shows the MIC values of PE, ethyl acetate, methanol, after incubating at 25◦C for 48 h. This experiment was con- and water extracts from the three plant materials against Sapro- ducted in triplicate, and the growth inhibition rate (IR; %) was legnia and Achlya klebsiana mycelium at 48 h and the MFCs calculated from mean values as IR = 100 × [(A − B)/(A − of PE extracts against spores at 72 h. The PE extracts exhibited C)], where A is mycelial growth in the control, B is mycelial the best antifungal activity against Saprolegnia and Achlya kleb- growth in the sample, and C is the average diameter of the siana, with MIC values of 62.5 mg/L or less. The PE extracts of rapeseeds. C. monnieri and A. lappa showed inhibition against Saprolegnia Spore germination test by the agar dilution method.—The PE spores with MFC values of 25 mg/L, whereas the MFC for M. extracts of the three plants (C. monnieri, M. officinalis, and A. officinalis was 12.5 mg/L. The PE extracts of C. monnieri and lappa) were tested for activity against fungal spores. The spore M. officinalis showed inhibition against Achlya klebsiana spores germination test was carried out by an agar dilution method with MFC values of 25 mg/L, while the MFC value for A. lappa as outlined by the National Committee for Clinical Laboratory was 12.5 mg/L. Standards (NCCLS 1997). The plates were prepared by adding The antifungal efficacy of PE extracts from C. monnieri, M. warm, liquid PDA with the required concentration of crude ex- officinalis, and A. lappa is presented in Table 3. The PE extract of tract (100, 50, 25, 12.5, 6.3, or 3.2 mg/L). Spores (10 µL; 1 × A. lappa was the most effective against Saprolegnia and Achlya 104 CFU/mL) were spotted in triplicate, and the plates were in- klebsiana mycelium, demonstrating the lowest EC50 values of cubated at 25◦C for 72 h. The minimum fungicidal concentration 11.3 and 26.1 mg/L, respectively. (MFC) was defined as the lowest concentration of extract that prevented visible growth or germination of spores; the presence of only one or two colonies was disregarded. DISCUSSION Statistical analysis.—The program SPSS (SPSS, Inc.) was In previous studies, many in vitro screening methods have used for all statistical analyses. The 50% effective concentration been described for the testing of new drugs against Saproleg- (EC50) at the 95% confidence level was calculated by probit nia. Stueland et al. (2005) developed a simple, rapid in vitro Downloaded by [Department Of Fisheries] at 20:47 19 August 2013 analysis (Finney 1971). screening method to test the fungistatic and fungicidal effects of chemical drugs against Saprolegnia. However, that method is not applicable to water-insoluble substances. Therefore, we RESULTS used a rapeseed microplate method, which can quickly and con- The anti-Saprolegnia activities of the 40 plant species are veniently evaluate the anti-Saprolegnia activity of some water- presented in Table 1. Plants that showed reproducible activity insoluble substances. The rapeseed microplate method may well at a concentration of 500 mg/L were considered for retesting. be useful for other purposes and for other oomycetes and fungi In this study, methanol extracts of 40 plants used in traditional as well, but this requires further investigation. Chinese medicine were screened. Of the 40 plants considered, The roots of A. lappa (Saussurea costus in India; also known 10 species (Cnidium monnieri, Scutellaria baicalensis, Men- as “muxiang” in China), which is officially listed in the Chi- tha haplocalyx, Acorus tatarinowii, Sabina vulgaris, Magnolia nese Pharmacopoeia (MOH 2005), have been widely used as a officinalis, Tripterygium wilfordii, Salvia miltiorrhiza, Aucklan- traditional Chinese medicine for the treatment of various kinds dia lappa, and Pseudolarix kaempferi) showed strong inhibition of disorders, such as asthma (Shah 1982; Sircar 1984), cough, against Saprolegnia and Achlya klebsiana. Three of those plants diarrhea, vomiting, indigestion, inflammation, and rheumatism (C. monnieri, M. officinalis, and A. lappa) exhibited the most (Shah 1982). The dried ripe fruit of C. monnieri (also known 472 HU ET AL.

TABLE 3. Antifungal efficacy of petroleum ether extracts from Cnidium monnieri, Magnolia officinalis,andAucklandia lappa (EC50 = 50% effective concentration, with 95% confidence interval in parentheses) against Saprolegnia sp. and Achlya klebsiana (y = probit(p); x = the log10 transformation of the concentration).

Species Against Regression equation EC50 (mg/L) χ2 Cnidium monnieri Saprolegnia y =−3.512 + 2.201x 39.422 (25.839–83.085) 2.216 Achlya klebsiana y =−2.673 + 1.607x 46.117 (39.223–54.599) 2.659 Magnolia ofﬁcinalis Saprolegnia y =−2.943 + 2.102x 25.136 (14.776–41.045) 0.097 Achlya klebsiana y =−2.372 + 1.424x 46.317 (38.748–55.870) 1.474 Aucklandia lappa Saprolegnia y =−2.274 + 2.158x 11.314 (4.640–17.640) 1.526 Achlya klebsiana y =−2.356 + 1.662x 26.134 (22.128–30.609) 6.591

as fructus cnidii; “she chuang zi” in Chinese) has been used for To date, no treatments have been identified as wholly effective the treatment of impotence, frigidity, and skin-related diseases, alternatives to malachite green. such as suppurative dermatitis and pudendal itching (Zheng et al. In this study, solvent extracts were evaluated for antifungal 1998; MOH 2005). Anthelmintic activity against Dactylogyrus activity. Additional research will be required to isolate the active intermedius on the gills of Goldfish Carassius auratus was also compounds and to find methods for addressing water solubility. observed (Wang et al. 2006, 2008). Cao et al. (2012) found In addition, field studies of these extracts are required to deter- that the water extract of C. monnieri had no inhibition against mine their practical use in fish culture, and the in vivo activity of Saprolegnia, with an MIC value of over 10.0 mg/mL. However, the extracts must be further investigated. In summary, we found we found that the PE extract of C. monnieri resulted in strong that the PE extracts of A. lappa, C. monnieri, and M. officinalis inhibition of Saprolegnia and an MIC value of 62.5 mg/L. The have the potential for use in developing a novel therapy for the difference between the two studies may result from geographic treatment of Saprolegnia infection. and ecological differences that produce within-species variation in C. monnieri and in Saprolegnia. In addition, the bioactivity of the extracts can be affected by the solvent and extraction tech- ACKNOWLEDGMENTS niques used. The bark of M. officinalis (referred to as “houpo” This work was supported by the National High Technology in Chinese) has been used in traditional Chinese medicine for Research and Development Program of China (863 Program; the treatment of abdominal distention and pains, dyspepsia, and number 2011AA10A216). asthmatic cough (MOH 2005). Many studies have demonstrated that M. officinalis has a wide variety of pharmaceutical properties, such as antispasmodic, antioxidant, anticancer, and an- REFERENCES tidepressant activities (Lee et al. 2011). However, very little Alderman, D. J. 1985. Malachite green: a review. Journal of Fish Diseases attention had been focused on the antimicrobial ability of M. of- 8:289–298. ficinalis, especially anti-Saprolegnia activity, prior to our study. Bangyeekhun, E., S. M. A. Quiniou, J. E. Bly, and L. Cerenius. 2001. Character- isation of Saprolegnia sp. isolates from Channel Catfish. Diseases of Aquatic The PE extracts of C. monnieri, M. officinalis, and A. lappa Organisms 45:53–59. showed highly inhibitory action against Saprolegnia and Achlya, Barnes, M. E., H. Stephenson, and M. Gabel. 2003. Use of hydrogen per- and this report provides the first documentation of antifungal ac- oxide and formalin treatments during incubation of landlocked fall Chi- Downloaded by [Department Of Fisheries] at 20:47 19 August 2013 tivity from A. lappa and M. officinalis. Some compounds that nook Salmon eyed eggs. North American Journal of Aquaculture 65:151– are alternatives to malachite green (e.g., formalin, hydrogen per- 154. Burka, J. F., K. L. Hammell, T. E. Horsberg, G. R. Johnson, D. J. Rainnie, oxide, and sodium chloride; Schreier et al. 1996; Barnes et al. and D. J. Speare. 1997. Drugs in salmonid aquaculture—a review. Journal of 2003; Gieseker et al. 2006) have been used in the treatment Veterinary Pharmacology and Therapeutics 20:333–349. of superficial fungal infections. Although these chemicals can Cao, H., W. Xia, S. Zhang, S. He, R. Wei, L. Lu, and X. Yang. 2012. Saprolegnia control the disease, they have some disadvantages in practice. pathogen from Pengze Crucian Carp (Carassius auratus var. Pengze) eggs Formalin was reported to be effective in treating Saprolegnia and its control with traditional Chinese herb. Israeli Journal of Aquaculture Bamidgeh [online serial] IJA 64.2012.685. infections on eggs, but there are safety concerns for the user Eguale, T., G. Tilahun, A. Debella, A. Feleke, and E. Makonnen. 2007. In vitro and the environment (Marking et al. 1994; Burka et al. 1997). and in vivo anthelmintic activity of crude extracts of Coriandrum sativum Hydrogen peroxide, which is approved in the USA to control against Haemonchus contortus. Journal of Ethnopharmacology 110:428– saprolegniasis on freshwater-reared finfish eggs, is strongly cor- 433. rosive and combustible, with effective concentrations as high Finney, D. J. 1971. Probit analysis, 3rd edition. Cambridge University Press, New York. as 1,000 mg/L (Schreier et al. 1996). Sodium chloride, despite Gieseker, C. M., S. G. Serfling, and R. Reimschuessel. 2006. Formalin treatment its safety, may be limited in applicability due to the high cost to reduce mortality associated with Saprolegnia parasitica in Rainbow Trout, of acquiring effective concentrations as high as 30,000 mg/L. Oncorhynchus mykiss. Aquaculture 253:120–129. COMMUNICATION 473

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North American Journal of Aquaculture Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/unaj20 Effects of Salmon Gonadotropin-Releasing Hormone Analog (GnRHa) on Reproductive Success and Egg Size in Rainbow Trout and Brook Trout a a b c Viktor W. Svinger , Tomáš Policar , Dennis M. Kallert & Jan Kouřil a Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, South Bohemian Research Centre of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology in Vodňany, Zátiší 728/II , 389 25 , Vodňany , Czech Republic b Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, H-1143 Budapest , 21 Hungaria krt , Hungary c Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, South Bohemian Research Centre of Aquaculture and Biodiversity of Hydrocenoses in Vodňany, Institute of Aquaculture, Husova třída 458/102 , 370 05 , České Budějovice , Czech Republic Published online: 06 Aug 2013.

To cite this article: Viktor W. Svinger , Tom Policar , Dennis M. Kallert & Jan Kouil (2013) Effects of Salmon Gonadotropin- Releasing Hormone Analog (GnRHa) on Reproductive Success and Egg Size in Rainbow Trout and Brook Trout, North American Journal of Aquaculture, 75:4, 474-486, DOI: 10.1080/15222055.2013.808299 To link to this article: http://dx.doi.org/10.1080/15222055.2013.808299

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ARTICLE

Effects of Salmon Gonadotropin-Releasing Hormone Analog (GnRHa) on Reproductive Success and Egg Size in Rainbow Trout and Brook Trout

Viktor W. Svinger* and Toma´sˇ Policar Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceskˇ eBud´ ejovice,ˇ South Bohemian Research Centre of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology in Vodnany,ˇ Zati´ sˇ´ı 728/II, 389 25 Vodnany,ˇ Czech Republic Dennis M. Kallert Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, H-1143 Budapest, 21 Hungaria krt, Hungary Jan Kourilˇ Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceskˇ eBud´ ejovice,ˇ South Bohemian Research Centre of Aquaculture and Biodiversity of Hydrocenoses in Vodnany,ˇ Institute of Aquaculture, Husova trˇ´ıda 458/102, 370 05 Ceskˇ eBud´ ejovice,ˇ Czech Republic

Abstract Rainbow Trout Oncorhynchus mykiss were treated with a 100- or 25-µg/kg dose of salmon gonadotropin-releasing hormone analog (sGnRHa) that was emulsified in Freund’s incomplete adjuvant (FIA). Controls were treated with a mixture of FIA and physiological saline. Latency (number of days from the first injection to the date of detected ovulation) was significantly shortened in treated groups compared with the control group. Egg weight, eyeing rate, and hatching rate were significantly reduced in treated groups; mortality after the eyed stage was significantly higher in treated groups. The relationship between latency and the eyeing rate or hatching rate was positive, but the relationship between latency and mortality after the eyed stage was negative. Brook Trout Salvelinus fontinalis were treated with a single injection of sGnRHa–FIA at 12.5 µg/kg, double acute injections of sGnRHa at 12.5 µg/kg administered 3 d apart, or a single acute injection of sGnRHa at 50 µg/kg. Controls were injected with FIA and physiological saline. Latency was significantly shortened in treated groups except the group that received the single acute injection of Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 50 µg/kg. Egg weight and alevin weight were significantly lower in treated groups. Egg mortality after the eyed stage was significantly higher in the double-injected group than in the 12.5-µg/kg sGnRHa–FIA group and the control group. Latency had a negative relationship with the eyeing rate and hatching rate. This result for Brook Trout is opposite that observed for Rainbow Trout. Latency and mortality after the eyed stage were not correlated. It is evident that the experimental designs currently used in salmonid research should be updated to determine reliable means of avoiding egg mortality, particularly in Rainbow Trout, after GnRHa treatment.

Gonadotropin-releasing hormone analogs (GnRHa) are used lows for accurate prediction of ovulation dates in many species. in salmonid reproduction to precisely control the timing of the Such treatments reduce the handling of broodstock, make brood- ﬁnal stages of the reproductive cycle. Application of GnRHa al- stock handling more ﬂexible over the season, may advance

*Corresponding author: [email protected] Received March 27, 2013; accepted May 18, 2013

474 SALMON GnRHa EFFECTS ON TROUT 475

ovulation by more than 5 weeks compared with the natural We conducted on-farm experiments with two of the most spawning time, and markedly shorten the spawning season farmed salmonid species in central Europe—Rainbow Trout (which often extends over a period of 3 months in salmonids). and Brook Trout—by using differing doses and administration Gonadotropin-releasing hormone analogs have been success- methods of D-Arg6Pro9NEt-sGnRHa, the superactive analog of fully applied in a wide range of salmonids, such as Rainbow native salmon GnRH (sGnRH). To identify potential side effects Trout Oncorhynchus mykiss (Breton et al. 1990; Arabacı et al. of using GnRHa in a hatchery, we determined ovarian fluid pH, 2004; Vazirzadeh et al. 2008), Arctic Char Salvelinus alpi- egg weight, eyeing rate, rate of mortality between eyeing and nus (Haraldsson et al. 1993; Jansen 1993; Gillet et al. 1996), hatch, hatching rate, weight of yolk sac fry (measured for Brook Atlantic Salmon Salmo salar (Crim et al. 1983a; Taranger Trout only), and rate of deformities in yolk sac fry. We chose et al. 1992; Vikingstad et al. 2008), Brown Trout Salmo trutta D-Arg6Pro9NEt-sGnRHa for use in this study because it is a (Mylonas et al. 1992; Noori et al. 2010), European Grayling Thy- standard active substance in many commercial hormone prepa- mallus thymallus (Kourilˇ et al. 1987; Mikolajczyk et al. 2008), rations used for induction of ovulation in fish (e.g., Ovaplant, Chinook Salmon O. tshawytscha (Olito et al. 2001), Sockeye OvaRH, Dagin, and Ovaprim). Salmon O. nerka (Slater et al. 1995), Chum Salmon O. keta (Park et al. 2007), Coho Salmon O. kisutch (Fitzpatrick et al. METHODS 1987), Brook Trout Salvelinus fontinalis (Svinger et al. 2013), Lake Trout Salvelinus namaycush (Erdahl and McClain 1987), Experimental Design Powan (European Whitefish) Coregonus lavaretus (Mikolajczyk Rainbow Trout.—For the Rainbow Trout experiment, 3-year- et al. 2005), and Northern Whitefish Coregonus peled (Svinger old females of a spring-spawning, eastern Bohemian strain and Kouril,ˇ in press). were used. One week prior to hormone injection, the Rainbow Most of these studies have focused on optimization of the hor- Trout broodstock were randomly divided into two experimen- mone administration method (acute versus sustained release of tal groups (ART and BRT) and a control group (CRT ) of similar GnRHa) or refinement of effective GnRHa doses. Other studies mean weight. The weight was 1,806 ± 57 g (mean ± SD) have assessed the endocrine response of salmonids to GnRHa for group ART , 1,924 ± 35 g for group BRT, and 1,861 ± injection outside the thermal optimum, especially at elevated 56 g for group CRT . Each group contained 20 individuals, which temperatures (Gillet et al. 1996; Pankhurst and Thomas 1998; were placed into three 4-m3 raceways supplied with water from Taranger et al. 2003; King and Pankhurst 2004, 2007; Vikingstad the Loucnˇ a´ River (part of the Elbe River system) in the Czech et al. 2008; Gillet and Breton 2009). There is limited informa- Republic. Water temperature was 9.5◦C at the beginning of the tion regarding the effect of hormone treatment on egg quality. trial and rose continuously, reaching 13.4◦C on the final day of Studies that have examined egg quality after hormone treatment the experiment. Temperature and oxygen were measured daily have produced inconsistent and controversial results. Crim et al. using a Handy Polaris oxygen meter (OxyGuard International (1983b, 1986), Crim and Glebe (1984), Taranger et al. (1992), A/S, Denmark). Oxygen content in the outlet was maintained at Haraldsson et al. (1993), Olito et al. (2001), and Noori et al. 9.5 mg/L throughout the experiment by using an aerator (Linn (2010) reported reduced fertility or a reduced eyeing rate in Geratebau¨ GmbH, Germany). The flow-through rate was main- salmonids that were treated with GnRHa doses of 100 µg/kg tained at 65 L/min. All fish were kept under the natural pho- of body weight (BW) or higher. Mylonas et al. (1992) reported toperiod, and feeding was ceased 4 d before the expected date of that fertility and eyeing were reduced when a double injection of stripping. Fish were not fed during the experimental period un- only 10 µg/kg BW was used, whereas other authors have found til they had been stripped. The experiment began with hormone no differences in eggs obtained from treated and untreated fe- injections on 24 February 2011 and continued until 17 May Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 males or from females treated with differing doses of GnRHa 2011, when all females in the control group had been stripped. (Jalabert et al. 1978; Donaldson et al. 1981; Billard et al. 1984; Fish were intraperitoneally injected with sGnRHa emulsified in Erdahl and McClain 1987; Slater et al. 1995; Arabacı et al. 2004; Freund’s incomplete adjuvant (FIA) as follows: group ART re- Vazirzadeh et al. 2008; Vikingstad et al. 2008). Regardless of ceived 100 µg of sGnRHa–FIA/kg of BW; group BRT received GnRHa dose, egg viability improved in GnRHa-treated females 25 µg of sGnRHa–FIA/kg of BW; and group CRT (the control) compared with naturally ovulating female Brook Trout (Svinger received 1:1 FIA and 0.9% physiological saline. et al. 2013) and steelhead (anadromous Rainbow Trout; Sower Brook Trout.—For the Brook Trout experiment, 3-year-old et al. 1984). females of the Czech Klatovy strain were used. One week be- In salmonids, the induction of ovulation several weeks prior fore hormone injection, broodstock were randomly divided into to their natural spawning time shortens the final stages of vitel- three experimental groups (ABT,BBT, and CBT) and a control logenesis, which may have negative consequences for eggs and group (DBT). The weight of fish was 587 ± 25 g (mean ± SD) fry. Reduced egg size after GnRHa treatment has been inci- in group ABT, 635 ± 28 g in group BBT, 587 ± 16 g in group dentally recorded in Coho Salmon (Donaldson 1981), Chinook CBT, and 568 ± 16 g in group DBT. Each group (n = 25) was Salmon (Olito et al. 2001), and recently in Northern White- placed in a separate 4-m3 raceway supplied with water from the fish (Svinger and Kouril,ˇ in press). No further detailed reports Loucnˇ a´ River. Water temperature was 11◦C at the beginning of describing GnRHa treatment effects on egg size are available. the trial and continually decreased to 4.9◦C on the last day of the 476 SVINGER ET AL.

experiment. Oxygen content and flow-through were maintained was measured using a WTW 3310 pH meter fitted with a Sen- at levels similar to those in the Rainbow Trout experiment and tix MIC-D microelectrode (WTW, Weilheim, Germany). From by using the same equipment. Fish were kept under the natural each female, 15 randomly chosen eggs were placed into a small photoperiod during the entire experimental period. The exper- sieve, and the remaining ovarian fluid was carefully removed iment was carried out from 4 October 2011 (when hormones by using paper towels. Egg weight was recorded to the nearest were injected) to 25 November 2011 (when all females in the 1 mg with a precision balance (KERN 572-31). A sample of control group had been stripped). Feeding was ceased 4 d be- 300 ± 10 eggs from each female was used for fertilization. Egg fore the expected date of stripping, and fish were not fed until samples were fertilized with an excess of sperm from three to they had been stripped. Females were intraperitoneally injected four males that had previously been assessed for sperm motil- with sGnRHa–FIA or received an acute injection of sGnRHa ity by a drop test (Linhart et al. 2011). A separate syringe was as follows: group ABT received a single injection of 12.5 µg used for sperm collection from each male, and sperm was not of sGnRHa–FIA/kg of BW; group BBT received double acute pooled until addition of the activation medium (6 g of NaCl, injections of 12.5 µg of sGnRHa/kg of BW, with injections 0.075 g of KCl, 0.15 g of CaCl2.2H2O, and 0.1 g of NaHCO3 administered 3 d apart; group CBT received a single acute injec- per liter) during egg fertilization. After fertilization, eggs were tion of 50 µg of sGnRHa/kg of BW; and group DBT (control) left 1 h for water hardening and then were placed into miniature received 1:1 FIA and 0.9% physiological saline. salmonid egg incubators with separate and controllable water Hormone doses in Brook Trout were chosen based on prelim- flow-through as described by Kallert (2009). inary findings. An sGnRHa–FIA dose of 25 µg/kg BW and an The final ovulating female Rainbow Trout in the 100-µg/kg acute double injection of 25 µg/kg BW were previously found sGnRHa (ART) group was detected at 18 d postinjection, when to be highly effective in synchronizing ovulation in Brook Trout the cumulative ovulation rate had reached 65%. The remaining (Svinger et al. 2013). These doses were selected to lower the seven females in group ART were sacrificed on day 49, when costs of treatment. Experiments were carried out under Czech no ovulation could be detected. A sample of 30 eggs was taken Republic law number 246/1992 and under the supervision of a from each of the seven females and was cleared in a mixture of person licensed for performing experiments with animals (Min- 6:3:1 ethanol : formaldehyde : glacial acetic acid to identify the istry of Agriculture license code CZ 00786). germinal vesicle position. Average water temperature during incubation was 9.4 ± ◦ ± ◦ Hormone Preparation 0.5 C for the Rainbow Trout trial and 6.0 0.5 C for the Brook 6 9 Trout trial. For both species, the oxygen level in incubators was Superactive analog D-Arg Pro NEt-sGnRHa was purchased ± from Bachem AG (Weil am Rhein, Germany). To pre- maintained at 11 1 mg/L and flow-through was maintained at approximately 0.25 L/min. Other water characteristics (e.g., pare sustained-release sGnRHa–FIA treatments for both trout − species, sGnRHa was dissolved in 0.9% NaCl and mixed with pH, Cl , Fe, NH3,NH4, nitrite [NO2], and nitrate [NO3]) were FIA (Sigma) at 1:1 (volume/volume) using an Ultraturrax Ika well below limits that have been shown to negatively impact T-10 homogenizer. Each female received a total volume of egg development. During incubation, dead (white) eggs were 0.5 mL of sGnRHa–FIA preparation, which was administered collected and counted. At the eyed stage, eggs were shocked by using a 1-mL insulin syringe fitted with a 0.7- × 30-mm needle. shaking to sort out and count the unfertilized or unprecipitated For acute treatments in Brook Trout, sGnRHa was diluted with dead eggs. Remaining eyed eggs were incubated until hatch, and 0.9% NaCl to achieve the required concentration. Females that eggs with dead embryos were removed and counted. Embryo were given acute injections received 1 mL of this solution per eyeing rate, hatching rate, mortalities between the eyed stage and hatch, and rate of deformities were calculated as a percentage Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 kilogram of BW. Acute treatments were administered by using a 1-mL insulin syringe fitted with a 0.33- × 12-mm needle. of the number of eggs that were placed into the incubator at the start of incubation. Weight of freshly hatched yolk sac fry in relation to hormone treatment was determined only for Brook Detection of Ovulation Trout. Ten individual hatchlings were randomly taken from the Females in both experiments were evaluated for ovulation clutch representing each female and were weighed to the nearest twice weekly by following the common procedures used in this 1mg. hatchery. Females were considered to have ovulated if eggs were released upon gentle pressure on the abdomen. Latency Statistical Analysis was counted as the mean number of days from the first sGnRHa All data were analyzed by using Statistica 9 Cz (StatSoft, injection until detection of ovulation. Tulsa, Oklahoma). Data describing the course of ovulation and the percentage of females that ovulated were explored by a Egg Quality Assessment survival analysis (Z-test). Differences in latency interval were Eggs from females that had ovulated were stripped into sep- analyzed using the nonparametric Kruskal–Wallis multiple arate dishes. Three egg samples were taken from each female, comparison test. Two-way ANOVA was used to characterize and each sample was placed in a glass beaker. Ovarian fluid pH differences in ovarian fluid pH, with treatment as a main factor. SALMON GnRHa EFFECTS ON TROUT 477

FIGURE 1. Course of ovulation (cumulative ovulation rate, %) in the respective groups of Rainbow Trout that were intraperitoneally injected with salmon gonadotropin-releasing hormone analog (sGnRHa) emulsiﬁed in Freund’s incomplete adjuvant (FIA; ART = 100 µg of sGnRHa–FIA/kg of body weight [BW]; BRT = 25 µg sGnRHa–FIA/kg BW; CRT = control, 1:1 FIA and 0.9% physiological saline). Groups without a letter in common are signiﬁcantly different (P < 0.05). The arrow indicates the date of sGnRHa administration.

Differences in egg weight, fry weight, and measures of embryo onset of peripheral migration to the animal pole. Latency was survival were assessed by one-way ANOVA. Arcsine transfor- 13.7 ± 0.7 d in group ART and 19.5 ± 2.5 d in group BRT; mation was applied for all percentage values. If significant dif- these latency periods were significantly shorter than the latency ferences were found by ANOVA, Tukey’s honestly significant of 52.8 ± 3.6 d for group CRT (P < 0.01). Latency in group difference test for unequal sample sizes was applied for de- ART was calculated only based on the 13 females that ovulated; tailed multiple comparison testing. The Kolmogorov–Smirnov the sacrificed females were not included in the calculation. test was used to confirm the normal distribution of data after Differences among treatments in ovarian fluid pH, egg Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 homoscedasticity verification by the Cochran–Bartlett test. Lin- weight, survival to the eyed stage, mortality between the eyed ear and nonlinear regression analyses were applied to correlate stage and hatch, hatching rate, and percentage of deformities in latency and embryonic survival variables. A significance level yolk sac fry are shown in Table 1. Regression analysis did not α of 0.05 was applied to all tests except where indicated. Data reveal a correlation between ovarian fluid pH and egg viability are presented as mean ± SE. characteristics. Egg weight was not predictive of egg viability variables or latency. Survival to the eyed stage plotted against latency showed a low but significantly positive correlation in RESULTS 2 group BRT (R = 0.22, P < 0.05; Figure 2). The lack of a corre- Rainbow Trout lation between survival to the eyed stage and latency for group In Rainbow Trout, both treatments significantly synchronized ART is likely due to the lower number of females included in ovulation and shortened the spawning period in comparison with the regression model, as the structure of the relationship was the control (P < 0.01). The cumulative percentage of females identical to that seen in group BRT (Figure 2). A significant cor- that ovulated during the study period is displayed in Figure 1. For relation between mortality after the eyed stage and latency was 2 all seven of the nonovulating females in group ART, the germinal found only for group ART (R = 0.50, P < 0.05) and was related vesicle remained in the center of the oocyte, with no sign of the to the lower number of fish in that group (Figure 3). Generally, 478 SVINGER ET AL.

TABLE 1. Mean ( ± SE) ovarian fluid pH, egg weight, eyeing rate, mortality between the eyed stage (ES) and hatch, hatching rate, and yolk sac fry deformity rate for Rainbow Trout (n = number of females in each group) after injection with salmon gonadotropin-releasing hormone analog (sGnRHa) that was emulsified in Freund’s incomplete adjuvant (FIA; ART = 100 µg of sGnRHa–FIA/kg of body weight [BW]; BRT = 25 µg sGnRHa–FIA/kg BW; CRT = control, 1:1 FIA and 0.9% physiological saline). Within a column, means with different lowercase letters are significantly different (one-way ANOVA followed by Tukey’s multiple comparison test for unequal sample sizes, P < 0.05).

Ovarian Egg weight Eyed eggs Mortality, ES Hatching Deformities Group n ﬂuid pH (mg) (%) to hatch (%) rate (%) (%)

ART 13 8.18 ± 0.06 z 55.26 ± 2.55 z 67.85 ± 6.12 z 11.28 ± 3.94 z 61.76 ± 6.42 z 0.12 ± 0.12 z BRT 20 8.02 ± 0.05 y 53.12 ± 2.06 z 71.34 ± 4.93 z 11.50 ± 3.18 z 64.58 ± 5.17 z 0.15 ± 0.09 z CRT 19 8.21 ± 0.05 z 62.22 ± 2.11 y 87.80 ± 5.06 y 1.60 ± 3.26 z 86.61 ± 5.31 y 0.18 ± 0.10 z

only eggs from treated specimens with the shortest latencies ovulation in Brook Trout and shortened the spawning period showed high mortality after the eyed stage (Figure 3). Hatch- compared with the control (P < 0.05; Figure 5). All treatments ing rate showed a low but significant positive correlation with showed low synchronization effects. The cumulative percentage 2 latency for group BRT (R = 0.33, P < 0.05; Figure 4). A low of females that ovulated over the study period is presented in hatching rate was found, especially in treated groups with the Figure 5. On November 25, 2011, when the experiment was ter- shortest latency (Figure 4). Regression analysis did not reveal a minated, the percentage of females that had been stripped was correlation between the percentage of yolk sac fry deformities 88% in group ABT, 68% in group BBT, 72% in group CBT, and and egg weight or latency. 76% in group DBT (Figure 5). All remaining Brook Trout females were lost due to saprolegniasis, a common phenomenon in Brook Trout Brook Trout during the spawning season. Mean latency reached With the exception of the single acute injection of 50 µg/kg 29.0 ± 2.8dingroupABT and 24.1 ± 3.3dingroupBBT; (group CBT), all treatments significantly induced and advanced these latencies were significantly shorter than the latency of Downloaded by [Department Of Fisheries] at 20:48 19 August 2013

FIGURE 2. Correlation between the percentage of eyed eggs (i.e., survival to the eyed stage [ES]) and latency (number of days from the ﬁrst injection to the date of detected ovulation) for each group of Rainbow Trout (groups are deﬁned in Figure 1). SALMON GnRHa EFFECTS ON TROUT 479

TABLE 2. Mean ( ± SE) ovarian fluid pH, egg weight, yolk sac fry weight, eyeing rate, mortality between the eyed stage (ES) and hatch, and hatching rate for Brook Trout (n = number of females in each group) after intraperitoneal injection with salmon gonadotropin-releasing hormone analog (sGnRHa) emulsified in Freund’s incomplete adjuvant (FIA) or with sGnRHa only (ABT = single injection of 12.5 µg of sGnRHa–FIA/kg of body weight [BW]; BBT = double acute injection of 12.5 µg sGnRHa/kg BW, with injections administered 3 d apart; CBT = single acute injection of 50 µg sGnRHa/kg BW; DBT = control, 1:1 FIA and 0.9% physiological saline). Within a column, means with different lowercase letters are significantly different (one-way ANOVA followed by Tukey’s multiple comparison test for unequal sample sizes, P < 0.05).

Ovarian Egg weight Fry weight Eyed eggs Mortality, ES Hatching rate Group n ﬂuid pH (mg) (mg) (%) to hatch (%) (%)

ABT 22 8.02 ± 0.02 z 39.21 ± 0.88 z 43.98 ± 0.99 z 82.05 ± 3.26 z 4.34 ± 0.88 z 77.71 ± 3.43 z BBT 17 8.07 ± 0.03 z 39.12 ± 1.00 z 43.93 ± 1.12 z 82.29 ± 3.71 z 7.12 ± 1.01 y 75.17 ± 3.90 z CBT 18 8.07 ± 0.03 z 38.46 ± 0.97 z 42.34 ± 1.09 z 74.53 ± 3.60 z 6.35 ± 0.98 zy 68.18 ± 3.79 z DBT 19 8.07 ± 0.02 z 44.74 ± 0.94 y 48.24 ± 1.06 y 79.44 ± 3.51 z 3.94 ± 0.95 z 75.50 ± 3.69 z

± < 2 42.6 2.2 d for group DBT (the control, P 0.01). Latency weight (r = 0.66 for group ABT, 0.82 for group BBT,0.77for ± for group CBT was 36.3 3.6 d and did not significantly differ group CBT, and 0.69 for group DBT; P < 0.01 for all groups). from that of any other group. No correlation between egg weight and latency was observed; Among-group comparisons of data on ovarian fluid pH, egg the exception was the control group, in which a small but signif- weight, yolk sac fry weight, survival to the eyed stage, mortality icant negative relationship was observed (r2 = 0.30; P < 0.05). after the eyed stage, and hatching rate are shown in Table 2. Re- Survival to the eyed stage was plotted against latency; although gression analysis did not reveal a correlation between ovarian groups ABT and DBT showed constant survival levels over the 2 2 fluid pH and egg viability variables. No correlation was found monitored period (ABT: r = 0.13, P = 0.10; DBT: r = 0.15, between egg weight, yolk sac fry weight, and egg viability mea- P = 0.10), a significant negative relationship was found for 2 2 sures. Egg weight was significantly predictive of yolk sac fry groups BBT and CBT (BBT: r = 0.42, P < 0.01; CBT: r = 0.55, Downloaded by [Department Of Fisheries] at 20:48 19 August 2013

FIGURE 3. Correlation between mortality (eyed stage to hatch) and latency for each group of Rainbow Trout (groups are deﬁned in Figure 1). 480 SVINGER ET AL.

FIGURE 4. Correlation between hatching rate and latency for each group of Rainbow Trout (groups are deﬁned in Figure 1).

P < 0.01; Figure 6). Mortality between the eyed stage and hatch final oocyte maturation. Treatments with GnRHa–FIA at doses showed no correlation with latency for groups ABT and CBT ranging from 25 to 50 µg/kg have reportedly been highly ef- (P > 0.05). For group BBT, the mortality value decreased with fective inducers and synchronizers of ovulation in Rainbow increasing latency, whereas in control group DBT the opposite Trout if administered some weeks prior to the spawning sea- trend was observed. However, the correlation was weak in both son (Arabacı et al. 2004; Vazirzadeh et al. 2008). This range of 2 2 groups (BBT: r = 0.36, P < 0.05; DBT: r = 0.24, P < 0.05; sGnRHa–FIA doses has also been shown to be effective in Brook Figure 7). A significant negative correlation between hatching Trout, European Grayling, and Northern Whitefish (Svinger and 2 rate and latency was found for group CBT (r = 0.56, P < 0.01; Kourilˇ 2012, in press). In Brown Trout Salmo trutta m. fario, Figure 8). No relationship between hatching rate and latency doses from 15 to 60 µg/kg were effective when D-Arg6Pro9NEt- Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 was observed in other groups, for which hatching rate remained sGnRHa was used (V. W. Svinger and D. M. Kallert, unpub- almost constant regardless of latency (Figure 8). No deformities lished data). Use of the high dose of FIA-emulsified sGnRHa of hatched Brook Trout yolk sac fry were observed. (i.e., as administered to group ART ) in salmonids has not been previously reported, although a number of studies have applied GnRH analogs at doses exceeding 100 µg/kg to Arctic Char DISCUSSION (Haraldsson et al. 1993), Atlantic Salmon (Weil and Crim 1983; All of the sGnRHa treatments in our study induced and/or ad- Crim et al. 1986; Taranger et al. 1992), Caspian Brown Trout vanced ovulation to a greater or lesser extent in both species; this Salmo trutta caspius (Noori et al. 2010), and Coho Salmon has been previously demonstrated for other salmonid species. (Donaldson et al. 1981). Since these studies differed in terms However, in Rainbow Trout, a high dose of sGnRHa during of study species, GnRH analogs, GnRHa delivery systems, and sustained-release FIA treatment (i.e., group ART ) was unex- experimental conditions, it is difficult to identify common de- pectedly less effective than a dose that was equivalent to 25% nominators. It can be stated that in these studies, high rates of of that level (group BRT ). Autopsy and subsequent examina- ovulation and spermiation were obtained when a high dose of tion of ova from the nonovulating individuals revealed that the GnRHa was applied, which is in contrast to our present results high-dose sGnRHa–FIA treatment did not result in induction of for Rainbow Trout. SALMON GnRHa EFFECTS ON TROUT 481

FIGURE 5. Course of ovulation (cumulative ovulation rate, %) in the respective groups of Brook Trout that were intraperitoneally injected with salmon gonadotropin-releasing hormone analog (sGnRHa) emulsiﬁed in Freund’s incomplete adjuvant (FIA) or with sGnRHa only (ABT = single injection of 12.5 µg of sGnRHa–FIA/kg of body weight [BW]; BBT = double acute injection of 12.5 µg sGnRHa/kg BW, with injections administered 3 d apart; CBT = single acute injection of 50 µg sGnRHa/kg BW; DBT = control, 1:1 FIA and 0.9% physiological saline). Groups without a letter in common are signiﬁcantly different (P < 0.05). Arrows indicate the date(s) of GnRHa administration.

Billard et al. (1984) reported that sustained-release treat- Nevertheless, it is likely that such cases may occur, and this ment with (des-Gly10, D-Ala6-NEt)-luteinizing hormone (LH)– should be studied in the future. releasing hormone analog in silicone rubber implants contain- The present results for Brook Trout expand on the ﬁndings ing a dose of 10–20 µg/kg delayed ovulation in Brown Trout of our previous study (Svinger et al. 2013) and clearly demon- if administered close to the natural spawning time—that is, strate that a single acute injection (i.e., group CBT) does not when simultaneous natural stimulation of LH pituitary secre- evoke high ovulation rates in this species if injected approxi-

Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 tion was likely to be taking place. In teleosts, Habibi (1991) mately 1 month prior to the natural spawning time. Generally, demonstrated that in vitro exposure of Goldfish Carassius au- a synchronization effect was not achieved with any of the treat- ratus pituitary to a continuous dose of native sGnRH or chicken ments, since the doses used in the sGnRHa–FIA treatment and GnRH-II resulted in desensitization and refractoriness of the the acute double-injection protocol were too low with respect pituitary to stimulation by these same hormones. The pituitary to injection time. Taranger et al. (1992) demonstrated that even desensitization phenomenon was later observed in Black Porgy a low acute GnRHa dose of 1 µg/kg BW can be effective in Acanthopagrus schlegeli (Yen et al. 2002). Whether treatment Atlantic Salmon if administered close to the natural spawning with continuous-release GnRHa at an unknown or inappropri- time. Thus, the treatments applied to Brook Trout might have ate physiological stage could impair LH secretion via pituitary been more effective if administered some weeks later. A sin- desensitization in vivo is unclear, especially since neither the gle acute injection of 25 µg/kg that was given approximately Billard et al. (1984) study nor our study addressed this ques- 3 weeks prior to the natural spawning period was effective in tion. It remains speculative whether the effects of high GnRHa up to 50% of Brook Trout females (Svinger et al. 2013). How- doses in sustained-delivery systems are influenced by specific ever, higher efficacy of the low-dose (12.5-µg/kg) sGnRHa–FIA physiological events during the period of hormone release. treatment compared with the single, acute high-dose (50-µg/kg) 482 SVINGER ET AL.

FIGURE 6. Correlation between the percentage of eyed eggs (i.e., survival to the eyed stage) and latency (number of days from the ﬁrst injection to the date of detected ovulation) for each group of Brook Trout (groups are deﬁned in Figure 5).

sGnRHa treatment underscores the greater importance of sus- egg wet weight tends to increase with postovulatory retention tained GnRHa delivery in salmonids relative to the dose of in the body cavity, together with ongoing overripening, due to GnRHa per kilogram of BW per se. increased water content (Hirose et al. 1977; Lahnsteiner 2000; The pH of ovarian fluid is considered a potential marker for Aegerter and Jalabert 2004; Mansour et al. 2008; D.M. Kallert, egg quality. Poor egg survival was seen when ovarian fluid pH unpublished data). In our study, less-extreme levels of ovarian was lower than 7.4 in Rainbow Trout (Wojtczak et al. 2007) fluid pH were detected. If we accept the quadratic relationship or when ovarian fluid pH was less than 8.1 in Lake Trout between the percentage of eyed embryos and the ovarian fluid (Lahnsteiner et al. 1999). Alteration of ovarian fluid pH due pH as suggested by Lahnsteiner et al. (1999), it is difficult to to the leaking of proteins into the fluid can indicate the first find a correlation when ovarian fluid pH lies within the optimum, Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 stages of egg degeneration (overripening), which may also be because survival to the eyed stage does not show a correlation simultaneously indicated by the presence of certain hydrolytic with ovarian fluid pH in the optimum range. enzymes in the ovarian fluid (Fauvel et al. 1993; Lahnsteiner In this study, egg weight upon ovulation was significantly et al. 1999; Lahnsteiner 2000). Ovarian fluid pH in our ex- lower with hormone injection in both species, regardless of periments was slightly reduced in Rainbow Trout group BRT , treatment or dosage. Our results represent the first record of egg whereas in Brook Trout the ovarian fluid pH was nearly equal diminution in Rainbow Trout after GnRHa treatment. Olito et al. among individuals and groups, regardless of sGnRHa treatment. (2001) suggested that GnRHa treatment may stimulate the pitu- No relationship between ovarian fluid pH and embryo survival itary to produce LH (gonadotropin hormone [GTH] II), which measures was found for either species, suggesting that over- stimulates final sexual maturation, but not follicle-stimulating ripening was not the cause of the decreased survival of embryos hormone (GTH-I), which is responsible for the uptake of egg from treated Rainbow Trout females. This is supported by the yolk precursors. This, along with deficiencies in maternal mes- fact that females were checked for ovulation twice weekly and senger RNA deposition into the ova, could pose a significant that egg wet weight was lower in treated females than in control problem in later development. Other possible reasons for lower females. In other species (nonsalmonids as well as salmonids), egg weight may be the early stripping of females, thereby SALMON GnRHa EFFECTS ON TROUT 483

FIGURE 7. Correlation between mortality (eyed stage [ES] to hatch) and latency for each group of Brook Trout (groups are deﬁned in Figure 5).

truncating the final phase of vitellogenesis or causing prema- Reduction in egg weight was followed by a decreased weight ture interruption of vitellogenesis via reduction of sex steroid of yolk sac fry in sGnRHa-treated Brook Trout. This agrees with production. Treated fish of both species, particularly groups BBT the results of other authors (Brown 1946; Fowler 1972; Wallace and CBT, showed reduced egg weight compared with untreated and Aasjord 1984; Hutchings 1991), who reported that salmonid fish, regardless of the similarity in latency. egg size is highly predictive of the size of hatched alevins. Sim- The reduction in egg weight was not the basis for lower em- ilar to the results of the present study, neither embryo survival bryo survival in Rainbow Trout, as suggested by Olito et al. nor the percentage of hatched alevins has been reported to be (2001), since no correlation between egg size and survival or correlated with egg size (Hutchings 1991; Jonsson´ and Svavars- percentage of deformities was detected in either species. Hence, son 2000). Nonetheless, in Brook Trout, larger eggs have been we can also assume that the reduction in egg weight was prob- shown to be positively related to survival throughout the first Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 ably not accompanied by the absence of specific proteins that 50 d of exogenous feeding (Hutchings 1991); furthermore, in Lahnsteiner (2007) strongly correlated with the percentage of Arctic Char, smaller eggs have been found to produce smaller eyed embryos in Brown Trout. On the other hand, it is not clear fry with lower initial feeding success and growth relative to whether the possible absence of these proteins is correlated larger fry (Wallace and Aasjord 1984). The size advantage may with reduced egg weight. The ability of GnRHa treatment to soon be counteracted by other environmental determinants of alter egg protein composition independent of egg size remains growth, and under good hatchery conditions, differential egg to be investigated. In addition, elevated mortality shortly be- size is not a primary determinant of reproductive success (see fore hatch was observed in Rainbow Trout groups ART and BRT review by Bromage et al. 1992). It is not clear whether a reduc- and in Brook Trout group BBT. This is in accordance with the tion in egg size induced by sGnRHa treatment could result in report by Bonnet et al. (2007) that the use of D-Arg6Pro9NEt- impaired growth potential of offspring; thus, further examina- sGnRHa to induce spawning in Rainbow Trout may result in tion is needed, especially if fry are intended for use in restocking egg defects that are characterized by embryo mortalities occur- activities in natural waters. ring after eyeing. However, the source of these defects remains Our results indicating that egg size is not predictive of embryo unclear. survival are in agreement with those of Springate and Bromage 484 SVINGER ET AL.

FIGURE 8. Correlation between hatching rate and latency for each group of Brook Trout (groups are deﬁned in Figure 5).

(1985) but are contrary to those of Craik and Harvey (1984), who Fitzpatrick et al. (1984), we suggest that reduced egg survival found a significant positive correlation between egg wet weight is chiefly caused by errors in determining the female’s oocyte and hatching. Inconsistency in results among studies consider- maturation status. There is evidence that Rainbow Trout eggs ing egg size as a measure of quality is common (Glebe et al. that are stripped immediately after ovulation have lower sur- 1979; Pitman 1979; Kato and Kamler 1983; Thorpe et al. 1984; vival rates than eggs that are stripped 4–10 d after ovulation Wallace and Aasjord 1984). This is mainly due to differences in (Sakai et al. 1975; Bry 1981; Springate et al. 1984). While con- the age and size of parent fish, variation in culture conditions, ducting experiments with GnRHa-synchronized ovulation, we and—probably a primary factor—variation in the ripeness stage endeavored to accurately detect the time of ovulation in exper- of the eggs used (Bromage et al. 1992). imental fish but without considering that underripe eggs might

Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 When the eyeing rate and hatching rate were plotted against be collected. In autumn 2012, we were able to attain synchro- latency in Rainbow Trout, females with the shortest latency had nized ovulation in Brown Trout S. trutta lacustris broodstock eggs with the lowest embryo survival. Pooling of the data from by using acute double injections of D-Arg6Pro9NEt-sGnRHa at the Rainbow Trout groups demonstrated a course of embryo sur- 25 µg/kg BW (our unpublished data). In some of the females, vival that was dependent on latency—nearly identical to findings ovulation was detected at 9 d postinjection, but manual stripping reported by Mylonas et al. (1992) for Brown Trout, Svinger and was delayed to 13 d postinjection. Ovulation in other females Kourilˇ (in press) for Northern Whitefish, and Fitzpatrick et al. was detected at 13 d postinjection, and those fish were immedi- (1984) for Coho Salmon. Mylonas et al. (1992) hypothesized ately stripped. Better fertilization rates were observed in females that GnRHa treatment caused a reduction in egg quality by dis- with the shortest latency but with delayed stripping (99% fertil- rupting the timing of two distinct processes: (1) final oocyte ization) than in females with longer latency that were stripped maturation controlled by maturation-inducing steroid 17α,20β- immediately after ovulation was detected (90% fertilization). P and (2) ovulation regulated by prostaglandins. We suggest Detailed follow-up comparison was not possible in this case, that this asynchrony is undoubtedly the primary cause of the and further assessments are underway. alteration in egg quality, but there is no conclusive evidence Variation in ripeness of the eggs being fertilized could also that either GnRHa treatment or dosage is the source. Similar to explain the inconsistency in results of previous studies that have SALMON GnRHa EFFECTS ON TROUT 485

investigated the influence of GnRHa treatments on egg quality. ing the post-ovulation period; effect of treatment with 17α-hydroxy-20β- There may also be differences among species, since the survival dihydroprogesterone. Aquaculture 24:153–160. of eggs from sGnRHa-treated Brook Trout is consistently equal Craik, J. C. A., and S. M. Harvey. 1984. Egg quality in Rainbow Trout: the relation between egg viability, selected aspects of egg composition, and time to or better than that of eggs from untreated control fish (Svinger of stripping. Aquaculture 40:115–134. et al. 2013), and the dependence of egg survival on latency in Crim, L. W., D. M. Evans, and B. H. Vickery. 1983a. Manipulation of the Brook Trout is opposite the pattern observed for Rainbow Trout. seasonal reproductive cycle of the landlocked Atlantic Salmon (Salmo salar) In the present study, the sGnRHa dose did not show any influence by LHRH analogues administered at various stages of gonadal development. on egg survival in either species. Canadian Journal of Fisheries and Aquatic Sciences 40:61–67. Crim, L. W., B. D. Glebe, and A. P. Scott. 1986. The influence of LHRH analog In summary, it is evident that the experimental designs that on oocyte development and spawning in female Atlantic Salmon, Salmo salar. are currently in use in salmonid research must be updated to Aquaculture 56:139–149. determine reliable means of avoiding egg mortality after Gn- Crim, L. W., A. M. Sutterlin, D. M. Evans, and C. Weil. 1983b. Accelerated RHa treatment. The gross egg mortality observed in our study ovulation by pelleted LHRH analogue treatment of spring-spawning Rainbow is unacceptable. For commercial breeders in particular, the sav- Trout (Salmo gairdneri) held at low temperature. Aquaculture 35:299–307. Donaldson, E. M., G. A. Hunter, and H. M. Dye. 1981. Induced ovulation in ings obtained by reduction of handling and better planning of Coho Salmon (Oncorhynchus kisutch): II. preliminary study of the use of stripping do not compensate for the potential risk of financial LH-RH and two high potency LH-RH analogues. Aquaculture 26:129–141. loss associated with low egg survival, investment in GnRHa Erdahl, D., and J. McClain. 1987. Effect of LH-RH analogue treatment on egg treatment, and reduced production of eyed eggs. maturation (ovulation) in Lake Trout brood stock. Progressive Fish-Culturist 49:276–279. Fauvel,C.,M.H.Omnes,` M. Suquet, and Y. Normant. 1993. Reliable assessment of overripening in Turbot (Scophthalmus maximus)byasimplepH ACKNOWLEDGMENTS measurement. Aquaculture 117:107–113. This work was supported by the following projects: Fitzpatrick, M. S., J. M. Redding, F. D. Ratti, and C. B. Schreck. 1987. Plasma CENAKVA CZ.1.05/2.1.00/01.0024, GAJU 074/2013/Z, and testosterone concentration predicts the ovulatory response of Coho Salmon Oveˇrenˇ ´ı technologie hormonaln´ ´ı synchronizace umelˇ eho´ vyt´ eruˇ (Oncorhynchus kisutch) to gonadotropin-releasing hormone analog. Cana- jikernacekˇ lososovitych´ ryb CZ.1.25/3.4.00/10.00314. Experi- dian Journal of Fisheries and Aquatic Sciences 44:1351–1357. Fitzpatrick, M. S., B. K. Suzumoto, C. B. Schreck, and D. Oberbillig. 1984. ments were conducted at the trout farm Ryba´rstvˇ ´ı Litomyslˇ Ltd., ◦ ◦ Luteinizing hormone-releasing hormone analogue induces precocious ovula- Czech Republic (49 53 3.104 N, 16 17 9.362 E). We thank the tion in adult Coho Salmon (Oncorhynchus kisutch). Aquaculture 43:67–73. hatchery management of Ryba´rstvˇ ´ı Litomyslˇ Ltd., especially Fowler, L. G. 1972. Growth and mortality of fingerling Chinook Salmon as Mirek Fabianek and Petr Trnka, for their excellent help. 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North American Journal of Aquaculture Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/unaj20 Use of Lethal Short-Term Chlorine Exposures to Limit Release of Nonnative Freshwater Organisms Christopher G. Ingersoll a , Eric L. Brunson a , Doug K. Hardesty a , Jamie P. Hughes a , Brittany L. King a & Catherine T. Phillips b c a U.S. Geological Survey, Columbia Environmental Research Center , 4200 New Haven Road, Columbia , Missouri , 65201 , USA b U.S. Fish and Wildlife Service, National Fish Hatchery and Technology Center , 500 East McCarty Lane, San Marcos , Texas , 78666 , USA c U.S. Fish and Wildlife Service, Panama City Fish and Wildlife Conservation Office , 1601 Balboa Avenue, Panama City , Florida , 32405 , USA Published online: 19 Aug 2013.

To cite this article: Christopher G. Ingersoll , Eric L. Brunson , Doug K. Hardesty , Jamie P. Hughes , Brittany L. King & Catherine T. Phillips (2013) Use of Lethal Short-Term Chlorine Exposures to Limit Release of Nonnative Freshwater Organisms, North American Journal of Aquaculture, 75:4, 487-494, DOI: 10.1080/15222055.2013.786008 To link to this article: http://dx.doi.org/10.1080/15222055.2013.786008

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ARTICLE

Use of Lethal Short-Term Chlorine Exposures to Limit Release of Nonnative Freshwater Organisms

Christopher G. Ingersoll,* Eric L. Brunson, Doug K. Hardesty, Jamie P. Hughes, and Brittany L. King U.S. Geological Survey, Columbia Environmental Research Center, 4200 New Haven Road, Columbia, Missouri 65201, USA Catherine T. Phillips1 U.S. Fish and Wildlife Service, National Fish Hatchery and Technology Center, 500 East McCarty Lane, San Marcos, Texas 78666, USA

Abstract Fish hatcheries and other types of aquatic facilities are potential sources for the introduction of nonnative species of fish or aquatic invertebrates into watersheds. Chlorine has been suggested for use to kill organisms that might be released from the effluent of a facility. While acute LC50s (concentrations lethal to 50% of organisms exposed for up to 96 h) for chlorine are available for some species, short-term LC100s for chlorine have not been determined. The objective of this study is to establish concentrations of chlorine that are lethal to 100% of organisms after brief (1-, 5-, or 15-min) exposures. A total of 22 species were exposed to total residual chlorine concentrations (TRC) of 1, 10, or 25 mg TRC/L for 1, 5, or 15 min under static conditions followed by a 24-h postexposure recovery period in water without the addition of chlorine. Concentrations of chlorine resulting in 100% lethality of organisms were established for all of the species tested except for four species of mollusks or for a beetle. Exposures for 5 to 15 min to 10–25 mg TRC/L were the lowest combined time–chlorine treatments under which all of the fish tested and the other invertebrates tested (17 species) exhibited 100% lethality by the end of the initial chlorine exposures or after the 24-h recovery period.

The introduction of nonnative aquatic organisms from production was also reported by Gozlan (2008). Release of non-

Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 sources such as culturing or toxicity testing facilities into a native aquatic organisms is also prevalent in the United States. watershed is an environmental concern, as invasive species can For example, Peterson et al. (2005) examined the occurrence alter existing habitats (Zhu et al. 2006), out-compete or replace of Nile Tilapia Oreochromis niloticus in the Pascagoula River native species (Meffe 1985; Fausch 1988; Walser et al. 2000), watershed of Mississippi likely released from two nearby aqua- introduce diseases (Hoffman and Schubert 1984), or change culture facilities. Populations of the freshwater amphipod Cran- the community composition (Lemly 1985). Furthermore, the gonyx pseudogracilis, native to the Great Lakes region and Gulf increase in freshwater aquaculture has increased the number of coastal plain, were found in Arizona and Nevada and were likely ﬁsh species introduced, with 624 species introduced worldwide the result of an accidental introduction by the Page Spring Fish (over a twofold increase of three decades ago; Gozlan 2008). Hatchery in Arizona (Zhang and Holsinger 2003). The inadver- A signiﬁcant positive relationship between the number of non- tent release of Brook Trout Salvelinus fontinalis was associated native introductions in a region and the level of aquaculture with acid rain toxicity testing conducted by our facility in the

*Corresponding author: [email protected] 1Present address: U.S. Fish and Wildlife Service, Panama City Fish and Wildlife Conservation Ofﬁce, 1601 Balboa Avenue, Panama City, Florida 32405, USA. Received December 20, 2012; accepted March 9, 2013 487 488 INGERSOLL ET AL.

early 1980s. Fortunately, a population of this coldwater species time that organisms might be exposed to chlorine in a treated of trout did not become established in the warmwater habitat effluent before reaching a receiving water with a lower chlo- of our watershed. Importantly, these types of introductions of rine concentration. A design of a small, inexpensive reservoir nonnative species by private, state, or federal aquatic facilities to maintain elevated concentrations of chlorine in effluents un- are preventable. der static renewal conditions or under low-flow conditions is Chlorine and ozone have been used to destroy taxa of con- also described for treating small volumes of water associated cern released into effluents generated from aquatic facilities. with laboratory culture or associated with a diluter used in a Acute chlorine or ozone toxicity median lethal concentrations laboratory toxicity test. (LC50s generated over a 24- to 96-h exposure) are available for some aquatic species (e.g., Wilde et al. 1983; Brooks and Bartos 1984; Huang et al. 1997; Leynen et al. 1998; Sellars et al. METHODS 2005; Sveceviciusˇ et al. 2005); however, LC100s representing Table 1 and Table S.1 (in the online version of this arti- 100% lethality, the concentration needed to destroy all aquatic cle) summarize test conditions that were used to conduct the organisms, have not been adequately established. chlorine toxicity tests. Organisms tested included 22 species of The objective of this study was to generate short-term LC100 fish, cladocerans, amphipods, midges, an oligochaete, a beetle, a chlorine toxicity data that can be used by facilities holding non- mussel, and snails commonly used to conduct laboratory toxicity native species obtained from outside of a local watershed. The tests or species that are periodically cultured for other purposes decision was made to focus on the use of chlorine as a toxi- (such as for study of invasive or listed species; Table S.1). The cant rather than ozone given occupational safety concerns with source of the test organisms was either from cultures maintained handling ozone (an odorless gas) and difficulties in generating, by the U.S. Geological Survey Columbia Environmental Re- maintaining, and analyzing concentrations of ozone in water. search Center (CERC) in Columbia, Missouri, or from cultures The decision was also made to generate short-term toxicity data maintained by the U.S. Fish and Wildlife Service (USFWS) (e.g., 1-, 5-, or 15-min exposure) to simulate a short period of National Fish Hatchery and Technology Center in San Marcos,

TABLE 1. Test conditions for conducting short-term chlorine toxicity tests with select aquatic organisms (adapted from ASTM 2012a). See Table S.1 for speciﬁc information on test temperature, water volume, age or size of test organisms, and chlorine exposure time and exposure concentrations.

Parameter Test conditions (1) Test type Chlorine toxicity test: 1-, 5-, or 15-min exposure (Table 2) followed by 24-h recovery period in clean water; source of chlorine: DuPont Pool Care Sanitizer 3/1 chlorinating tablets C610CR (active ingredient 99% [Trichloro-S-Triazinetrione], available chlorine 90%) (2) Temperature (◦C) Typically 23 (Table S.1) (3) Lighting quality Wide-spectrum ﬂuorescent lights (about 500 lx) on a 16 h light : 8 h dark cycle (4) Test chamber volume 300-mL glass beaker (test organisms exposed in 160-mL inner chamber consisting of a glass tube with stainless steel screen [0.25-mm opening] located at the bottom [Figure 1 in Miao et al. 2010]); 1,600-mL glass chamber (stainless steel screen [1-mm opening] covering three 3-cm holes located at the lower side of the chamber; or a dip net [Table S.1]) (5) Water volume (mL) 160–1,000 (6) Test water Well water diluted with deionized water to a hardness of 100 mg/L, an alkalinity of 90 mg/L Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 (as CaCO3) and pH of 8.2 (mg/L: Ca 28, Mg 10, K 1.0, Na 10, Cl 12, SO4 19, and dissolved organic carbon 0.4) (7) Age of organisms Typically juveniles (Table S.1) (8) Organisms per replicate Typically 5–10 (Table S.1) (9) Replicates per treatment Typically 3–4 with fewer replicates tested if there were limited numbers of test organisms available (Table S.1) (10) Nominal chlorine exposure 0 (control) and two chlorine treatments (1, 10, or 25 mg total residual chlorine [TRC]/L concentrations (mg/L) [Table 2]) (11) Water quality Hardness, alkalinity, pH, dissolved oxygen, and conductivity at start of exposures; temperature daily and dissolved oxygen after the end of the 24-h recovery period; chlorine at the start and end of the exposures (Table S.1) (12) Test duration and endpoints Survival after 1, 5, or 15 min of the initial chlorine exposure and after a 24-h recovery period from the chlorine exposure (13) Test acceptability 90% survival of control organisms LETHAL SHORT-TERM CHLORINE EXPOSURES 489

TABLE 2. Summary of toxicity data for short-term exposures of select freshwater aquatic organisms to chlorine. Gray highlighted cells designate treatments with 0% survival. ND = not determined, NA = not applicable, TRC = total residual chlorine; asterisks indicate invasive species.

Survival (%) Lowest chlorine (TRC/L) and 1min + 5min + 15 min time treatment Treatment 24 h 24 h + 24 h with no Species (TRC; mg/L) 1 min recovery 5 min recovery 15 min recovery survival Fish Fathead Minnow Control ND ND 100 100 100 100 5 min at 10 mg Pimphales promelas 10 ND ND 0 0 0 0 25 ND ND 0 0 0 0 Channel Catﬁsh Control ND ND ND ND 100 100 5 min at 10 mg Ictalurus punctulatus 1 ND ND 100 100 0 13 10 ND ND 0 0 0 0 Rainbow Trout Control ND ND 100 100 100 97 15 min at 10 mg Oncorhynchus mykiss 10 ND ND 100 73 100 0 or 5 min at 25 ND ND 100 036 0 25 mg Bull Trout Control ND ND 100 100 100 100 15 min at 10 mg Salvelinus conﬂuentus 10 ND ND 100 100 100 0 or 5 min at 25 ND ND 100 0 100 0 25 mg White Sturgeon Control ND ND 100 100 100 100 15 min at 10 mg Acipenser transmontanus 10 ND ND 100 100 100 0 or 5 min at 25 ND ND 100 0 0 0 25 mg Fountain Darter Etheostoma fontinalis Large Control ND ND 100 95 100 100 5 min at 10 mg 1 ND ND 100 100 100 95 10 ND ND 0 0 0 0 Small Control 100 100 100 100 ND ND 5 min at 10 mg 1 100 100 100 100 ND ND 10 95 95 0 0NDND Large Control 100 100 100 100 ND ND 5 min at 10 mg 1 100 90 90 90 ND ND 10 70 60 0 0NDND Devils River Minnow Dionda diaboli Small Control ND ND 100 100 100 95 5 min at 10 mg

Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 1 ND ND 100 95 75 75 10 ND ND 0 0 0 0 Large Control 100 100 100 100 ND ND 5 min at 10 mg 1 100 100 100 100 ND ND 10 80 50 0 0NDND Green Sunﬁsh Control 100 100 100 100 ND ND 5 min at 10 mg Lepomis cyanellus 10 95 95 0 0NDND 25 25 25 0 0NDND Desert Pupﬁsh Control ND ND 100 100 100 100 5 min at 10 mg Cyprinodon macularius 10 ND ND 100 0 100 0 25 ND ND 0 0 0 0 Bighead Carp Control ND ND 100 100 100 100 15 min at 10 mg Hypophthalmichthys 10 ND ND 100 60 0 0 or 5 min at nobilis* 25 ND ND 35 0 0 0 25 mg (Continued on next page) 490 INGERSOLL ET AL.

Survival (%) Lowest chlorine (TRC/L) and 1min + 5min + 15 min time treatment Treatment 24 h 24 h + 24 h with no Species (TRC; mg/L) 1 min recovery 5 min recovery 15 min recovery survival Cladocerans Daphnia magna Control ND ND 100 100 100 100 5 min at 10 mg 10 ND ND 0 0 0 0 25 ND ND 0 0 0 0 Ceridaphnia dubia Control ND ND 100 100 100 100 5 min at 10 mg 10 ND ND 0 0 0 0 25 ND ND 0 0 0 0 Amphipods Gammarus pseudolimnaeus Control ND ND 100 100 100 100 5 min at 10 mg 10 ND ND 100 0 100 0 25 ND ND 100 0 0 0 Hyalella azteca Control ND ND 100 100 100 100 15 min at 25 mg 10 ND ND 30 70 27 17 25 ND ND 13 27 0 0 Midges Chironomus dilutus Control ND ND 100 100 100 100 15 min at 25 mg 10 ND ND 67 23 30 6.7 25 ND ND 20 6.7 0 0 Chironomus riparius Control ND ND 100 100 100 100 15 min at 10 mg 10 ND ND 63 23 0 0 or 5 min at 25 ND ND 75 0 0 0 25 mg Oligochaete Lumbriculus variegatus Control ND ND 100 100 100 100 5 min at 10 mg 10 ND ND 100 0 0 0 25 ND ND 0 0 0 0 Beetle Microcylloepus pusillus Control ND ND NA NA 100 100 >15 min at 1 ND ND 100 100 100 100 10 mg 10 ND ND 100 100 100 100 Mussel Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 Fatmucket Control ND ND 100 100 100 100 >15 min at Lampsilis siliquoidea 10 ND ND 10 83 83 83 25 mg 25 ND ND 30 40 43 43 Snails Lymnaea stagnalis Control ND ND 100 100 100 100 >15 min at 10 ND ND 100 90 100 93 25 mg 25 ND ND 100 70 100 50 Red-rim melania Control 100 90 100 100 ND ND >5 min at 25 mg Melanoides tuberculatus* 10 100 100 100 100 ND ND 25 100 100 100 100 ND ND Quilted melania Control 100 100 100 100 ND ND >5 min at 25 mg Tarebia granifera* 10 100 100 100 95 ND ND 25 100 100 100 100 ND ND LETHAL SHORT-TERM CHLORINE EXPOSURES 491

Texas (provided to CERC using an overnight delivery service). smaller-sized fish or invertebrates, (2) 1,600-mL glass jars Test organisms were acclimated for at least 24 h in test water at for medium-sized fish, or (3) dip nets for larger-sized fish the selected test temperature before the start of the exposures. (Table S.1). The 300-mL exposure chambers contained a 160- Fish were not fed for 24 h before the start of the exposures, mL inner exposure chamber consisting of a glass tube with and invertebrates were provided with a small amount of culture stainless steel screen (0.25-mm opening) located at the bottom food during the acclimation period. The test water was well wa- (Figure 1 in Miao et al. 2010). The 1,600-mL exposure cham- ter diluted with deionized water to a hardness of 100 mg/L (as bers had three 3-cm holes covered with stainless steel screen CaCO3; Table 1). (1-mm opening) located at the lower side of the chamber to The source of chlorine in the toxicity tests was chlorine facilitate mixing of water inside and outside the chambers. tablets (DuPont Pool Care Sanitizer 3/1 chlorinating tablets At start of the exposures conducted in the 300-mL cham- C610CR; Wilmington, Delaware; active ingredient [Trichloro- bers, a first set of 300-mL replicate chambers were filled with S-Triazinetrione], 99%; available chlorine, 90%). Use of these test water without the addition of chlorine. Test organisms were chlorine tablets as a source of chlorine was chosen due to the then placed into the test water within the inner chamber of each low cost of the tablets. In addition, the chlorine tablets provide replicate. The 160-mL inner chambers containing test organisms a simple means of continuously maintaining concentrations of were then gently lifted and immediately submerged into a sec- chlorine above a target effluent concentration. ond set of 300-mL replicate exposure chambers containing the Preliminary chlorine acute toxicity tests were conducted un- appropriate exposure concentrations of chlorine. The 160-mL der flow-through conditions with the amphipod Hyalella azteca inner chambers were then lifted three times without impinging and the oligochaete Lumbriculus variegatus using methods de- the test organisms onto the screen at the bottom of the inner scribed by Wang et al. (2007). Two sources of chlorine were used chambers to mix the chlorine water within the inner chambers. in the toxicity tests: chlorine as reagent-grade sodium hypochlo- After the desired chlorine exposure period (i.e., 1, 5, or 15 min), rite (NaOCl, available chlorine 10–13%; Aldrich, Milwaukee, the 160-mL inner chambers were removed and submerged in a Wisconsin) and chlorine as obtained from the chlorine tablets. third set of 300-mL chambers containing no added chlorine and Results of this study indicated that chlorine obtained from the each inner chamber was lifted three times. The 160-mL inner chlorine tablets exhibited similar acute effect concentrations chambers were then removed and submerged into a fourth set of compared with chlorine tested as NaOCl. For H. azteca,the 300-mL replicate chambers containing no added chlorine and 48-h LC50 was 0.14 mg (confidence limits of 0.11–0.16 mg) were lifted three times to start the 24-h recovery period without TRC/L with tablets and 0.15 mg (0.12–0.19 mg) TRC/L with the addition of food. NaOCl as the source of chlorine. For L. variegatus, the 48-h A similar procedure was followed for the 1,600-mL chambers LC50 was 0.24 mg (0.20–0.27 mg) TRC/L with tablets and except that 40-L chambers containing 28 L of test water were 0.31 mg (0.27–0.34 mg) TRC/L with NaOCl. used to dose the test organisms contained within the 1,600-mL Concentrations of chlorine were relatively stable over 15- replicate chambers. Small fish were transferred into the 1,600- min exposure periods (Table S.1); hence, static conditions were mL chambers using small, plastic weigh boats. A nylon dip net used to maintain chlorine concentrations in the toxicity tests was used to contain larger fish during the chlorine exposures (Table 2). The chlorine exposure waters were prepared by grind- in the 40-L chambers, followed by holding fish for the 24-h ing a portion of a chlorine tablet to a fine powder with a ceramic recovery period in 15 L of water in a 19-L glass jar with gentle mortar and pestle and then adding the appropriate amount of the aeration. Table S.1 describes which exposure chamber was used powder to 28 L of test water contained in a 40-L glass chamber. for each species (i.e., a 160-mL inner chamber, a 1,600-mL The test water was then mixed manually for up to 5 min until chamber, or a dip net). Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 all of the chlorine powder visually dissolved. Survival of test organisms was determined at the end of each Test organisms were exposed to a control test water without chlorine exposure and at the end of a 24-h recovery period addition of chlorine and to two of three chlorine concentrations by counting the survivors. Snails or mussels were transferred (target concentrations of 1, 10, or 25 mg TRC/L) for two of from the 160-mL chambers into 50-mL glass beakers containing three time periods (1, 5, or 15 min), followed by a 24-h recovery 20 mL of water to determine survival (foot movement within a period in water without the addition of chlorine to determine if 5-min observation period) under a dissecting microscope. Other there is recovery of organisms after the initial chlorine exposure smaller test organisms were transferred from the 160-mL cham- (Table 2). Initially, exposures were conducted at 10 and 25 mg bers to a glass tray placed on a light table to determine survival TRC/L for 5 and 15 min, but due to the observed frequent 100% (based on movement within a 5-s period in response to a gentle lethality to the majority of the test organisms with a treatment touch with a blunt probe). of 25 mg TRC/L for 15 min, the decision was made to test 10 Concentrations of dissolved oxygen, pH, conductivity, hard- and 25 mg TRC/L for 1 and 5 min for some of the additional ness, alkalinity, and ammonia were determined at the start of test organisms (Table 2). the exposures, and dissolved oxygen was determined at the end Three types of exposure chambers were used to con- of the 24-h recovery period (Eaton et al. 2005). Water sam- duct the chlorine toxicity tests: (1) 300-mL glass beakers for ples for chlorine measurement were analyzed immediately after 492 INGERSOLL ET AL.

sample collection. Total residual chlorine was measured in exposures to a variety of chemicals (e.g., Ward and DeGraeve water samples at the start and end of the exposures with a Hach 1980; Wang et al. 2007; ASTM 2012a). Hence, standard meth- spectrophotometer using Method 8370 with a target concentra- ods for conducting toxicity tests with mussels recommend in- tion of 1 mg TRC/L and Method 10102 for samples with target cluding a 24-h recovery period at the end of an exposure to chlorine concentrations of 10 or 25 mg TRC/L (Model DR 2800; determine if mussels may be avoiding exposure to a chemical Hach, Loveland, Colorado). by closing the shell (ASTM 2012b). Bighead Carp were imported in the 1970s for aquaculture and use in sewage treatment, escaped from captivity, and by RESULTS AND DISCUSSION the late 1990s were abundant in the Mississippi and Missouri Levels of hardness, alkalinity, pH, conductivity, ammonia, drainages (Kolar et al. 2005). Chlorine was effective in killing and dissolved oxygen were all relatively consistent across toxi- this species; however, the carp were somewhat less sensitive to city tests (Table S.1). Concentrations of dissolved oxygen after chlorine (100% lethality with either a 15-min exposure to 10 mg the 24-h recovery periods ranged from 89% to 115% of the TRC/L or with a 5-min exposure to 25 mg TRC/L) compared concentrations at the start of the initial chlorine exposures, indi- with the majority of the other fishes or invertebrates tested (e.g., cating stable conditions in the static chambers used to hold test 100% lethality with a 5-min exposure to 10 mg TRC/L; Table 2). organisms after the initial chlorine exposures. Two of the snails tested (red-rim melania and quilted melania) Concentrations of chlorine at the start of the initial exposures are also invasive species, likely released by the aquarium and were within the following ranges: (1) 0.9–1.3 mg/L for the target ornamental trade into North America in the 1930s and 1940s, exposure concentration of 1 mg TRC/L, (2) 9.5–10.8 mg/L for and are able to out compete or eradicate native populations of the target exposure concentration of 10 mg TRC/L, and (3) snails (Karatayev et al. 2009). 23.0–28.0 mg/L for the target exposure concentration of 25 mg Concentrations of chlorine resulting in 100% lethality to the TRC/L (Table S.1). Concentrations of chlorine at the end of mollusks or the beetle could likely be established by either in- the 1- to 15-min exposure periods typically dropped by <5%. creasing the exposure concentration to chlorine or by increasing Concentrations of chlorine were below detection (<0.05 mg the exposure time. However, given the ability of mollusks to TRC/L; Wang et al. 2007) in the control waters and in the avoid short-term exposure to toxicants (e.g., by mussels closing waters in the chambers at the start of the 24-h recovery periods valves or by snails closing the operculum), alternate approaches after the initial chlorine exposures. may be needed to reduce the likelihood of releasing these types Survival of test organisms met the test acceptability require- of nonnative species into receiving waters (e.g., use of a physical ment of 90% control survival in all of the chlorine toxicity tests barrier such as sand or a mesh screen sock). Additionally, mol- (Table 2). Concentrations of chlorine resulting in 100% lethal- lusks, including invasive zebra mussels Dreissena polymorpha, ity were established for all of the organisms tested except for are relatively tolerant in longer exposures to chlorine. For exam- the mollusks (fatmucket or three species of snails [pond snail, ple, zebra mussels (10-mm shell length) required an exposure red-rim melania, and quilted melania]) and the riffle beetle. Ex- to 3 mg TRC/L for 10 d to exhibit 100% lethality (Rajagopal posures for 5 to 15 min to 10 and 25 mg TRC/L were the lowest et al. 2002). Studies are ongoing at our facility to evaluate the combined time–chlorine treatments under which all of the fish use of sodium chloride, sodium hydroxide, calcium hydroxide, tested and the other invertebrates tested (17 species) exhibited or hydrogen peroxide as alternative chemicals to chlorine for 100% lethality by the end of the initial chlorine exposures or use in killing nonnative mollusks, including New Zealand mud after the 24-h recovery period. snails Potamopyrgus antipodarum (B. Watten, U.S. Geological Size or life stage of organisms tested did not influence the Survey, personal communication). Of course, culturing or test- Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 lowest time–chlorine treatment resulting in 100% lethality for ing of taxa under static renewal conditions would also reduce either Fountain Darter or Devils River Minnow (Table 2). After a the risk of release of nonnative species into a watershed. 1-min exposure, the concentration of chlorine required to result A 35-L chlorine reservoir was designed to maintain the target in 100% lethality of test organisms was not established for any of concentration of chlorine in the effluent generated from inter- the species tested (i.e., LC100 at 1 min greater than the highest mittent proportional diluters used to conduct laboratory toxicity concentrations tested of 10–25 mg TRC/L; Table 2). tests with fish or aquatic invertebrates (e.g., Wang et al. 2007). For some of the species tested, organisms that were initially The reservoir was 28 cm deep × 25 cm wide × 50 cm long alive at the end of the chlorine exposures subsequently died with a plastic baffle located 8 cm from the bottom of the reser- during the 24-h recovery period (i.e., Rainbow Trout, White voir. The baffle subdivided the reservoir and directed water flow Sturgeon, Desert Pupfish, Bighead Carp, Gammarus pseudolim- through the bottom of the reservoir to increase residence time naeus, Chironomus dilutus, C. riparius, and Lumbriculus var- of test organisms within the reservoir. A 1/3-hp pump (little- iegatus; Table 2). In contrast, mussels not exhibiting foot move- giantpump.com) was used to help mix the chlorine released ment after the initial chlorine exposure periods recovered during from the tablets within the reservoir. the 24-h period in water without the addition of chlorine. Mus- The reservoir system was designed to treat the relatively sels or snails have been observed to be able to avoid short-term small volumes of water (e.g., about 500 L/d) typical of smaller LETHAL SHORT-TERM CHLORINE EXPOSURES 493

hatchery operations, or specific to treatment of isolated sources of TRC (Brungs 1973), the concentrations of TRC relative to of effluent from raceways or ponds that contain nonnative the amount of chlorine added can be influenced by the presence species. Treatment of larger volumes of water would be possible of dissolved organic carbon or particulate organic carbon. Chlo- but would require a larger chlorine reservoir to account for rine toxicity may also be influenced by life stage of organisms retention time and would likely require direct additions of tested (e.g., Valenti et al. 2006). Hence, site-specific toxicity chlorine rather than use of chlorine tablets to deliver the desired testing with chlorine is advisable with the life stage(s) of the concentration of chlorine in the reservoir. species of interest using a test water representative of the water Chlorine concentrations of >60 mg TRC/L were maintained quality of the effluent at a facility. Longer exposures to higher in a 35-L reservoir containing three chlorine tablets that received concentrations of chlorine would be needed for tolerant species 16 volume additions/d of clean water from the diluter (e.g., the or tolerant life stages, but the volume of the chlorine reservoir 12-L diluter cycling every 30 min [average water addition of would likely need to be increased from the 35-L reservoir de- 400 mL/min]). However, with only one chlorine tablet in the scribed above to increase residence time of the organisms in the reservoir, concentrations of chlorine were maintained at only reservoir. >20 mg TRC/L. The tablets are designed to slowly dissolve in water, releasing a consistent amount of chlorine for mass ACKNOWLEDGMENTS of tablet remaining in the water. Concentrations of chlorine in We would like to thank the members of Toxicology Branch the reservoir were relatively consistent over time as long as for technical support; Julia Towns for literature support; Zach- daily rate of effluent addition to the reservoir was constant and ery Shattuck for assistance with collection of species obtained approximately the same amount of tablets was maintained in from the USFWS; and Tom Brandt, Robin Calfee, Holly Puglis, the reservoir. In our case we kept three tablets in the reservoir and three anonymous reviewers for providing comments on an at any one time, new tablets being added about weekly. earlier draft of the paper. Reference to trade names does not Assuming a 30-min cycle of water addition to the chlorine imply endorsement by the U.S. Government. reservoir, it is likely that the minimum time an organism might be contained within the reservoir would be at least 30 min. Hence, the duration of the chlorine toxicity data generated from REFERENCES the 1- to 15-min exposures (Table 2) would be applicable for ASTM (American Society for Testing and Materials). 2012a. Annual book of standards, volume 11.06: ASTM E729-96 (2007) standard guide for conduct- establishing target chlorine concentration in this static-renewal ing acute toxicity tests on test materials with fishes, macroinvertebrates, and reservoir. A reservoir could be larger to increase residence time amphibians. ASTM, West Conshohocken, Pennsylvania. of water or the rate of water added to the reservoir could be ASTM (American Society for Testing and Materials). 2012b. Annual book of increased, but a study would be needed to determine the number standards, volume 11.06: ASTM E2455-06 standard guide for conducting lab- of tablets required to maintain a target concentration of chlorine oratory toxicity tests with freshwater mussels. ASTM, West Conshohocken, Pennsylvania. in the reservoir. 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We are currently evaluating the use of a 100 µm-mesh American Public Health Association, American Water Works Association, screen sock placed on the influent to increase residence time of Water Environment Federation, Washington, D.C. Downloaded by [Department Of Fisheries] at 20:48 19 August 2013 organisms that might enter the reservoir. Fausch, K. D. 1988. Tests of competition between native and introduced Concentrated solutions of acid must not come in con- salmonids in streams: what have we learned? Canadian Journal of Fisheries and Aquatic Sciences 45:2238–2246. tact with high concentrations of chlorine in water to pre- Gozlan, R. E. 2008. Introduction of non-native freshwater fish: is it all bad? Fish vent the occupational safety hazard of toxic chlorine gas be- and Fisheries 9:106–115. ing released into the atmosphere (http://www.osha.gov/SLTC/ Hoffman, G. L., and G. Schubert. 1984. Some parasites of exotic fishes. Pages healthguidelines/chlorine/recognition.html). Treatment with 233–261 in W. R. Courtenay Jr. and J. R. Stauffer Jr., editors. Distribution, sodium thiosulfate can be used to neutralize the chlorine added biology, and management of exotic fishes. Johns Hopkins University Press, Baltimore, Maryland. to the water before an effluent leaves a facility (e.g., at a propor- Huang, J. L., L. Wang, N. Q. Ren, X. L. Liu, R. F. Sun, and G. L. Yang. 1997. tion of about 60 mg/L chlorine to 120 mg/L sodium thiosulfate; Disinfection effect of chlorine dioxide on viruses, algae and animal planktons Brungs 1973; ASTM 2012a). in water. Water Research 31:455–460. The current study generated toxicity data for select life stages Karatayev, A. Y., L. E. Burlakova, V. A. Karatayev, and D. K. Padilla. 2009. of test organisms in one type of water with a low concentration of Introduction, distribution, spread, and impacts of exotic freshwater gastropods in Texas. Hydrobiologia 619:181–914. particulate organic carbon and dissolved organic carbon (e.g., Kolar, C. S., D. C. Chapman, W. R. Courtenay Jr., C. M. Housel, J. D. dissolved organic carbon of about 0.4 mg C/L). While water Williams, and D. P. Jennings. 2005. Asian carps of the genus Hypoph- quality characteristics do not generally affect the acute toxicity thalmichthys (Pisces, Cyprinidae): a biological synopsis and environmental 494 INGERSOLL ET AL.

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North American Journal of Aquaculture Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/unaj20 Sparing Fish Oil with Beef Tallow in Feeds for Rainbow Trout: Effects of Inclusion Rates and Finishing on Production Performance and Tissue Fatty Acid Composition Brian R. Gause a & Jesse T. Trushenski a a Center for Fisheries, Aquaculture, and Aquatic Sciences and Departments of Zoology and Animal Science, Food and Nutrition , Southern Illinois University Carbondale , 1125 Lincoln Drive, Life Science II, Room 173, Carbondale , Illinois , 62901 , USA Published online: 29 Aug 2013.

To cite this article: Brian R. Gause & Jesse T. Trushenski (2013) Sparing Fish Oil with Beef Tallow in Feeds for Rainbow Trout: Effects of Inclusion Rates and Finishing on Production Performance and Tissue Fatty Acid Composition, North American Journal of Aquaculture, 75:4, 495-511 To link to this article: http://dx.doi.org/10.1080/15222055.2013.811134

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ARTICLE

Sparing Fish Oil with Beef Tallow in Feeds for Rainbow Trout: Effects of Inclusion Rates and Finishing on Production Performance and Tissue Fatty Acid Composition

Brian R. Gause and Jesse T. Trushenski* Center for Fisheries, Aquaculture, and Aquatic Sciences and Departments of Zoology and Animal Science, Food and Nutrition, Southern Illinois University Carbondale, 1125 Lincoln Drive, Life Science II, Room 173, Carbondale, Illinois 62901, USA

Abstract Sparing fish oil with alternative lipids in aquafeeds generally results in the loss of long-chain polyunsaturated fatty acids (LC-PUFAs) from farmed fish tissues; however, finishing feeds can be used to augment LC-PUFA levels prior to harvest. We reared Rainbow Trout Oncorhynchus mykiss (initial weight, 47.0 ± 0.2 g [mean ± SE]) in a recirculation aquaculture system (190-L tanks; 16.1 ± 0.0◦C water temperature) on grow-out diets containing fish oil (FO; 100% FO), beef tallow (BT; 100% BT), or blends of these lipids (50% BT, 75% BT) in combination with 4, 8, or 12 weeks of finishing with the 100% FO feed (three replicate tanks per feeding regimen; N = 3). After 31 weeks, production performance was unaffected by feeding regimen: weight gain = 1,151 ± 30%, feed conversion ratio = 1.50 ± 0.03, specific growth rate = 1.15 ± 0.01% body weight/d, and feed intake = 2.19 ± 0.02% body weight/d. Replacing fish oil with beef tallow altered tissues in favor of tallow-associated monounsaturated fatty acids at the expense of fish-oil-associated LC-PUFAs. Finishing had a corrective effect on these distortions, with longer finishing periods resulting in more comprehensive restoration. The extent of profile distortion and plasticity of the fillet, liver, intraperitoneal fat, eye, gill, and brain tissues appeared to be a function of physiological demand for certain fatty acids, lipid class composition, and tissue turnover rates; however, for all tissues, profile similarity between the control (fish fed the 100% FO feed exclusively) and experimental regimens varied with cumulative fish oil intake. Our results indicate fish oil sparing with beef tallow is an effective approach to reduce the costs of Rainbow Trout production and, if combined with finishing to augment the LC-PUFA content of the edible tissues, produce more nutritious seafood while minimizing the reliance on fish oil as a feedstuff. Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 Increasing demand for nutrient-dense, industrially com- et al. 2008a, 2011a, 2011c, 2013a; Fountoulaki et al. 2009; pounded aquafeeds has led aquaculture to a near monopoly Geurden et al. 2009; Pettersson et al. 2009; Bell et al. 2010; Ng over the global fish oil supply (FAO 2012), with greater than et al. 2010; Thanuthong et al. 2011). In these studies, production 70% of the annual fish oil production being consumed by the performance was largely unaffected by lipid substitution; how- aquafeed industry. In order for aquaculture to continue grow- ever, in each case the fatty acid profile of the tissues was altered. ing, over-reliance on fish oil as a principal source of energy Specifically, fish oil sparing is typically associated with a sig- and essential fatty acids in aquafeeds must be avoided (Naylor nificant loss of 20:5(n-3) (eicosapentaenoic acid [EPA]; in the et al. 2009). Fish oil sparing using various plant-derived lipids, numerical fatty acid notation the number to the left of the colon as well as fat and oil products from the animal-rendering indus- is the number of carbon atoms in the molecule, the number im- try, has proven successful in a number of fish taxa (Caballero mediately to the right of the colon is the number of double bonds, et al. 2002; Turchini et al. 2003; Figueiredo-Silva et al. 2005; and the number after the hyphen indicates the position of the first Fonseca-Madrigal et al. 2005; Bureau et al. 2008; Trushenski double bond from the methyl end), 22:6(n-3) (docosahexaenoic

*Corresponding author: [email protected] Received May 5, 2013; accepted May 24, 2013 495 496 GAUSE AND TRUSHENSKI

acid [DHA]), and other long-chain polyunsaturated fatty acids Moreover, in the beef tallow experiment, fillets of fish fed fish (LC-PUFAs; C20 and C22 fatty acids with three or more double oil or fish oil–beef tallow blends were also equivalent in terms bonds) (Bell et al. 2001; Turchini et al. 2003; Menoyo et al. 2004; of consumer acceptance (Trushenski et al. 2011c). Thus, the use Wonnacott et al. 2004; Mourente et al. 2005; Wassef et al. 2009) of SFA- and MUFA-rich lipids in Rainbow Trout feeds appears that are abundant in marine-origin lipids but absent or present quite promising, and, based on the low cost of beef tallow (as only in trace amounts in terrestrial-origin lipids. Long-chain of April 2013, US$880/metric ton versus $2,275/metric ton for polyunsaturated fatty acids are considered valuable dietary con- fish oil [FAO 2013; USDA/AMS 2013]), fish oil sparing with stituents for their myriad of functions linked to maintaining hu- this ingredient is attractive from a feed cost perspective. How- man health and well-being (Connor 2000; Aranceta and Perez-´ ever, a number of questions regarding the use of beef tallow in Rodrigo 2012). Sparing fish oil with alternative lipids is nec- Rainbow Trout feeds remain, such as whether greater sparing or essary to ensure the economic and environmental sustainability complete replacement of fish oil with this lipid is advisable, what of the aquaculture industry; however, the associated effects on duration of finishing is needed to correct for any diet-induced fillet fatty acid composition have led some to question whether losses of LC-PUFAs, and whether these manipulations influ- the public can look to aquaculture as a source of these critical ence consumer acceptance of the resultant fillets. Accordingly, nutrients in the future (Hunter and Roberts 2000; Tocher 2009). we evaluated the production performance, tissue composition, It has been suggested that tissue fatty acid profile change and consumer acceptability of Rainbow Trout raised on grow- in fish generally follows a simple dilution model, whereby the out feeds containing graded levels of fish oil and beef tallow in tissues change following a change in fatty acid intake to reflect combination with finishing in the final weeks prior to harvest. the profile of the new diet (Jobling 2003; Robin et al. 2003). Accordingly, many have investigated finishing as a means of METHODS modifying fillet composition and augmenting LC-PUFA con- Feed formulation, manufacturing, and analysis.—Feed for- tent prior to harvest. This approach aims to conserve fish oil by mulations were based on those used by Trushenski et al. (2011c) feeding reduced or fish-oil-free feeds for most of grow out and in an experiment screening various rendered fats as alternatives restricting the use of LC-PUFA-rich feeds to the final weeks of to fish oil in Rainbow Trout feeds (Table 1). Feeds contained the production cycle in order to tailor fillet profile just prior to menhaden Brevoortia spp. fish oil (FO; 100% FO), beef tallow harvest. Numerous studies have shown the fillets of fish raised on (BT; 100% BT), or blends of these lipids (50% BT, 75% BT). All alternative-lipid-based grow-out feeds can be modified with fin- feeds contained 200 g/kg menhaden fish meal and were formu- ishing feeds (Bell et al. 2003; Glencross et al. 2003; Izquierdo lated to contain 470 g/kg protein and 140 g/kg lipid. Ingredients et al. 2005; Lane et al. 2006; Trushenski et al. 2008a, 2009; were mixed with a cutter-mixer (model CM450; Hobart Corpo- Trushenski and Boesenberg 2009); however, complete restora- ration, Troy, Ohio), pelleted in a food grinder (1.5 hp electric tion of fillet (n-3) LC-PUFAs is less commonly reported. More grinder; Cabela’s, Sydney, Nebraska) and dried at 38◦C (100◦F) recent research has demonstrated that the fatty acid compo- with a commercial-grade food dehydrator (Harvest Saver R-5A; sition of the grow-out feed and the associated “prefinishing” Commerical Dehydrator Systems, Eugene, Oregon) to approx- tissue profile can have an overriding influence on subsequent imately 93% dry matter. Feeds were stored at −20◦Cforthe profile restoration and finishing success. Specifically, medium- duration of the feeding trial. Triplicate samples of feed were chain polyunsaturated fatty acids (MC-PUFAs; C18 fatty acids analyzed to confirm proximate (Table 1) and fatty acid compo- with two or more double bonds), especially 18:2(n-6), appear sition (Table 2). Samples were lyophilized (Freezone 6; Lab- to “outcompete” LC-PUFAs for tissue deposition and inter- conco Corporation, Kansas City, Missouri) to determine mois- fere with profile restoration during finishing, whereas saturated ture content and then pulverized. Protein (LECO FP-528; LECO Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 fatty acids (SFAs; fatty acids with no double bonds) and mo- Corporation, St. Joseph, Michigan) and ash (muffle furnace, nounsaturated fatty acids (MUFAs; fatty acids with one dou- 600◦C for 4 h) content were determined for each sample. Lipid ble bond) appear to enhance LC-PUFA retention and profile content was determined gravimetrically following chloroform– restoration during finishing (Trushenski et al. 2008a, 2009). methanol extraction (Folch et al. 1957). Reserved crude lipid Many studies have demonstrated greater LC-PUFA deposition samples were subjected to acid-catalyzed transmethylation per- among fish fed SFA- and MUFA-rich feeds in comparison with formed overnight at 50◦C (Christie 1982). The resultant fatty those fed MC-PUFA-rich feeds (Laporte and Trushenski 2011; acid methyl esters (FAMEs) were separated using a gas chro- Trushenski et al. 2011a, 2011b, 2011d, 2013a, 2013b; matograph equipped with a flame-ionization detector fitted with Turchini et al. 2011; Ramezani-Fard et al. 2012; Trushenski a permanently bonded polyethylene glycol, fused silica capil- and Kanczuzewski 2013). lary column (Omegawax 250, 30 m × 0.25 mm inner diameter, In two recent trials with Rainbow Trout Oncorhynchus 0.25-µm film; Supelco, Bellefonte, Pennsylvania). The injec- mykiss, we found that hydrogenated soybean oil (rich in SFAs) tion volume was 1.0 µL, helium was the carrier gas (30 cm/s, and beef tallow (rich in SFAs and MUFAs) could spare 50% of 205◦C), and the injector temperature was 250◦C. A split injec- the dietary fish oil without impacting production performance tion technique (100:1) was used, and the temperature program or fillet LC-PUFA content (Trushenski et al. 2011a, 2011c). was as follows: 50◦C held for 2 min, increased to 220◦Cat BEEF TALLOW IN RAINBOW TROUT FEEDS 497

TABLE 1. Feed formulations and proximate composition. Feeds contained ﬁsh oil (100% FO), beef tallow (100% BT), or blends of these lipids (50% BT, 75% BT). All values represent grams per kilogram; proximate composition data are reported as least-squares means ± SE of triplicate samples.

Ingredients and composition 100% FO 50% BT 75% BT 100% BT Corn gluten meala 250.00 250.00 250.00 250.00 Menhaden ﬁsh mealb 200.00 200.00 200.00 200.00 Soybean mealc 200.00 200.00 200.00 200.00 Blood meald 50.00 50.00 50.00 50.00 Wheat bran 149.80 149.80 149.80 149.80 Menhaden ﬁsh oilb 91.00 45.50 22.75 Beef tallowd 45.50 68.25 91.00 Carboxymethyl cellulose 20.00 20.00 20.00 20.00 Dicalcium phosphate 15.00 15.00 15.00 15.00 Sodium phosphate 15.00 15.00 15.00 15.00 Choline chloride 6.00 6.00 6.00 6.00 Vitamin premixe 1.20 1.20 1.20 1.20 Stay-C (35%)f 1.00 1.00 1.00 1.00 Mineral premixg 1.00 1.00 1.00 1.00 Proximate compositionh Dry matter 939 ± 7 923 ± 18 926 ± 14 931 ± 12 Protein 477 ± 2 472 ± 2 478 ± 6 475 ± 5 Lipid 138 ± 4 142 ± 6 147 ± 5 138 ± 1 Ash 87 ± 590± 394± 395± 4

aGrain Processing Corporation, Muscatine, Iowa; 60% protein. bOmega Protein, Houston, Texas. cSeimer Enterprises, Teutopolis, Illinois; 47% protein soybean meal. dDarling International, Irving, Texas. eFormulated to contain 25.000% L-ascorbyl-2-polyphosphate, 14.000% RRR-alpha tocopheryl acetate, 13.160% vitamin K, 12.500% inositol, 12.500% nicotinic acid, 7.500% riboﬂavin, 6.250% calcium pantothenate, 2.500% pyridoxine hydrochloride, 1.250% thiamine mononitrate, 1.000% vitamin A palmitate, 0.500% cyanocobalamin, 0.450% folic acid, 0.125% biotin, and 0.010% cholecalciferol in a cellulose base. fArgent Laboratories, Redmond, Washington. gFormulated to contain 24.897% zinc oxide, 14.933% ferrous sulfate, 3.470% manganese oxide, 0.967% cupric carbonate, 0.262% potassium iodide, 0.060% sodium selenate, and 0.030% cobalt carbonate in a cellulose base. hProtein, lipid, and ash are reported as grams per kilogram dry matter.

4◦C/min, and held at 220◦C for 15 min. Individual FAME were • 100% BT + 4 weeks, 100% BT + 8 weeks, 100% identified by reference to external standards (Supelco 37 Com- BT + 12 weeks: fish were fed the 100% BT feed in ponent FAME Mix, PUFA-1, and PUFA-3; Supelco, Bellefonte, combination with 4, 8, or 12 weeks of finishing with Pennsylvania). the 100% FO feed. Experimental design.—The aforementioned feeds were used in combination to generate 10 feeding regimens to assess the ef- Rainbow Trout (initial weight, 47.0 ± 0.2 g [mean ± Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 fects of dietary fish oil–beef tallow inclusion rates and finishing- SE]; Crystal Lakes Fisheries, Ava, Missouri) were stocked period duration on production performance, tissue composition, into 30 tanks (16 fish/tank) of a recirculation system con- and consumer acceptance of Rainbow Trout. Replicate groups sisting of 190-L circular tanks with associated mechanical of fish were fed according to the following regimens (Figure 1): (sand filter, Pentair Pool Products, Sanford, North Carolina; PolyGeyser bead filter, Aquaculture Systems Technologies, • 100% FO only: fish were fed the 100% FO feed for the New Orleans, Louisiana) and biological filtration (trickle-down duration of the 31-week experiment. biofilter bed), supplemental aeration, and thermal control (Aqua • 50% BT + 4 weeks, 50% BT + 8 weeks, 50% BT + Logic, San Diego, California). Each of the feeding regimens 12 weeks: fish were fed the 50% BT feed in combina- described above were randomly assigned to triplicate tanks tion with 4, 8, or 12 weeks of finishing with the 100% (N = 3). Fish were fed assigned feeds once daily to appar- FO feed. ent satiation. Water quality was monitored throughout the trial • 75% BT + 4 weeks, 75% BT + 8 weeks, 75% BT + (temperature and dissolved oxygen measured daily, YSI 550 12 weeks: fish were fed the 75% BT feed in combina- temperature–oxygen meter, Yellow Springs Instruments, Yel- tion with 4, 8, or 12 weeks of finishing with the 100% low Springs, Ohio; other water quality parameters measured FO feed. weekly, Hach DR 2800 portable spectrophotometer, digital 498 GAUSE AND TRUSHENSKI

TABLE 2. Fatty acid composition (g/100 g fatty acid methyl esters) of feeds. Values represent least-squares means ± SE; SE < 0.1 are reported as “0.0”.

Fatty acid(s) 100% FO 50% BT 75% BT 100% BT 14:0 7.5 ± 0.0 5.3 ± 0.0 4.5 ± 0.0 3.5 ± 0.0 15:0 0.6 ± 0.0 0.6 ± 0.0 0.6 ± 0.0 0.5 ± 0.0 16:0 18.7 ± 0.0 21.5 ± 0.0 23.7 ± 0.0 25.4 ± 0.0 17:0 0.5 ± 0.0 0.8 ± 0.0 1.0 ± 0.0 1.2 ± 0.0 18:0 3.5 ± 0.0 10.1 ± 0.0 13.7 ± 0.0 17.2 ± 0.0 20:0 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 SFAsa 31.1 ± 0.0 38.6 ± 0.0 43.7 ± 0.0 48.0 ± 0.1 14:1 0.3 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 16:1(n-7) 9.5 ± 0.0 6.3 ± 0.0 4.9 ± 0.0 3.4 ± 0.0 18:1(n-7) 2.9 ± 0.0 3.2 ± 0.0 1.5 ± 0.0 1.1 ± 0.0 18:1(n-9) 9.0 ± 0.0 19.7 ± 0.0 24.1 ± 0.0 29.0 ± 0.0 20:1(n-9) 0.9 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 0.3 ± 0.0 MUFAsb 22.5 ± 0.0 29.9 ± 0.0 31.1 ± 0.0 34.1 ± 0.0 16:2(n-4) 1.1 ± 0.0 0.6 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 16:3(n-4) 1.3 ± 0.0 0.7 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 18:3(n-4) 0.4 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 18:2(n-6) 9.2 ± 0.1 9.8 ± 0.0 10.4 ± 0.0 10.6 ± 0.1 18:3(n-6) 0.4 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 20:2(n-6) 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 20:3(n-6) 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 20:4(n-6) 1.0 ± 0.0 0.6 ± 0.0 0.5 ± 0.0 0.3 ± 0.0 (n-6)c 11.0 ± 0.1 11.0 ± 0.0 11.4 ± 0.0 11.2 ± 0.1 18:3(n-3) 1.7 ± 0.0 1.3 ± 0.0 1.2 ± 0.0 1.0 ± 0.0 18:4(n-3) 2.7 ± 0.0 1.5 ± 0.0 1.0 ± 0.0 0.4 ± 0.0 20:3(n-3) 0.2 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 20:4(n-3) 1.4 ± 0.0 0.8 ± 0.0 0.5 ± 0.0 0.2 ± 0.0 20:5(n-3) 12.9 ± 0.0 7.2 ± 0.0 4.7 ± 0.0 1.9 ± 0.0 22:5(n-3) 2.3 ± 0.0 1.3 ± 0.0 0.9 ± 0.0 0.4 ± 0.0 22:6(n-3) 11.5 ± 0.0 6.7 ± 0.0 4.6 ± 0.0 2.3 ± 0.0 (n-3)d 32.6 ± 0.1 19.0 ± 0.0 12.8 ± 0.0 6.2 ± 0.0 PUFAse 46.4 ± 0.0 32.5 ± 0.0 25.2 ± 0.0 17.9 ± 0.1 LC-PUFAsf 29.5 ± 0.0 16.9 ± 0.0 11.3 ± 0.0 5.2 ± 0.0 MC-PUFAsg 14.3 ± 0.1 13.1 ± 0.0 13.0 ± 0.0 12.3 ± 0.0 (n-3):(n-6) 3.0 ± 0.1 1.8 ± 0.0 1.1 ± 0.0 0.6 ± 0.0

aSaturated fatty acids; sum of all fatty acids with no double bonds. b

Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 Monounsaturated fatty acids; sum of all fatty acids with a single double bond. cSum of all (n-6) fatty acids; includes 20:2(n-6), 20:3(n-6), and 20:4(n-6) in addition to other individually reported (n-6) fatty acids. dSum of all (n-3) fatty acids. ePolyunsaturated fatty acids; sum of all fatty acids with two or more double bonds; includes 20:2(n-6), 20:3(n-6), and 20:4(n-6) in addition to other individually reported PUFAs. f Long-chain polyunsaturated fatty acids; sum of all C20 and C22 fatty acids with three or more double bonds; includes 20:3(n-6) and 20:4(n-6) in addition to other individually reported LC-PUFAs. g Medium-chain polyunsaturated fatty acids; sum of all C18 fatty acids with two or more double bonds.

titrator, and reagents, Hach Company, Loveland, Colorado) and Illinois University Carbondale Institutional Animal Care and was maintained within ranges suitable for Rainbow Trout pro- Use Committee (Animal Care and Use Protocol #08–042). duction: temperature = 16.1 ± 0.0◦C, dissolved oxygen = Baseline sample collection.—After approximately 19 weeks 8.1 ± 0.1 mg/L, total ammonia-N = 0.1 ± 0.0 mg/L, nitrite- of culture, ﬁsh were removed from each tank for use in a sep- N = 0.1 ± 0.0 mg/L, nitrate-N = 11.6 ± 0.7 mg/L, and arate thermal tolerance experiment (data not discussed here), alkalinity = 144 ± 4 mg/L as CaCO3. All husbandry prac- for collection of baseline tissue samples, and to reduce stocking tices, as well as the sampling procedures described below, were rates and cull underperforming individuals. Three representative conducted with the guidance and approval of the Southern ﬁsh per tank were transferred to another recirculation system for BEEF TALLOW IN RAINBOW TROUT FEEDS 499

FIGURE 1. Schematic illustrating experimental design and sampling schedule.

the thermal tolerance experiment. Three additional representa- any treatment. Remaining fish were fed for an additional 12 tive fish per tank were collected, euthanized via an overdose of weeks according to the feeding regimens previously described MS-222 (tricaine methanesulfonate, Finquel, Argent Chemical (Figure 1). Laboratories, Redmond, Washington; >200 mg/L for 10 min Harvest sample collection.—After 31 consecutive weeks, the after cessation of opercular movement), individually weighed, remaining fish were collected from each tank and euthanized in a and dissected to remove samples of fillet, liver, intraperitoneal salted ice water slurry. Each fish was individually weighed and fat, gill, brain, and eye tissues for determination of fatty acid dissected. Boneless, skinless, J-cut fillets, livers, and visceral profiles (see below). All samples were kept frozen at −80◦C masses were removed from each fish to determine dress out, until analysis. Additional fish were then removed to reduce hepatosomatic index (HSI), and viscerosomatic index (VSI), stocking rates to six fish per tank to accommodate fish growth and prevent overcrowding for the remainder of the trial. Small, subordinate fish were selectively removed in the course of this Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 culling event. Most of these fish were not effectively competing for feed and had gained less than 200% of their stocking weight at the time of baseline sampling (weight gain of representative baseline fish was greater than 600% at this time point) (Fig- ure 2). Although removal of underperforming fish artificially improved production performance metrics, maintaining these individuals in the population would have resulted in artificially depressed production performance. Further, we were concerned that samples collected from these individuals would not fully represent dietary effects on tissue fatty acid profiles. The number of culled fish exhibiting less than 100% weight gain or less than 200% weight gain did not vary significantly among dietary treatments, indicating that there was no effect of grow-out feed FIGURE 2. Weight distribution of Rainbow Trout removed at the baseline on the occurrence of small, subordinate fish and the culling time point for collection of tissue samples (black bars) and culled to remove did not disproportionately influence production performance in underperforming individuals and generally reduce stocking rates (white bars). 500 GAUSE AND TRUSHENSKI

respectively (see below). Tissue samples were collected as be- where Pij is the mean percent content of fatty acid “i”inthe fore for analysis of fatty acid profile. Remaining fillets were control treatment (100% FO only) and Pih is the mean percent stored separately at −80◦C for use in a taste test (see below). content of fatty acid “i” in an experimental treatment. The total Production performance.—Standard production perfor- number of fatty acids included in each calculation (n)varied mance metrics were calculated according to the following for- among tissue types, but, in all cases, only major individual fatty mulas: acids (>1% of total quantified FAME; no fatty acid groupings, e.g., SFA, MUFA) were used in the calculation of Djh. Weight gain (%) = 100 average individual final weight − average individual initial weight Fillet color and taste test.—Whole frozen fillets were re- × , ∼ ◦ average individual initial weight moved from storage, placed in a refrigerator ( 4 C), and al- average individual feed consumption (dry matter) lowed to thaw for 24 h. Color analysis was conducted on Feed conversion ratio (FCR) = , average individual weight gain whole, thawed fillets using a Hunterlab MiniScan EZ colorime- ter (Hunter Associates Laboratory, Reston, Virginia), which Specific growth rate (SGR; % body weight [BW]/d) = 100 quantifies color in terms of lightness (L*; white: 100, black: − × loge (final weight) loge(initial weight) , 0), red–green (a*; green: a* < 0, red: a* > 0), and yellow–blue days of feeding (b*; blue: b* < 0, yellow: b* > 0) parameters. Fillets were then Feed intake (%BW/d) = 100 portioned into pieces approximately 4 cm × 4cm × 2cm / × 0.5 × × × total dry matter intake (initial individual weight final individual weight) , (length width thickness) and stored refrigerated until the days of feeding taste test 2 d later. Immediately before the taste test, samples fillet weight were pan seared in neutral-flavored cooking oil and then baked Dress out (%) = 100 × , ◦ ◦ BW in a convection oven at 177 C (350 F) until portions reached liver weight ◦ ◦ HSI = 100 × , and an internal temperature of 63 C (145 F). Cooked portions were BW transferred to numerically labeled disposable plates and assem- total viscera weight VSI = 100 × . BW bled into three-portion groups for standard triangle tests (see below). Average individual values were calculated by dividing tank val- To simplify the taste test and accommodate a relatively small ues by the number of fish in the tank at the time; parameters number of panelists, we decided to evaluate only one experi- associated with feed consumption were based on average indi- mental group in comparison with the 100% FO only control vidual values calculated on a daily basis (i.e., average consump- group. We chose to evaluate fillets from the experimental group tion values were calculated daily and summed over the course that consumed the least amount of fish oil but had levels of of the trial). Feed consumption data were used, along with di- (n-3) LC-PUFAs equivalent to fillets from the control group: etary composition and feedstuff pricing information (FAO 2013; based on feeding data, dietary fish oil content, and fillet fatty USDA/AMS 2013) to determine cumulative intake of fish oil acid profile, the experimental group selected for the evaluation and beef tallow as well as “lipid feeding costs” (average costs was 100% BT + 12 weeks. The taste test was conducted as incurred as a result of cumulative fish oil and beef tallow intake part of a presentation, “Consumer Acceptance of Sustainable over the course of the trial expressed as dollars per fish). Fish,” given at the Foodservice Educators Learning Community Tissue fatty acid profile analysis.—Fillet samples were Annual Summit (March 31, 2012; Charlotte, North Carolina). lyophilized, pulverized, and extracted in the same manner as Sixteen participants, including 11 males and 5 females com- described above for feed samples. All other tissue samples were prising a broad age range (individuals in their 20s, 30s, 40s, and 50s), attended the presentation and participated in the taste test.

Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 homogenized directly in solvent (Power Gen 1000 homogenizer; Fisher Scientific, Pittsburgh, Pennsylvania) before lipid Before starting the taste test, participants were given verbal and extraction. Resultant crude lipid samples were transmethylated written instructions regarding the purpose of the test and the and analyzed as described for feeds. Following fatty acid pro- general concept of a triangle test and signed required consent file analysis, milligrams of EPA + DHA per 85-g fillet portion forms. Participants were then provided with water and specific (3 oz.) were calculated for each treatment based on mean fillet instructions (i.e., waiting a few minutes between tests, cleans- dry matter and lipid content, fatty acid composition, and a fatty ing their palates with water between samples, how to fill in the acid–lipid mass conversion factor of 0.93 g fatty acids/g lipid questionnaires, etc.) and presented with the fish portions for (Weihrauch et al. 1977). Coefficient of distance (Djh) values their triangle test. All taste test methods, including the detailed (Turchini et al. 2006), comparing overall fatty acid profiles be- procedures described below, were conducted according to the tween the experimental groups and the 100% FO only group, standards of the Southern Illinois University Human Subjects were calculated for each tissue type as follows: Committee under protocol #09590. Each triangle test was designed to compare fillets from the / + n 1 2 100% BT 12 weeks group with fillets of fish from the 100% Djh = (Pij − Pih)2 , FO only control group. Thus, the 100% FO only was randomly i−1 assigned to one portion or two portions within each triangle BEEF TALLOW IN RAINBOW TROUT FEEDS 501

test and the 100% BT + 12 weeks treatment was assigned to replicates; mean values are presented for illustration purposes the other portion(s). For each triangle test, participants were only. asked to taste each portion (retasting was allowed) and then fill out a short questionnaire, asking which of the portions was different. In addition, participants were asked to complete a RESULTS survey asking their willingness on a scale of 1–5 (1 = not willing Production performance did not vary among the dietary treat- at all; 5 = very willing) to pay a higher price for fish that were ment groups after 31 weeks of culture: survival = 91.5 ± 1.3% considered sustainably raised (those that come from sources, (grand mean ± SE; range = 85.4–97.9%), final weight = farmed or wild, that can maintain or increase production in the 587.7 ± 13.1 g (range = 503.5–642.8 g), weight gain = long term without jeopardizing the affected ecosystems) or local 1,151 ± 30% (range = 986–1,277%), FCR = 1.50 ± 0.03 or regional fish (those that are available 644 km [400 mi] or less (range = 1.39–1.69), SGR = 1.15 ± 0.01% BW/d (range = from their origin). They were also asked if they were willing to 1.08–1.19% BW/d), feed intake = 2.19 ± 0.02% BW/d pay a premium for (n-3)-enriched foods including fish and, if (range = 2.13–2.30% BW/d), dress out = 27.9 ± 0.5% so, how large a premium they would accept ($0.50–2.00/lb). (range = 26.4–30.0%), HSI = 1.06 ± 0.02 (range = 0.96– Statistical analysis.—Production performance and tissue 1.20), and VSI = 12.6 ± 0.3 (range = 11.5–13.7). These re- fatty acid data were analyzed using one-way analysis of variance sults were consistent with previous observations of Rainbow (ANOVA) with post hoc Tukey’s honestly significant difference Trout performance in our laboratory (Table 3). Stocking densi- pairwise comparison tests for parameters exhibiting significant ties varied over the course of the experiment as fish grew and treatment effects. These procedures were completed using the were culled or removed to collect baseline tissue samples. Ini- PROC GLIMMIX function of SAS 9.2 (SAS Institute, Cary, tial stocking densities ranged from 3.8 to 4.1 kg/m3 (mean ± North Carolina). In all cases, replicate tanks served as experi- SD, 4.0 ± 0.1 kg/m3); after baseline sampling and culling, den- mental units (N = 3 for control group, N = 9 for experimental sities ranged from 6.3 to 13.8 kg/m3 (approximation based on groups for data collected at 19 weeks; N = 3 for all groups size of sampled, not culled, fish; 10.5 ± 1.7 kg/m3); harvest for data collected at 31 weeks) and differences were considered densities ranged from 11.6 to 22.8 kg/m3 (17.7 ± 2.6 kg/m3). significant at P < 0.05. Coefficient of distance, taste test data, Significant differences in cumulative fish oil versus beef tallow and mean fillet EPA + DHA content values were not subjected consumption were observed, and these differences gave rise to formal statistical analysis because of insufficient numbers of to differences in oil feeding costs, indicating all experimental

TABLE 3. Production performance and fillet characteristics of Rainbow Trout fed diets containing different levels of beef tallow in conjunction with finishing periods of different durations. Values represent least-squares means; pooled standard error (PSE) and P-values are also reported. Means with common letter labels are not significantly different (P > 0.05).

50% BT 75% BT 100% BT

100% +12 +8 +4 +12 +8 +4 +12 +8 +4 Parameter FO only weeks weeks weeks weeks weeks weeks weeks weeks weeks PSE P-value Survival (%) 91.7 95.8 97.9 89.6 89.6 89.6 91.7 85.4 91.7 91.7 6.3 0.7812 Initial weight (g) 47.4 46.8 47.5 47.6 46.4 47.9 46.4 46.7 47.3 46.2 1.0 0.6561 Final weight (g) 583.9 554.3 604.0 638.0 603.3 571.3 503.5 642.8 605.2 570.8 59.0 0.4941 Weight gain (%) 1,134 1,085 1,174 1,241 1,201 1,091 986 1,277 1,184 1,137 137 0.6549 FCR 1.5 1.5 1.5 1.4 1.4 1.6 1.7 1.4 1.5 1.5 0.1 0.7051 Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 SGR (% BW/d) 1.1 1.1 1.2 1.2 1.2 1.1 1.1 1.2 1.2 1.1 0.0 0.6081 Feed intake (% BW/d) 2.1 2.2 2.2 2.2 2.1 2.2 2.3 2.2 2.2 2.2 0.1 0.9480 Total fish oil 70.7 z 55.6 y 51.8 y 46.7 xy 50.0 xy 40.1 x 27.3 w 46.7 xy 28.2 w 13.8 v 3.0 <0.0001 consumption (g/fish) Total beef tallow 0.0 u 13.7 y 21.8 w 29.8 x 21.4 w 33.4 x 42.7 y 29.0 x 44.4 y 56.0 z 1.6 <0.0001 consumption (g/fish) Oil feeding cost ($/fish) 0.16 z 0.14 yz 0.14 yz 0.13 y 0.13 y 0.12 xy 0.10 wx 0.13 y 0.10 wx 0.08 w 0.01 <0.0001 HSI 1.0 1.0 1.0 1.0 1.1 1.1 1.0 1.1 1.1 1.2 0.1 0.5135 VSI 12.8 11.8 13.1 13.0 12.0 11.5 12.0 13.7 13.1 12.7 1.2 0.7037 Dress out (%) 27.6 27.6 26.2 30.0 29.9 26.7 27.6 26.4 28.1 28.8 2.5 0.7979 Fillet characteristics 17.8 12.8 14.0 13.9 16.6 14.2 15.4 12.1 15.8 13.6 2.8 0.6518 Lipid (%) Dry matter (%) 27.5 27.7 27.6 28.2 30.2 28.4 25.5 27.3 28.5 29.4 2.9 0.9167 Fillet pH 6.6 6.6 6.6 6.7 6.7 6.6 6.6 6.6 6.6 6.6 0.1 0.7273 L* 49.8 48.2 49.9 50.3 48.7 49.4 49.1 48.4 47.6 50.4 2.3 0.9516 a* 13.8 14.4 14.3 15.0 12.0 13.4 11.3 13.1 12.5 12.9 2.0 0.7134 b* 31.9 31.5 34.7 35.0 32.2 32.8 28.0 30.3 28.7 32.6 2.8 0.2754 EPA + DHA/3 oz. 774 605 630 545 610 536 500 514 545 354 portion (mg)a aStatistical analysis was not performed as only one data point was generated for each treatment. 502 GAUSE AND TRUSHENSKI

TABLE 4. Fatty acid composition (g/100 g fatty acid methyl esters) of baseline Rainbow Trout fillet samples collected after 19 weeks. Values represent least- squares means of major fatty acids (≥1% of total fatty acid methyl esters); pooled standard error (PSE) and P-values are also reported. Where PSE values varied among means because of unequal replication among treatment groups, ranges are reported; PSE < 0.1 are reported as “0.0”. Means with common letter labels are not significantly different (P > 0.05). See Table 2 footnotes for fatty acid definitions.

Fatty acid(s) 100% FO 50% BT 75% BT 100% BT PSE P-value 14:0 5.8 z 4.5 y 3.7 x 3.1 w 0.1 <0.0001 16:0 21.4 x 22.6 y 23.0 y 23.7 z 0.2–0.3 <0.0001 18:0 4.9 w 7.5 x 8.5 y 9.6 z 0.1–0.2 <0.0001 SFAs 32.1 x 34.6 y 35.2 y 36.3 z 0.3–0.4 <0.0001 16:1(n-7) 8.2 z 6.5 y 5.5 x 4.9 w 0.1 <0.0001 18:1(n-7) 3.5 z 3.4 z 3.5 z 2.7 y 0.1 <0.0001 18:1(n-9) 13.1 w 23.3 x 28.0 y 34.2 z 0.4–0.6 <0.0001 20:1(n-9) 1.1 x 1.4 y 1.5 y 1.8 z 0.1 <0.0001 MUFAs 26.0 w 34.6 x 38.5 y 43.6 z 0.1 <0.0001 18:2(n-6) 8.6 9.0 9.1 8.9 0.1–0.2 0.1901 (n-6) 10.5 10.7 10.8 10.9 0.1–0.2 0.2284 18:3(n-3) 1.3 z 1.0 y 0.8 x 0.6 w 0.0 <0.0001 18:4(n-3) 1.5 z 0.9 y 0.6 x 0.3 w 0.0 <0.0001 20:4(n-3) 1.4 z 0.8 y 0.5 x 0.2 w 0.0 <0.0001 20:5(n-3) 7.1 z 3.9 y 2.5 x 1.2 w 0.1–0.2 <0.0001 22:5(n-3) 2.5 z 1.4 y 0.9 x 0.4 w 0.0 <0.0001 22:6(n-3) 17.7 z 12.2 y 10.2 x 6.4 w 0.4–0.5 <0.0001 (n-3) 31.5 z 20.1 y 15.5 x 9.2 w 0.5–0.7 <0.0001 PUFAs 42.0 z 30.8 y 26.3 x 20.1 w 0.6–0.8 <0.0001 LC-PUFAs 30.0 z 19.4 y 15.3 x 9.6 w 0.5–0.7 <0.0001 MC-PUFAs 11.4 z 10.9 yz 10.4 y 9.8 x 0.2 <0.0001 (n-3):(n-6) 3.0 z 1.9 y 1.4 x 0.8 w 0.0–0.1 <0.0001

regimens ($0.08–0.13/fish) offered significant cost savings version of this article). In general, finishing reversed the accu- over the 100% FO only regimen ($0.16/fish) except 50% mulation of MUFAs and loss of LC-PUFAs observed among BT + 8 weeks and 50% BT + 12 weeks (both $0.14/fish) fish fed the beef-tallow-based feeds, though complete profile (Table 3). restoration was not observed for all tissue types in all treatment After 19 weeks, tissue fatty acid profiles largely reflected groups (Table 6, Supplementary Tables 6–10). The most overt fatty acid intake (Table 4, Supplementary Tables 1–5 in the on- differences and profile changes were observed among fish in the line version of this article), with tissues of fish fed less fish 100% BT + 12 weeks regimen (Figure 3). Tissue samples var- oil and more beef tallow generally containing higher levels of ied in terms of their response to dietary manipulation; however, MUFAs, particularly 18:1(n-9), and lower levels of LC-PUFAs, the plasticity rankings were not the same as those observed at Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 particularly 20:5(n-3) and 22:6(n-3), though statistically signifi- 19 weeks: maximum Djh values for eye (22.9) and intraperi- cant differences were not noted at each level of fish oil sparing in toneal fat (26.2) tissues remained high, whereas values for gill all tissue types. These general trends were observed in all of the (11.0) and fillet (16.7) tissues were more consistent with the tissues; however, some appeared to be more strongly influenced lower values observed for liver (8.0) and brain (11.1) tissues by dietary fatty acid profile than others: maximum Djh values (Table 5). However, in all cases, tissue fatty acid profiles varied were higher for the gill (23.1), fillet (25.7), intraperitoneal fat primarily as a function of cumulative fish oil consumption (Fig- (26.1), and eye (26.2) tissues, suggesting a higher degree of ure 4): fish that consumed greater amounts of fish oil had tissue compositional plasticity, whereas brain (11.2) and liver (16.3) profiles that were more similar to those of fish fed the 100% were apparently less affected by diet (Table 5). FO feed exclusively, regardless of whether fish oil consumption After 31 weeks, the tissues continued to reflect grow-out occurred primarily during grow out or finishing. Fillet levels of feed composition to a certain degree; however, tissue fatty acid EPA and DHA also reflected cumulative fish oil consumption, profiles increasingly reflected the composition of the 100% FO ranging from 354 mg EPA + DHA/portion among fish fed the feed, particularly among groups that were finished for longer pe- least amount of fish oil to 774 mg EPA + DHA/portion among riods of time (Table 6, Supplementary Tables 6–10 in the online those fed the most (Table 3). BEEF TALLOW IN RAINBOW TROUT FEEDS 503 Downloaded by [Department Of Fisheries] at 22:46 29 August 2013

FIGURE 3. Fatty acid composition of harvest (A) ﬁllet, (B) brain, (C) liver, (D) eye, (E) gill, and (F) intraperitoneal fat samples collected from Rainbow Trout in the 100% BT + 12 weeks regimen after 31 weeks. Data are expressed as a fraction of tissue fatty acid composition of ﬁsh in the 100% FO only treatment group. Values were calculated from relative fatty acid methyl ester composition (% FAME) as% FAME100% BT + 12 weeks /FAME100% FO only. Based on this calculation, a value of 1 represents equality between the treatments (indicated by the grey line). For purposes of comparison, corresponding data generated from baseline tissue samples are also shown (dashed line). 504 GAUSE AND TRUSHENSKI

TABLE 5. Coefﬁcient of distance values for Rainbow Trout tissues collected after 19 weeks (baseline samples) or 31 weeks (harvest samples). Values were calculated using mean fatty acid compositional data, thus statistical analysis was not performed as only one data point was generated for each treatment.

50% BT 75% BT 100% BT 100% +12 +8 +4 +12 +8 +4 +12 +8 +4 Tissues FO only weeks weeks weeks weeks weeks weeks weeks weeks weeks Baseline Fillet 0.0 12.6 18.2 25.7 Brain 0.0 6.2 9.0 11.2 Liver 0.0 6.3 9.2 16.3 Eye 0.0 13.7 20.0 26.2 Gill 0.0 11.2 17.0 23.1 Intraperitoneal fat 0.0 14.0 19.6 26.1 Harvest Fillet 0.0 2.0 4.5 6.3 7.7 7.9 9.2 4.8 10.5 16.7 Brain 0.0 4.0 4.8 5.2 5.7 5.7 7.2 6.7 8.1 11.1 Liver 0.0 1.8 2.8 4.4 2.9 4.1 2.3 1.1 4.0 8.0 Eye 0.0 5.0 8.6 10.9 10.8 12.8 17.1 11.3 17.6 22.9 Gill 0.0 2.2 3.4 5.4 5.1 5.8 9.4 4.5 7.2 11.0 Intraperitoneal fat 0.0 6.3 9.9 12.1 12.9 13.4 20.0 11.9 19.4 26.2

Fillet pH (6.62 ± 0.01 [grand mean ± SE], range = 6.56– majority of panelists (69%) participating in the taste test were 6.69) and color (L* = 49.2 ± 0.4, range = 47.6–50.4; a* = unable to differentiate between portions from the 100% FO only 13.3 ± 0.4, range = 11.3–15.0; b* = 31.8 ± 0.6, range = and 100% BT + 12 weeks groups, indicating that ﬁllet taste and 28.0–35.0) were unaffected by dietary treatment (Table 3). A acceptability are not likely to be affected by the dietary regimens

TABLE 6. Fatty acid composition (g/100 g fatty acid methyl esters) of harvest Rainbow Trout fillet samples collected after 31 weeks. Values represent least- squares means of major fatty acids (≥1% of total fatty acid methyl esters); pooled standard error (PSE) and P-values are also reported; PSE < 0.1 are reported as “0.0”. Means with common letter labels are not significantly different (P > 0.05). See Table 2 footnotes for fatty acid definitions.

50% BT 75% BT 100% BT

Fatty 100% +12 +8 +4 +12 +8 +4 +12 +8 +4 acid(s) FO only weeks weeks weeks weeks weeks weeks weeks weeks weeks PSE P-value

14:0 5.4 z 5.0 yz 4.2 xy 4.2 xy 4.5 xyz 4.0 wxy 3.7 wx 4.2 xy 3.8 xw 3.0 x 0.3 <0.0001 16:0 23.3 22.7 22.4 23.3 23.2 22.9 22.5 23.2 22.5 22.8 0.6 0.6575 18:0 5.8 w 6.0 w 6.8 wxy 7.3 xy 7.0 xy 7.2 xy 7.5 y 6.6 wx 7.5 xy 8.7 z 0.3 <0.0001 SFAs 34.6 33.7 33.3 34.8 34.6 34.1 33.8 34.0 33.7 34.5 0.7 0.5181 16:1(n-7) 8.1 z 7.3 yz 6.6 xy 6.5 xy 7.1 xyz 6.4 xy 5.9 wx 6.4 xy 6.0 wxy 5.1 w 0.4 <0.0001 Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 18:1(n-7) 3.6 z 3.6 yz 3.5 yz 3.5 yz 3.6 yz 3.6 yz 3.5 yz 3.4 yz 3.3 yz 3.2 y 0.1 0.0298 18:1(n-9) 16.1 w 16.4 w 19.5 wx 21.5 xy 22.7 xy 23.1 xy 24.1 xy 20.1 wx 25.4 y 30.6 z 1.4 <0.0001 20:1(n-9) 1.4 yz 1.3 y 1.4 y 1.5 yz 1.6 yz 1.5 yz 1.4 y 1.5 yz 1.5 yz 1.8 z 0.1 0.0164 MUFAs 29.3 w 28.6 w 30.8 wx 33.0 wxy 35.0 xy 34.6 xy 35.0 xy 31.4 wxy 36.2 yz 40.8 z 1.5 <0.0001 18:2(n-6) 8.6 8.8 8.5 8.3 8.8 8.6 9.3 8.2 8.9 8.9 0.3 0.0983 (n-6) 10.5 10.8 10.6 10.4 10.7 10.6 11.4 10.5 11.2 11.3 0.3 0.1057 18:3(n-3) 1.2 z 1.1 yz 1.0 xyz 0.9 x 1.0 xy 0.9 x 0.9 x 0.9 x 0.9 x 0.7 w 0.0 <0.0001 18:4(n-3) 1.2 z 1.0 yz 0.9 xy 0.7 x 0.8 xy 0.7 x 0.7 wx 0.8 xy 0.7 x 0.4 w 0.1 <0.0001 20:4(n-3) 1.2 z 1.1 yz 1.0 xyz 0.9 xy 0.9 x 0.8 x 0.7 x 0.9 xy 0.7 x 0.4 w 0.1 <0.0001 20:5(n-3) 5.4 z 5.4 z 4.5 yz 3.8 y 3.7 y 3.8 y 3.3 y 4.5 yz 3.5 y 2.1 x 0.3 <0.0001 22:5(n-3) 2.1 z 2.1 z 1.8 yz 1.5 xy 1.5 xy 1.5 xy 1.4 xy 1.7 xyz 1.3 x 0.8 w 0.1 <0.0001 22:6(n-3) 14.6 yz 16.2 z 16.1 z 13.8 yz 11.7 xy 13.0 xyz 12.8 xyz 15.2 yz 11.8 xy 9.1 x 1.2 0.0002 (n-3) 25.6 yz 27.0 z 25.2 xyz 21.7 wxyz 19.6 w 20.8 wxy 19.9 wx 24.1 wxyz 18.9 vw 13.5 v 1.5 <0.0001 PUFA 36.2 yz 37.8 z 35.9 xyz 32.2 wxyz 30.4 vwx 31.3 wxy 31.2 wxy 34.6 wxyz 30.1 vw 24.7 v 1.6 <0.0001 LC-PUFAs 24.7 yz 26.2 z 24.9 yz 21.6 xyz 19.2 wx 20.5 xy 19.8 xy 24.1 xyz 19.0 wx 14.1 w 1.5 <0.0001 MC-PUFAs 10.9 11.0 10.4 10.0 10.6 10.2 10.9 9.9 10.5 10.0 0.4 0.1018 (n-3):(n-6) 2.4 z 2.5 z 2.4 yz 2.1 wxyz 1.8 wxy 2.0 wxyz 1.8 vwx 2.3 xyz 1.7 vw 1.2 v 0.2 <0.0001 BEEF TALLOW IN RAINBOW TROUT FEEDS 505 Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 FIGURE 4. Relationships between Rainbow Trout cumulative ﬁsh oil consumption and Djh values by tissue type. Solid lines represent linear regressions; regression equations and r2 values are provided.

we evaluated. Panelists indicated that they were willing to pay DISCUSSION higher prices for fish that were considered “sustainable” or from In the present work, Rainbow Trout grew well and efficiently, “local or regional” sources. All 16 panelists responded with a regardless of dietary lipid source or finishing-period duration. value of “3” (on a scale of 1–5) or higher (seven panelists Thus, it appears that in feed formulations similar to the one responded with “5”) when asked their willingness to pay higher we used beef tallow may be used to partially or completely re- prices for sustainably raised fish. Fourteen of the 16 panelists place fish oil without affecting the production performance of responded with a “3” or higher (five panelists responded with Rainbow Trout. Rainbow Trout have been successfully reared “5”) when asked their willingness to pay a higher price for on diets containing fish, soybean, rapeseed (canola), linseed locally raised seafood. Half of the respondents (8 out of 16) (flaxseed), palm, olive, cottonseed, coconut, and sunflower oils indicated that they would be willing to pay a $1.00/lb premium and blends or derivatives of these oils (e.g., partially or fully for (n-3)-enriched foods, including Rainbow Trout. hydrogenated oils), as well as beef tallow, pork lard, poultry fat, 506 GAUSE AND TRUSHENSKI

and restaurant grease, without significant growth impairment, accurate in predicting lipid digestibility in these other taxa (Hua though some variation in growth efficiency has been reported and Bureau 2009). Collectively, these results suggest that SFAs (Yu et al. 1977a, 1977b; Greene and Selivonchick 1990; Ca- are poorly utilized by Rainbow Trout, particularly at colder wa- ballero et al. 2002; Fonseca-Madrigal et al. 2005; Richard et al. ter temperatures. However, several groups have reported little to 2006; Drew et al. 2007; Bureau et al. 2008; Geurden et al. 2009; no effect of sparing fish oil in Rainbow Trout feeds with various Pettersson et al. 2009; Turchini and Francis 2009; Ng et al. alternative lipids, including SFA- and MUFA-rich lipids such as 2010; Thanuthong et al. 2011; Trushenski et al. 2011a, 2011c). pork lard, beef tallow, and hydrogenated fats (Yu et al. 1977b; Similar results have been reported in studies of fish oil sparing Bureau et al. 2008; Trushenski et al. 2011a, 2011c). In fact, in other salmonid fishes (Guillou et al. 1995; Bell et al. 2001, negative effects of fish oil sparing with beef tallow and other 2002, 2003, 2010; Turchini et al. 2003). Taken together, these SFA-rich rendered animal fats have been reported only rarely results are consistent with the significant body of literature on for Rainbow Trout or various other cold-, cool-, or warmwa- fish oil sparing in aquaculture feeds (see Turchini et al. 2011) inter fishes (Trushenski and Lochmann 2009). Further, Reinitz dicating that fish growth is typically unaffected by dietary lipid (1980) observed that Rainbow Trout grow equally well on diets source, so long as essential fatty acid requirements are met. containing fish oil or blends of fish oil, beef tallow, and pork Although there is some debate as to whether Rainbow Trout lard, regardless of whether the fish were cultured at 11◦Cor may perform better on diets containing intact LC-PUFAs (Lee 20◦C. Similarly, Ng et al. (2004) reported that diets contain- et al. 1967; Yu and Sinnhuber 1972; Takeuchi and Watanabe ing blends of fish oil, rapeseed oil, and crude palm oil (rich in 1976; Watanabe and Takeuchi 1976), it is widely reported that SFAs and MUFAs) were all well digested by Atlantic Salmon only 18:2(n-6) and 18:3(n-3) are truly essential for this species and yielded equivalent growth performance, regardless of crude (Castell et al. 1972a, 1972b, 1972c; NRC 2011). Our diets con- palm oil inclusion rate or declining water temperature (11–6◦C). tained at least 0.1% 18:3(n-3), 0.5% 20:5(n-3) + 22:6(n-3), and Saturated-fatty-acid-rich lipids may be less well digested than 1.2% 18:2(n-6) (dry matter basis, calculated from data reported unsaturated-fatty-acid-rich lipids, particularly at colder temper- in Tables 1 and 2 and a conversion factor of 0.93 g FAMEs/g atures; however, utilization does not appear to be so poor as to lipid), thus meeting the reported Rainbow Trout requirements significantly impair growth or justify avoiding these lipids in of 1.0% 18:3(n-3) or 0.5–1% 20:5(n-3) + 22:6(n-3) and 1.0% the formulation of Rainbow Trout feeds. 18:2(n-6) (NRC 2011). Although each of the regimens yielded Whereas changing from one dietary lipid source to another equivalent production performance, estimated production costs may not impact growth performance of fishes, one can expect associated with these feeds and feeding strategies varied based tissue fatty acid profiles to be distorted by sparing or replac- on cumulative fish oil versus beef tallow consumption: assuming fish oil with alternative fats and oils. The compositional ing current prices for fish oil and beef tallow, lipid feeding plasticity of fish tissues has been well documented in numer- costs ranged from as little as $0.08/fish for the 100% BT + 4 ous species (see Tocher 2003; Trushenski et al. 2006; Turchini weeks treatment to as much as $0.16/fish for the 100% FO only et al. 2009, 2011), including Rainbow Trout (Yu et al. 1977a; treatment. Caballero et al. 2002; Fonseca-Madrigal et al. 2005; Drew et al. Beyond the concerns of meeting essential fatty acid require- 2007; Trushenski et al. 2011a, 2011c). The restorative effects of ments, results of the present experiment suggest that diets high finishing on tissue levels of LC-PUFAs are similarly well estab- in SFAs and MUFAs are well utilized by Rainbow Trout for lished (Turchini et al. 2006; Trushenski et al. 2008a; Benedito- growth. This is contrary to early reports that suggested that Palos et al. 2009; Fountoulaki et al. 2009; Thanuthong et al. SFA-rich lipids were less well digested by salmonids than 2011). In this broad context, our results are unsurprising: re- unsaturated-fatty-acid-rich lipids (Tunison and McCay 1935; placing fish oil with beef tallow altered tissue composition in Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 Austreng et al. 1980) and therefore SFA-rich alternative lipids favor of beef-tallow-associated MUFAs at the expense of fish- are less suitable than other lipids for Rainbow Trout feeds. Gen- oil-associated LC-PUFAs, whereas finishing had a corrective erally, these authors alluded to the higher melting points of effect on these profile distortions, with longer finishing periods SFA-rich lipids as the principal cause for reduced digestibility, resulting in more comprehensive profile restoration. However, i.e., these lipids would be solid fats at typical water tempera- our results are also consistent with more recent research sup- tures for salmonid culture and would presumably be less readily porting the “omega-3 sparing effect” of alternative lipids in digested than liquid oils. Using a model based on data from which MC-PUFAs are in relatively low abundance (Trushenski Rainbow Trout and Atlantic Salmon Salmo salar fed different et al. 2008a, 2011a, 2013a, 2013b; Turchini et al. 2011; dietary lipid sources, Hua and Bureau (2009) determined that Ramezani-Fard et al. 2012; Trushenski and Kanczuzewski lipid digestibility can be predicted based on water temperature 2013). Although the purported mechanisms continue to be de- and the proportion of SFAs in the diet, with colder water temper- bated, these trials have demonstrated that alternative lipids rich atures and higher SFA levels being associated with reduced lipid in SFAs and, in some cases, MUFAs impact tissue fatty acid digestibility. The model was subsequently validated using other profiles less than alternative lipids containing MC-PUFAs, par- datasets from digestibility trials with Rainbow Trout, Atlantic ticularly 18:2(n-6). This paradigm can be explored by compar- Salmon, and various other nonsalmonid fishes and was highly ing the degree of profile modification observed when fish are BEEF TALLOW IN RAINBOW TROUT FEEDS 507

FIGURE 5. Coefficient of distance (Djh) values calculated for experimental treatment groups in the present experiment and four other experiments assessing fish oil sparing in Rainbow Trout feeds. In each case, values were calculated to compare the experimental groups with the control groups identified in each experiment. Only individual fatty acids (no fatty acid groupings) representing ≥1% of total fatty acid methyl esters were included in the calculations. The control groups varied but generally involved grow-out feeds containing fish oil as the primary lipid source. The experimental groups also varied but generally involved replacing fish oil in the grow-out feed with an alternative lipid or lipid blend as indicated by the treatment designations listed. In the present experiment, the control group was 100% FO only. In Trushenski et al. (2011a, 2011c), the control group was fed a diet containing 100% menhaden fish oil. In Caballero et al. (2002), the control group was fed a diet containing 100% Capelin Mallotus villosus oil. In Thanuthong et al. (2011) and Fonseca-Madrigal et al. (2005), the control groups were fed a diet containing 100% marine fish oil (species of origin not identified by the authors). Two experiments involved finishing periods (Trushenski et al. 2011c; present experiment), and in these cases the experimental groups were offered the fish-oil-based control feed for the number of weeks indicated by the treatment

Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 designations. As noted, two experimental groups were fed identical feed formulations and were ﬁnished for the same period of time.

fed diets containing different dietary lipid sources; in Figure 5 cant MUFA enrichment and LC-PUFA depletion were observed we have highlighted variation in diet-induced tissue modifica- in the present study, the magnitude of profile distortion we ob- tion using Djh values for several experiments, including the served using beef tallow is considerably smaller than is likely to present work investigating the effect of alternative lipids on the have occurred had we used soybean oil, canola oil, or another composition of Rainbow Trout fillets. Admittedly, these exper- MC-PUFA-rich lipid. iments involved different types of fish oil, basal dietary formu- Certain fish tissues are known to have “signature” fatty lations, trial lengths, etc. Regardless, two clear trends emerge acid profiles (Tocher and Harvie 1988; Haliloglu˘ et al. 2004; from this collection of data: (1) greater levels of fish oil sparing Trushenski et al. 2008a), which reflect lipid class composition expectedly cause greater profile distortion, and (2) MC-PUFA- and tissue-level demand for particular fatty acids, as well as rich lipids, such as soybean, canola, and sunflower oils, have a dietary intake. First, regarding the influence of lipid class com- disproportionately large effect on fillet profile compared with position, certain fatty acids are more commonly associated with SFA- and MUFA-rich lipids, such as rendered animal fats or phospholipids because of the affinity of enzymes and binding coconut, palm, and hydrogenated plant oils. Although signifi- proteins involved in phospholipid synthesis for fatty acids with 508 GAUSE AND TRUSHENSKI

specific chain lengths and degrees of unsaturation (Jolly et al. of the trial. The gill, liver, and fillet tissues did exhibit such 1998); in fish, phospholipid moieties often preferentially in- a transformation, with maximum Djh values decreasing conclude C16 and C18 fatty acids as well as LC-PUFAs (Lie and siderably between 19 and 31 weeks. Although these tissues do Lambertsen 1991; Dey et al. 1993). Accordingly, tissues that contain a considerable amount of neutral lipid that is presumably contain higher levels of phospholipids may be expected to more subject to the slower accumulative processes of new lipid depo- strongly reflect these preferred fatty acids and to be somewhat sition, they are also subject to different rates of tissue turnover less responsive to changes in dietary fatty acid composition (Mommsen 2001) that are presumably greater than that of adi- (Trushenski et al. 2008b). Conversely, tissues containing higher pose tissue, perhaps allowing for more comprehensive profile proportions of neutral lipid may be expected to be more com- remodeling. Tissue turnover is a function of synthesis and degra- positionally plastic and subject to diet-induced changes in fatty dation, and liver and gill tissues appear to outrank muscle tissue acid profile. Castledine and Buckley (1980) confirmed these as- in both (Houlihan et al. 1986, 1988). This pattern seems to be sumptions in Rainbow Trout undergoing positive or negative reflected in the magnitude of profile change observed between nutrient flux. They reported selective incorporation (fed fish) or 19 and 31 weeks in the trial, which suggests a somewhat greater retention (fasted fish) of 22:6(n-3) and other fatty acids within degree of turnover and apparent plasticity in the gill (maximum the polar lipid fractions of fish independent of dietary content Djh ∼23 →∼11) and liver (maximum Djh ∼16 →∼8) tissues but nonselective accumulation (fed fish) or catabolism (fasted compared with the fillet tissue (maximum Djh ∼26 →∼17). It fish) of consumed fatty acids within the neutral lipid fractions, is puzzling that Rainbow Trout eye tissue, which we expected to which tended to reflect dietary composition more directly. Sec- mirror brain tissue based on previous research (Brodtkorb et al. ond, regarding the influence of tissue-level demand for fatty 1997; Trushenski et al. 2012, 2013b) and the functional and acids, some fatty acids play unique structural or physiological compositional attributes of optic tissues, was most similar to the roles that may cause them to become enriched in specific tissues. intraperitoneal fat in terms of compositional plasticity. How- For example, tissues in which eicosanoids are routinely synthe- ever, our fish exhibited a significant amount of overlying ocular sized may be expected to maintain higher levels of 20:4(n-6) adipose tissue, which may have contributed to this unexpected (physiological role) (Zhou and Nilsson 2001). Also, brain and result. Stoknes et al. (2004) observed the lipid class and fatty eye tissues may be expected to contain higher levels of 22:6(n-3) acid composition of fish eyeballs to vary somewhat from that of because of its unique, biomechanical properties (Gawrisch et al. the surrounding tissues, with the surrounding tissues generally 2003) and the critical stabilizing or supportive role it appears to containing more neutral lipid and less polar lipid. It is possible play in neuronal and retinal membranes undergoing biophysi- that incomplete removal of overlying ocular adipose tissue af- cal changes during signal reception and transduction (structural fected our eye fatty acid profiles. However, excluding the eye role) (Crawford et al. 1999; Salem et al. 2001). Again, these tissues, the response of Rainbow Trout tissues to dietary manip- generalizations have been confirmed in Rainbow Trout, which ulation appears consistent with the interrelated effects of tissue typically deposit significant amounts of 20:4(n-6) in kidney tis- lipid class composition, structural and physiological demand for sue (Castell et al. 1972a) and 22:6(n-3) in eye and brain tissues fatty acids, and tissue turnover rates. (Tocher and Harvie 1988). Dietary recommendations regarding fatty acids vary, but the In this dual context of tissue-level lipid class composition consensus is that most human populations do not consume ad- and fatty acid demand, it is unsurprising that the lipid fraction equate amounts of (n-3) LC-PUFAs to ensure optimal health of Rainbow Trout brain tissue, which is comprised principally of (Aranceta and Perez-Rodrigo´ 2012). The Dietary Guidelines phospholipids and undoubtedly requires 22:6(n-3) and perhaps for Americans 2010 recommends an average daily intake of other fatty acids to maintain structural integrity and physiologi- 250 mg EPA + DHA (USDA/USDHHS 2011). Based on the Downloaded by [Department Of Fisheries] at 22:46 29 August 2013 cal function, was consistently less responsive to dietary manip- lipid content and fatty acid composition of Rainbow Trout fil- ulation than that of the other tissues (maximum Djh values ≈ 11 lets reported herein, consumers would have to eat two to five 3 at 19 and 31 weeks). Along these same lines, it is unsurprising oz. portions per week to achieve the this benchmark. Although that intraperitoneal fat tissue, which is primarily comprised of the fillets of fish fed beef-tallow-based feeds may contain less neutral lipids and is unlikely to exhibit particularly strict mem- EPA and DHA, the nutritional value of Rainbow Trout can be re- brane structural demands, became highly distorted among fish stored by augmenting fillet (n-3) LC-PUFA levels with finishing fed the beef-tallow-based feeds (maximum Djh values ≈ 26 at feeds. Although the results of the taste test are limited by sample 19 and 31 weeks). Although one might expect neutral lipids size and the absence of complete demographic information and to respond quickly to finishing and for intraperitoneal fat Djh should be considered preliminary, it is interesting that the pan- values to decline prior to harvest, one must recognize that neu- elists participating in our taste test expressed a willingness to tral lipid profile change is most likely a function of accretion, pay premium prices for (n-3)-enriched, sustainable, and locally not turnover (Castledine and Buckley 1980), and the fatty acid or regionally sourced seafood; a more statistically robust evalu- pool established within the intraperitoneal fat tissue during the ation of hybrid Striped Bass (White Bass Morone chrysops × first 19 weeks of the trial was perhaps too large to be dramat- Striped Bass M. saxatilis) yielded similar responses (Smith et al. ically affected by new lipid deposition during the final weeks 2013). This suggests a marketing advantage for farm-raised fish, BEEF TALLOW IN RAINBOW TROUT FEEDS 509

including Rainbow Trout, which can be raised close to popu- Brodtkorb, T., G. Rosenlund, and Ø. Lie. 1997. Effects of dietary levels of 20:5n- lation centers using production methods tailored to meet the 3 and 22:6n-3 on tissue lipid composition in juvenile Atlantic Salmon (Salmo consumer expectations regarding nutritional value and sustain- salar) with emphasis on brain and eye. Aquaculture Nutrition 3:175–187. Bureau, D. P., K. Hua, and A. M. Harris. 2008. The effect of dietary lipid and ability. Regardless of consumer preference and willingness to long-chain n-3 PUFA levels on growth, energy utilization, carcass quality, pay for sustainable and (n-3)-enriched foods, fish oil sparing and immune function of Rainbow Trout, Oncorhynchus mykiss. Journal of with beef tallow in Rainbow Trout grow-out feeds appears to be the World Aquaculture Society 39:1–21. an effective approach to reduce the costs of production (cutting Caballero, M. J., A. Obach, G. Rosenlund, D. Montero, M. Gisvold, and lipid feeding costs by as much as 50%) and, if combined with M. S. Izquierdo. 2002. Impact of different dietary lipid sources on growth, lipid digestibility, tissue fatty acid composition and histology of Rainbow finishing to correct for the loss of beneficial fatty acids from Trout, Oncorhynchus mykiss. Aquaculture 214:253–271. the edible tissues, produce high-quality, nutritionally valuable Castell, J. D., D. J. Lee, and R. O. Sinnhuber. 1972a. Essential fatty acids in seafood while minimizing reliance on fish oil as a feedstuff. the diet of Rainbow Trout (Salmo gairdneri): lipid metabolism and fatty acid composition. Journal of Nutrition 102:93–99. Castell, J. D., R. O. Sinnhuber, D. J. Lee, and J. H. Wales. 1972b. Essential fatty acids in the diet of Rainbow Trout (Salmo gairnderI): physiological ACKNOWLEDGMENTS symptoms of EFA deficiency. Journal of Nutrition 102:87–92. We thank Crystal Lakes Fisheries for providing the Rain- Castell, J. D., R. O. Sinnhuber, J. H. Wales, and D. J. Lee. 1972c. Essential fatty bow Trout used in this trial. We also thank Jennifer Johnson, acids in the diet of Rainbow Trout (Salmo gairdneri): growth, feed conversion Matt Young, Justin Rosenquist, Kenson Kanczuzewski, Chris and some gross deficiency symptoms. Journal of Nutrition 102:77–85. Castledine, A. J., and J. T. Buckley. 1980. Distribution and mobility of omega Bowzer, John Bowzer, Curtis Crouse, Frank Woitel, and Bon- 3 fatty acids in Rainbow Trout fed varying levels and types of dietary lipid. nie Mulligan for their assistance in tissue collection and feed- Journal of Nutrition 110:675–685. ing. We also thank Sylvia Smith, Rocky Jokbengboon, and Christie, W. W. 1982. Lipid analysis, 2nd edition. Pergamon, Oxford, UK. Aneesha Hargrave for their assistance with the taste test, as Connor, W. E. 2000. Importance of n-3 fatty acids in health and disease. Amer- well as all of our participants. 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