Iodine nutrition and toxicity in Atlantic cod (Gadus morhua) larvae

Cod larvae rearing

The experiment was performed at the Institute of Marine Research (IMR), Austevoll Research Station, Norway. This study was carried out within the Norwegian animal welfare act guidelines (code 750.000) at an approved facility. As this trial was assumed to be a nutrition trial based on all available studies up to the date of the trial, no specific permit was required under the guidelines. Naturally spawned and fertilised Atlantic cod eggs were obtained from in house second generation brood stock. Prior to incubation, eggs were disinfected with 200 mg L^{- 1} glutaraldehyde for 9 min at 6 °C and eggs were incubated with a standard protocol as described in Penglase et al. (2010). Upon 100% hatching (16 dpf, 99 day degrees), larval density in the incubators was measured via tube sampling and ranged from 2000 to 3000 larvae L⁻¹. Three days post 100% hatching (dph), larvae (65 ± 2 µg DW fish⁻¹, n = 2 where n is a pool of 428 or 520 fish, and 4.9 ± 0.2 mm fish^{- 1}, n = 30 (mean ± SD), measured 5 dph) were transferred into the experimental tanks.

Larvae were stocked at an estimated density of 50 000 larvae (120 larvae L⁻¹) in each of the nine 500 L (400 L water volume) experimental tanks (n = 3), using volumes of larvae taken from incubators based on the initial larval density measurements. The larval tanks, including colour, material, water inlets, filter size, cleaning procedures and algal additions were as described previously (Penglase et al., 2010). Water inflow to each tank (temperature 8.0 °C, salinity 34.8 ± 0.2‰, 20- µm sand/lamella filtered, degassed, from 160 m depth) started at 0.8 L min^{- 1} at larval transfer and increased over time to reach 4 L min^{- 1} by 39 dph. The water temperature in larval tanks at transfer was 8.0 °C (3 dph), and gradually increased and then maintained at 11.5 °C from 27 dph. Oxygen saturation (75–102%) and temperature were measured once daily in the outlet pipe of each tank. Dim light was provided continuously.

Rotifer culture and enrichment

Rotifers (Brachionus plicatilis. ‘Cayman’, adult lorica length 184 ± 10 µm, width 134 ± 11 µm) were batch cultured in 500 L tanks and washed as previously described (Penglase et al., 2011) (Section 2.2.5 and 2.2.6) with the exception that algae paste (Chlorella sp., Docosa, SV12, Japan) was used as the culture feed. After washing, rotifers were enriched with either a control or treatment diet. The control enrichment was 250 mg Ori-green (Skretting, Norway) million^{- 1} rotifers. The treatment enrichment (I+rotifers) was as per controls, but in addition 60 mg L^{- 1} of sodium iodide (NaI; VWR, Belgium art. no. 27915.297) was added to the water at the start of rotifer enrichment. Ori-green was prepared to manufacturer’s directions, while NaI was dissolved in cold tap water, prior to addition to rotifer enrichment tanks. Rotifers were enriched for 2 h at densities between 1000 to 2000 mL^{- 1} in water (as for rotifer culturing) with continual aeration and oxygenation (oxygen saturation was kept above 80%). After 1.5 h of this enrichment, an antibacterial (Pyceze, Novartis, Switzerland) was added to both control and treatment enrichment tanks at a rate of 0.2 ml L^{- 1}. Pyceze was used to lower rotifer bacterial numbers and thus control for any antibacterial effect of I enrichment. After enrichment, rotifers were washed, concentrated to 2000–4500 rotifers mL^{- 1}, transferred to storage tanks with aeration, and cooled rapidly (<10 min) to 8.5 °C. To maintain I concentrations in the I+rotifers, 60 mg I L^{- 1} (as NaI) was added to the treatment rotifer holding tank. Rotifers samples for element analysis were collected from rotifer storage tanks on 4 separate days during the larvae feeding trial. Rotifers were collected on 62 µm mesh, washed for 5 min with 12 °C saltwater, placed in 25 mL containers and stored at −20 °C.

Larval cod feeding trial

The feeding trial started at 4 dph, using rotifers as prepared in Section 2.2. Larvae were fed control or I+rotifers (HI+rotifers) or a mixture of both (80:20, control:I+, MI+rotifers). Each tank received increasing quantities of rotifers with time, starting from 3.5 million rotifers tank^{- 1} day^{- 1} at 4 dph increasing to 6 million rotifers tank^{- 1} day^{- 1} by 39 dph. The same quantity of rotifers was fed to each tank, and larvae were assumed to be fed to satiation. The rotifers were fed daily to larvae in two batch feedings of equal rotifer quantity at 10:00 and 15:00. Rotifers were poured gently into larval tanks in a circular motion to ensure even rotifer distribution and minimal larvae disturbance. Control larvae and HI+larvae were fed only control or treatment rotifers respectively. The MI+larvae were fed I+ and control rotifers at 10:00, and only control rotifers at 15:00 resulting in the overall feeding ration consisting of 80:20 control: I+rotifers.

For later analysis of skeletal deformities, fish were reared on identical diets from 40 to 124 dph. Larvae were co-fed Artemia (OK performance cysts, INVE, Belgium) and rotifers from 40 to 44 dph. Both Artemia and rotifers were enriched with Ori-green as per directions. Fish were fed only Artemia from 45 to 68 dph, co-fed Artemia and formulated diet (AgloNorse-EX1, Trofi, Tromsø, Norway) from 69 to 91 dph. Only formulated feed was fed from 92 dph (EX1; 92–94 dph, EX1 and 2; 95–103 dph, EX2; 104–115 dph, EX3; 116–124 dph). Formulated feed was administered continuously for 24 hrs day^{- 1} by belt feeders. Flow rate was increased from 4 L min^{- 1} at 30 dph to 8 L min^{- 1} at 108 dph, while water current speed in tanks was minimized by letting water enter through a 32 mm diameter inlet tube. Along with the increased water flow rate, oxygen saturation (64–96%) was maintained by removal of fingerling cod when required.

Sampling

Larvae were sampled for weight and length at 5, 9, 19, 30, and 124 dph, element and thyroid hormone analysis at 5, 9, 19 and 30 dph, thyroid follicle morphology at 19, 30 and 37 dph and for analysis of skeletal deformities at 124 dph. All fish were euthanised with an overdose of tricaine methane sulfonate (MS 222) upon sampling.

Larvae sampled for weight, minerals and thyroid hormones were collected on mesh (62 µm), briefly rinsed with ddH₂0, and patted dry from underneath with paper towel. Larvae were then placed in pre weighed tubes and frozen immediately in liquid nitrogen and stored at −80 °C until analysis. All tubes were then reweighed to determine sample wet weights. Tubes sampled for weight determination were thawed and larvae were counted (n = 20–100) to determine wet weight per larvae. Dry weight was determined from dry matter, which in turn was determined from tubes weighed before and after lyophilising. The standard length of the larvae was measured according to Hamre et al. (2008a) on 10 larvae tank^{- 1}. Larval densities in tanks were measured at 30 dph as described by Penglase et al. (2010).

Larvae for investigation of thyroid follicle morphology (3 fish tank^{- 1}) were selectively sampled to be similar in length, thus representing similar levels of morphological development (Sæle & Pittman, 2010). Larvae were placed in individual tubes containing 1 ml of 4% paraformaldehyde in PBS buffer at pH 7.2. Samples were left overnight and then transferred to separate tubes containing 70% ethanol, where they remained at 4 °C until embedding.

For analysis of skeletal deformities, cod juveniles (124 dph, n = 50 per tank) were measured for length and weight, frozen flat and subsequently stored in individual labelled plastic bags at −20 °C until analysis. Survival in one HI+ tank decreased to zero prior to this sampling, so data represents the mean ± SD n = 2 for the HI+fish at 124 dph.

Analytical methods

Calculations

Larval survival was adjusted using a linear individual probability timeline for each tank to calculate the probable survival of sampled larvae had they remained in the tanks until density measurements at 30 dph, using the following equation; ${\displaystyle \text{Estimated survival of sampled larvae at time point }Y=\left(\right(100-\left(\right(100-S)/T_1)\ast T_2)/100)\ast C_2}$ where S is the measured survival % at 30 dph, T₁ equals the time period in days from the start (5 dph) and end (30 dph) survival measurements (25 d), T₂ equals the number of days time point Y is from the end survival measurement and X equals the number of larvae sampled at time point Y. This equation was used to produce two numbers, one for the sampling at 9 (T₂ = 21) and one at 19 (T₂ = 11) dph, and along with the number of larvae sampled on day 30, were added to the larvae measured in tanks from density measurements taken after sampling at 30 dph. Specific growth rates (SGR) of cod larvae were calculated with the following equation SGR =  (e^∧((lnW₁−lnW₀)/(t₂−t₁))−1) × 100 where W₀ and W₁ are the initial and final dry weights (tank means) respectively, and t₂–t₁ is the time interval in days between age t₁ and t₂ (Ricker, 1958). Fulton’s condition factor (FC) was calculated using FC = Weight (g)∗100/Length (cm)³. The I concentration ratio (CR) between larvae and feed was CR = Larval I content (mg kg^{- 1} DW)/rotifer I content (mg  kg^{- 1} DW).

Data analyses

Statistica software (Statsoft Inc., 2008, Tulsa, USA, Ver.9) was used for statistical analysis of data except when GraphPad Prism (GraphPad Software, San Diego, CA, USA, Ver. 5) was used to model fit the I concentration of cod larvae versus rotifers, and on data from 124 dph, as the loss of one replicate in the HI larvae group prevented ANOVA analysis between all three groups. Data analysed with Statistica were checked for homogeneity of variances using Levene’s test before having significance tested with one-way ANOVA followed with Fisher’s least significant difference (LSD) homogeneity post-hoc test at each time point. Data with significantly different variance between treatments according to Levene’s test (p < 0.05) was log transformed before analysis. As the density of cod larvae has a large effect on growth (Koedijk et al., 2010), growth data during the larval stage was analysed with ANCOVA with the final larval density in tanks included as a continuous predictor. Data from 124 dph were analysed using regression, and tested against the null hypothesis that rotifer I content had no effect on outcome. Differences among means were considered significant at p < 0.05.

Cod larvae growth

No statistically significant differences in cod larvae length (p > 0.08) or dry weight (p > 0.25) occurred between treatments, although high variation between tanks may have masked effects (Fig. 1). On average, cod larvae grew from 4.9 ± 0.2 mm to 6.8 ± 0.5 mm in length, and 0.065 ± 0.002 mg to 0.27 ± 0.07 mg fish^{- 1} in dry weight from 5 to 30 dph (Fig. 1), representing a specific growth rate of 6.3% day^{- 1} during this period.

Survival

There were no statistical differences (p > 0.39) in the cod larval survival adjusted for sampled larvae (see Section 2.6) between treatments at 30 dph which were 28 ± 2, 39 ± 20 and 30 ± 15% for controls, MI and HI groups respectively, while the average for all groups was 32 ± 13%. Survivals based solely on densities in tanks at 30 dph without taking into account sampled larvae were 12 ± 2, 19 ± 12 and 12 ± 10% for controls, MI and HI groups respectively.

Iodine and other essential element concentrations in rotifers and cod larvae

Control rotifers contained 0.60 ± 0.33 mg I kg^{- 1} DW while HI+rotifers contained 129 ± 101 mg I kg^{- 1} DW (Table 1). Whole body I levels in cod larvae were significantly different between groups (p < 0.01, Fig. 2). Cod larvae (5 dph) had a starting concentration of 4.0 ± 0.3 mg I kg^{- 1} DW. Between 9 and 30 dph average I concentrations were 1.6 ± 0.3 mg I kg^-1 DW for control larvae, while MI larvae had 3 fold higher levels (4.9 ±2.4 mg I kg^{- 1} DW), and HI larvae 7 fold higher levels (11.0 ± 3.3 mg I kg^-1 DW) than controls. Other element concentrations were also affected by treatment in both rotifers and cod larvae. HI+rotifers contained less Fe and Mn than controls (Table 1), while HI larvae contained more Mn, Fe and Cu than controls and MI larvae at one or more time points (Figs. 2b–2c and 2e). Both HI and MI larvae contained higher levels of Co than controls (Fig. 2d), while larval Zn and Se concentrations were unaffected by treatment (Figs. 2f–2g). Rotifer macro mineral concentrations were unaffected by treatment (Table 1), but increased levels of Ca and Mg, and lower levels of P and K were observed in HI larvae in comparison to controls and/or MI larvae during the rotifer feeding period (Fig. 3).

Iodine concentration in cod larvae versus rotifers

The rate of increase in cod larvae I concentrations decreased as dietary I levels (rotifer I concentration) increased, and thus the I concentration ratio between cod larvae and rotifers displayed a negative trend (Fig. 4; p < 0.01). The age of the cod larvae did not effect their I concentration ratio (p = 0.96). The model predicts that the ratio of I in cod larvae versus rotifers equals 1 when rotifers have 3.5 mg I kg^{- 1} DW (Fig. 4).

Thyroid hormones and thyroid follicle morphology

There were no differences in thyroid hormone levels or ratios between treatments (Fig. 5). Data was normalised to aid interpretation, as T₃ results were higher than obtained previously (Penglase et al., 2010) and the high result was validated by the analysis of T₃ with a second method (see methods). The total volume of thyroid follicle was 1.3 fold lower and the total epithelium volume was 1.4 fold lower per fish in HI versus MI larvae, but not controls, at 30 dph (Figs. 6a and 6c). The thyroid follicle colloid to epithelium ratio was higher in HI larvae than controls at 19 (1.7 fold) and 37 (1.8 fold) dph, while MI larvae did not differ from controls (Fig. 6d). No statistically significant differences were observed between groups in colloid volume or total number of thyroid follicles (Figs. 6b and 6e). Images of thyroid follicle sections from control and HI larvae at 37 dph are shown in Fig. 7.

Weight, length, condition factor and rate of skeletal deformities at 124 dph

There were no significant differences in the weights (average; 2.50 ± 0.19 g), lengths (6.14 ± 0.16 cm) or condition factors (1.05 ± 0.03) between groups at 124 dph (Table 2, n = 400). Neck axis angle became closer to normal (180 ± 3 degrees) with increasing I levels in rotifers, but there were no differences in any of the other skeletal deformity measurements (Table 2).