Effects of diflubenzuron on Northern shrimps (Pandalus borealis) at ambient and future climate conditions

Increasing use of the chitin synthesis inhibitor diflubenzuron (DFB) as anti-parasitic drug in salmon aquaculture has raised concerns over its impact on marine ecosystems. We have exposed Northern shrimp (Pandalus borealis) to DFB medicated feed under current (C: 7.0°C, pH 8.0) and future global change conditions (Ocean Acidification and Warming; OAW: 9.5°C and pH 7.6). WP1. Shrimp larvae. Two weeks exposure to DFB medicated feed caused significantly increased mortality. The effect of OAW and DFB on mortality of shrimp larvae was additive; 10% mortality in C, 35% in OAW, 66% in DFB and 92% in OAW+DFB. In OAW+DFB feeding and swimming activity was reduced for stage II larvae and none of the surviving larvae developed to stage IV. Due to shorter intermoult period at elevated temperature, the OAW+DFB larvae were exposed throughout two instead of one critical pre-moult period. This can explain the more serious sub-lethal effects for OAW+DFB than DFB larvae. A single day exposure at 4 days after hatching did not affect DFB larvae, but high mortality was observed for OAW+DFB larvae, possibly because they were exposed closer to moulting when crustaceans are more sensitive to chitin synthesis inhibitors. WP2. Adult shrimp. Ovigerous shrimp were exposed to DFB medicated salmon feed for two weeks before their post-hatch moult. The shrimp were exposed to DFB under the same current and future environmental conditions as the shrimp larvae in WP1. The mortality of DFB exposed shrimp was approximately 60% higher than mortality of control shrimp, and while more than 60% of the control shrimp moulted successfully, only 2-7% of DFB exposed shrimp survived moulting. Most of the mortality occurred during moulting in the depuration period, and even after four weeks of depuration, when the mean tissue concentration was still 33 ng/g w/w, mortality was higher for DFB exposed shrimp. High mortality was observed for adult shrimp after consumption of approximately 0.1 g of DFB medicated feed. Mortality was also higher in OAW than in C, but the combined effect of DFB and OAW was less than additive. In an additional experiment, where adult shrimp without eggs were exposed to DFB medicated feed, high moult-related mortality was observed already after four days exposure. Some exposed shrimp did, however, moult successfully after two-three weeks of depuration. WP3. The transcriptome for P. borealis has been generated, and the reference transcriptome results have been deposited in the Short Read Archive data base in NCBI (National Center for Biotechnology Information). To investigate the effect of exposing shrimp to DFB and OAW+DFB on gene expression, qPCR was performed on five genes selected from the reference transcriptome. Gene expression of peroxiredoxin in DFB larvae, and GAPDH, DD9B and lipase in OAW+DFB larvae, were all significantly down regulated relative to gene expression in C larvae. Conclusions from the laboratory experiments. High mortality was observed for adult shrimp and shrimp larvae exposed to DFB medicated feed, indicating that the use of DFB as an anti-parasitic drug is a threat to benthic and planktonic crustaceans living in areas with salmon farms. The additive effect of DFB (local driver) and OAW (global drivers) on survival of shrimp larvae emphasize the importance of managing the local drivers (reducing pollution) to slow down the detrimental impact of future global environmental changes. The precautionary principle should be applied to reduce the discharge of pesticides used as anti-parasitic medicines in salmon aquaculture. A good solution for the environment would be to culture salmon in semi-closed cages, where effective protection against sea lice has been documented. WP4. A population model was run for different scenarios of timing of DFB application (spring and/or autumn) and for different percentages of the shrimp population that was affected (adults 5-50% and larvae 0.5-5%). The DFB exposure had a negative effect on modelled abundances of both larvae and adults in all scenarios, based on the effects observed on shrimp in the laboratory. Under current environmental conditions, the long-term abundance of DFB-exposed populations decreased by 10-50%, compared to the long-term abundance of the control population. The timing of the DFB treatment had consequences for both abundance and stage structure, since the different stages dominate in different seasons. Future global change conditions had in general a stronger effect on abundances than the DFB treatment: the OAW population usually had abundance below 30% of the control population. This must be considered like a severe "worst case scenario" since no adaptation to future global change conditions was considered. The combined effects of OAW and DFB treatment were generally additive, which was consistent with the individual-level effects observed in the experiments.

Increasing use of the chitin synthesis inhibiting pesticide diflubenzuron (DFB) against salmon lice (Lepeophtheirus salmonis) in marine aquaculture has raised concerns over its environmental impacts. In the proposed project we will perform laboratory expe riments to study the effects of DFB on Northern shrimp (Pandalus borealis). Predicted future climate conditions (increased temperature and ocean acidification) may indirectly and directly change the effect of DFB on shrimps. We will study the combined eff ects of DFB exposure and climate change on different life stages of shrimps in laboratory experiments and use population modelling to find out if DFB treatment of sea lice infestation can reduce local shrimp populations. In addition to this, an important aim is to develop a genetic marker for exposure to chitin synthesis inhibitors in P. borealis. The planktonic shrimp larvae will be exposed to low concentrations of resuspended DFB contaminated sediment in addition to particulates from DFB medicated pelle ts at ambient (7°C, pH 8.0) and predicted future climate conditions (10°C and pH 7.6). The selected endpoints for the larvae are mortality (especially related to moulting), deformities of the exoskeleton, development time, growth, feeding rate, respiratio n rate and swimming behaviour. In the field adult shrimps can be exposed to DFB from feeding on medicated pellets, contaminated sediments and faeces from treated fish in addition to exposure to resuspended particulates. We will expose ovigerous shrimps to DFB from contaminated sediment and from medicated pellets at ambient and future climate conditions. The main endpoints in this experiment are hatching success for shrimp embryos and post-hatch moulting for adult female shrimps, in addition to deformities of the exoskeleton, feeding rate, respiration rate and shrimp behaviour.