Interactive effects of warming and microplastics on metabolism but not feeding rates of a key freshwater detritivore

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concentrations only inhibited metabolism at the highest temperatures.

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To address this critical gap in microplastic research, we experimentally tested the 112 independent and combined impacts of microplastics and warming on the energy demand 113 (metabolism) and energy intake (feeding) of an important and widely distributed freshwater 114 detritivore -the amphipod, Gammarus pulex. We hypothesised that there would be: (1) an 115 increase in metabolic and feeding rates with increasing temperature; (2) a reduction in 116 metabolic and feeding rates with increasing microplastic concentration; and (3) weaker 117 effects of microplastics on metabolic and feeding rates at higher temperatures. Gammarus pulex is a ubiquitous benthic shredder in European running waters that breaks 122 down coarse particulate organic matter, channelling the associated energy to predators such as fish. By converting terrestrial litter inputs into the fine particulate and dissolved organic 124 matter, these shredders also convey these resources to other invertebrates, especially in 125 upland streams (Wallace and Webster 1996). The species is commonly used as a model  For the feeding rate experiments, we exposed amphipods to ten concentrations of (1) 238 Here, R 0 is the metabolic or feeding rate at T 0 , M is dry body mass (mg), b R is an allometric 239 exponent, E R is the activation energy of the biochemical reactions underpinning R (eV), k is 1, exploring the main effects of temperature and body mass on metabolic or feeding rate. We 244 then mass-corrected the response variables by dividing metabolic or feeding rate by M bR .

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To determine the effect of microplastics on our mass-corrected response variables 246 (R M ), we first calculated the change in metabolic or feeding rate (∆R M ) relative to the 247 microplastic-free control treatment. We subtracted the mean mass-corrected metabolic or feeding rate in the control at each temperature from the individual replicate measurements 249 containing microplastics at the corresponding temperature. A positive value of ∆R M indicates 250 an increase, while a negative value of ∆R M indicates a decrease in metabolic or feeding rate. 251 We performed a multiple linear regression exploring the main and interactive effects of There was a significant log-linear increase in respiration rate with both body mass and 263 temperature (F 2,40 = 10.64; p = 0.001; r 2 = 0.31; Table 1), supporting our first hypothesis. The 264 respiration rate of G. pulex increased with body mass with an allometric exponent of 0.45 ± 265 0.33 (mean ± 95% CI; Figure 1a) and with temperature with an activation energy of 0.23 ± 266 0.13 eV (mean ± 95% CI; Figure 1b). There was a significant main effect of microplastic 267 concentration on respiration rate (Table 2), with a reduction in respiration rate relative to the 268 control as microplastic concentration increased (Figure 1), supporting our second hypothesis.

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There was also an interactive effect of temperature and microplastic concentration on the 270 change in respiration rate relative to the microplastic-free controls (F 3,29 = 5.73; p = 0.003; r 2 271 = 0.31; Table 2). Here, there was an increase in respiration rate relative to the controls at the 272 coolest temperature, but a decrease in respiration rate relative to the controls at both 15 and respiration rates.   on the change in amphipod metabolic rates relative to microplastic-free control (see Table 2).

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The lines of best fit show the effect of microplastic concentration on the response variable at 293 each of the three temperatures. There was a significant log-linear increase in feeding rate with both body mass and 297 temperature (F 2,120 = 11.89; p < 0.001; r 2 = 0.15; Table 1), supporting our first hypothesis.

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The feeding rate of G. pulex on leaf litter increased with body mass with an allometric 299 exponent of 0.72 ± 0.70 (mean ± 95% CI; Figure 3a) and with temperature with an activation 300 energy of 0.57 ± 0.25 eV (mean ± 95% CI; Figure 3b). There was no significant main effect    Figure 3. Body mass and temperature dependence of amphipod feeding rates. Ln feeding rate 318 increased significantly with both (a) body mass and (b) temperature (see Table 1). Note that   Figure 4. Experimental warming and microplastic concentrations had no effect on the change 324 in amphipod feeding rates relative to microplastic-free control (see Table 2). The lines of best acclimation also did not immediately lead to reduced feeding rates, suggesting either a 352 delayed response in the latter, or that feeding rate may be more directly influenced by the rate 353 of gastric digestion than oxygen consumption (Wallace 1973). There was also no change in 354 the feeding rate of G. pulex, or its congeneric G. fossarum, after exposure to microplastics,     temperature -Microplastics pollution reduces metabolic rates but not feeding rates -Experimental warming alters the effect of microplastics on metabolic rates -Increased microplastics concentrations enhance metabolism at the coolest temperature, but inhibit metabolism at the highest temperatures