I agree with the new title proposed by authors in their reply “Effects of acoustic pollution on Daphnia magna fitness: the importance of noise type.”

However, this is not the title included in the change tracking files (wich is "No evidence for an effect of chronic boat noise on the 1 fitness of reared water fleas"). I think that “Effects of acoustic pollution on Daphnia magna fitness: the importance of noise type.” should be kept.

Apart from that point, authors improved the clarity of their article, which doesn't deserve further modification.

Dear Editor,

This revised version is fully satisfying to me. I guess that the Morse running issue can be delt with independently, and doesn't deserve further consideration in the context of the present article.

For this 2nd round of review, authors have significantly taken into account the comments of the reviewers. The results description and discussion have been globally improved, and the representativity of these results have been slightly mitigated in light of the suboptimal (not to say bad) physiological condition of their daphnia used to respond to their problematic.

However, I don’t understand the choice to change the title of the article for this second round from “A freshwater zooplankton in the face to boat noise pollution” to “Chronic boat noise does not alter the fitness of Daphnia magna”

I strongly disagree with the new proposed title - I don’t consider that the main conclusion of this study can be that chronic boat noise does not alter the fitness of Daphnia magna – and this should not be the ‘take home message’ of this article. Due to the bad physiological condition of daphnia in this study (more than 50% mortality before maturity), I don’t think that authors can generalize their observation at this point, this would be an overinterpretation. This is even more reinforced by the fact that the causes of this suboptimal physiological state are not identified and controlled by the authors. To my point of view, the message that can be derived from this study is that, at similar suboptimal physiological condition, impacts on fitness differs between two scenari of noise exposure (from the current article and Prosnier, Rojas, et al. (2022)). But the lack of fitness impact of chronic boat noise on D.magna cannot be established as an absolute conclusion based on this single study.

Specific comments:

L95: mJ is not a usual metrics to describe daphnia food intake. Could you provide some (at least one) references to estimate the adequacy of this diet/mJ amount for D. magna ?

L193: “differed from previous results obtained” – to help reader, please provide the ref of the previous results you refer to.

L205: “Although noise had no statistical effect on the survival and fecundity…” – It is not clear that you refer to the current article in this beginning of sentence, as it directly follow a reference to Prosnier, Rojas, et al. (2022). Please clarify to help understanding of your argumentation.

L248 (“As a perspective”): As your data do not provide direct insight toward multi-stress, I think this paragraph isn’t a perspective of your study, but an additional interesting consideration. 

The article entitled “A freshwater zooplankton in the face to boat noise pollution”, by L. Prosnier, E. Rojas and V. Medoc focuses on an interesting and understudied topic: the consequence of chronic irregular noise exposure on Daphnia magna survival and reproduction, as proxy for fitness. This study is based on valuable individual data of daily survival and reproduction under two conditions of noise exposure.

It is well written, and the context, the objectives and working hypotheses are well explained.

The experimental design is well described, even if it could be profitable to provide picture of the device (L96). This could allow to figure out some points: are all the 18 microcosms at the same distance of the loudspeaker? Is it possible that some microcosm disturbs the noise transmission to other ones? 

There are some missing information to fully describe the methodology:

L. 88: the physicochemical parameters of the used tap water is not provided

L89: further than algae mass, it could be interesting/important to consider the amount of carbon, and thus to precise the amount of C in 0.05g of dry alguae.

L105-106: Please provide the rate of mortality that caused the need of individuals replacement. Is it a normal phenomenon? Where came from the alternative individuals? (same brood? Which raising condition until integration in the experimental device?). This is a really important point, as this could lead to a major source of variability in the data (eg : missing window of exposure in some individuals…).

Fig. 1 caption: please define “SPL”. “visuals category” should be clarified.

I am not qualified to judge the suitability of sound exposure setup and suitability of sound signal treatment.

The experimental design is really interesting, and seems suitable to provide interesting insight on chronic irregular noise exposure on Daphnia magna.

However, results are far too briefly described, and there is a lack of description of reproductive and survival parameters in control individuals – there is to few data and statistical description and details in the results section. Lethal times for 50% of individuals are represented on a scale with intervals much too large to be graphically read (fig 2a), and their numerical value isn’t given in the text. This would really help the reader to consider this major element = the survival rate of control individuals drops down to 50% within only 5 days, that is, I guess likely before the first reproductive cycle. This may explain the low total number of clutches during lifetime. 

In addition, the number of individuals considered in each condition is provided only for the “number of clutches”, were it should be provided for all parameters.

These interesting individual data of daily survival and reproduction should also be further exploited (eg: providing age at first clutch). Given that the number of surviving individuals, and thus the number of individuals able to reproduce, decreases with time, it would be interesting to represent the cumulated production of neonates per daphnia in relation to age, and to include the number of considered individuals for each clutch. This would allow to figure out the representativity of mean values for each clutch, and the evolution of clutch size during lifetime which is supposed to be observed in D. magna. 

The survival rate drops down to 50% within only 5 days, and the number and size of clutch seems really low compared to other studies (eg: Parisot et al, Aquatic Toxicology 163 (2015) 27–36). This raises major questions about the suitability of rearing conditions during the time of the experiment, and this could suggest that the considered individuals (whatever the noise exposure) are in suboptimal physiological condition. In my point of view, this should be one of the main points of discussion of the article, as this may limit, or at least open another perspective on the representativity of the results: are individuals unaffected because their physiological performances are already at their lower point? The lack of discussion on that point weaken the conclusion of the article. It is to note than this problem of physiological condition is also found on the article of the same authors (Prosnier et al 2022, non peer-reviewed preprint) that is used as the main comparison point in the discussion.

Thus, I think that this article deserve more work on results description and discussion to provide valuable insight into anthropogenic noise impact on Daphnia magna.

As indicated in its title « A freshwater zooplankton in the face to boat noise pollution », the preprint submitted by Loïc Prosnier and his collaborators to PCI-Ecotoxicology, reports on a study of the impact of anthropogenic noise to aquatic invertebrate life. Like light, noise is an emerging environmental issue of great concern, which has been sofar totally neglected in ecotoxicology. Given that such insidious pollutions are expected to amplify with the human population growth and associated activities and to combine with other stressors and pollutants,  it is urgent to start to assess their impacts to natural ecosystems. In this respect, the present report, by addressing this issue in Daphnia magna (a zooplanktonic species widely used as model in standard ecotoxicity testing) experimentally exposed to boat noises, is of strong intrinsic value.

Not being an expert in noise treatment and effects, I focus my review on general characteristics of the design, and on result analysis and discussion. My only request on noise pollution is the extent that the intensity used in the laboratory is representative or not of real levels of environmental exposure. Did the authors check for it and how did they do it ? It might also be important to recall that in the « field », noise may not be uncoupled from vibrations, which was not simulated in the experiment.

The experimental design is globally well described, yet additional details would be helpful to the reader.

Mortality is said to be corrected at the beginning, by replacing dead juveniles. Please indicate the exact period concerned by this treatment. It seems that mortality was quite high at start (50 % by day 5, whatever the treatment), which may lead to ask why not delaying exposure to day 10 or so, as it would have allowed to avoid replacing dead juveniles during the course of the experiment (which should avoided, as much as possible).

The experiment duration is not specified, and only accessible on Fig dealing with survival. It is a pity, because apparently data are not censored (traits measured until the death of all animals), which is rare enough (in ecotoxicity testing) to be mentionned. If so, this should be clearly specified.

Next, the dataset (individual-level survival and reproduction) is appropriate to the estimation of population growth (using Euler-Lotka’s equation), and is advocated to have a better idea of fitness, as fitness impact was targeted by the authors (not only fitness-related traits taken separately).

Also, it is not clear if maternal mortality was accounted for in reproduction data. I suggest using a model suited to deal with toxicity over time (as implemented in MORSE package, see  Baudrot & Charles, 2021). I am quite curious to know about the findings based on such analysis.

Results do not indicate fitness effects of the noise treatment applied. The authors consider them as contradictory to those obtained in their previous study (Prosnier et al. 2022), yet noise conditions differed (constant vs intermittent application, intensity). Thus, to me, the contradiction may only be appearant, and they authors should better explain why they view discrepancies between the two studies as contradictory.

Next, discussion should extend to development, for two reasons : development is a particularly sensitive period in an organism life and noise and it would be interesting to know if newborns were well developed, if aborted embryos occurred, etc. Even if only qualitatively noted throughout the experiment, this information is critical to assess wether fitness is affected or not.

Finally, as perspective, the authors mention  foodweb-level experiments, as a way to go further into noise effect assessment, yet I would rather suggest that functional approaches dealing with mechanoreceptors and genes potentially involved in noise perception, would be more relevant in a « non hearing » organism. At least, this would deserve mention. It is surprising that the following reference was lacking (Popper, A., Salmon, M. & Horch, K. Acoustic detection and communication by decapod crustaceans. J Comp Physiol A 187, 83–89 (2001). https://doi.org/10.1007/s003590100184/). I also believe that discussed could be enriched by considering terrestrial invertebrates, among which several groups are acutely sensitive to noise. (see e.g., Morley, E.L., Jones, G., Radford, A.N., 2014. The importance of invertebrates when considering the impacts of anthropogenic noise. Proceedings of the Royal Society B: Biological Sciences 281, 20132683. https://doi.org/10.1098/rspb.2013.2683

Another issue that might be mentionned as perspective is that noise ability to act synergistically with other stressors present in daphnid's environment could be worth testing. 

Other minor comments :

X-mesh : specify mesh-size

L71 : « Additionally, an important zooplankton predator (Chaoborus flavicans) increased anti-predatory defence behaviour when exposed ». This sentence is a little confusing. Is the behaviour observed on Chaoborus’s prey ? If yes, specify species (or organism).

L97-99. Four tanks were used, each containing 18 individual 150ml vessels. Please specify that this was per treatment (otherwise 18x4 = 72 microcosms are not sufficient to get 57+58 experimental units). Note also that the tank effect should be included as random factor in the test design. Instead of a t –test, please use a linear mixed effect model (lmer if normal, glmer if not, with lme4 package).

Fig.1. Please indicate why you measured noise only on half of the tanks.

Discussion: I am not sure to understand what "hearing vertebrates" really means.