The Effect of Turbidity on the Behavior of Bleak (Alburnus alburnus)

Round 1

Reviewer 1 Report

the MS aims to investigate the effect of turbidity on the behavior of bleak Alburnus alburnus under experimental conditions. Although the topic is really interesting, I found the MS very confusing, and control was missed in some experiments. Starting from the conclusions, they are forced and based on statements that are not connected with the real findings of the MS.

The introduction in general is well written and only lacks literature references in the description of the bleak biology (see pdf attached for details comments).

Materials and methods are really confusing, and details about the experiments are missed (for example the duration of the experiments). It is not clear why 8 groups of 5 individuals were selected (no details about the criteria used) and if all the groups were tested at the same time. It is not clear which other parameters were measured together with turbidity although temperature is one of the parameters that was considered in the model (line 165). Oxygen demand is a crucial parameter for fish, especially under experimental conditions. It is not clear if this parameter was measured and considered during the experiment but the effect of a potential oxygen decrease (due to the addition of clay in a low level of water 10 cm) could have affected the behavior of the fish bringing them to an increasing of vertical movements (of all the individuals together with a consequent decrease of the inter-individual distance)  that could have been wrongly registered as a ''shoaling'' behaviors. 

The number of individuals that have been examined is also quite low and it is not clear the criterion used to set this minimum sample size.

Morever it is not clear if the sex of the fish was also recorded (given that the capacity of create group with individual sof the same sex is one of the main characterists of cyprinids. About this, it is not clear in which season the fish were collected.

The lack of fish SL in the model is a big problem because the difference between the fish is affected also by the fish size itself. the average size of the individuals is reported but the descriptive statistic of the SL of the sample is not clear (minimum, maximum, and standard deviation are not given). So the fish size should be considered in the analyses. The same lack is also reported for the Temperature that was measured but only the mean was given in the MS. 

Figure 1 reports the difference between the groups but it is not specified what the error bars are (SD, SE, variance?)

Discussions are not well supported by literature and the findings are not appropriately discussed.

One of the main concerns is that it should be considered that the experiment was carried on in 10 cm of water, and the addition of clay could have also affected the quantity of oxygen in the aquarium putting the fish under stress. This stress could be the cause of their change in behavior. For example, if the movement of the fish after the addition of clay was vertical it is clear that the fish were all 'looking'' for fresh and oxygenated water given that bleak is a cyprinid that has a preference for oxygenated water. Thus, also the direction of the movement (together with the fish size) after the addition of clay should be considered as a variable in the model.

     In general, it is not clear whether the protocols used were designed on purpose or where protocols are known from the literature (and tested for other species) but modified. 

For all these reasons the MS can not be accepted and should be reconsidered only after major revision (control missing in some experiments). 

Please find all the detailed comments and suggestions in the pdf attached.

Comments for author File: Comments.pdf

Moderate editing of English language required

Author Response

Thank you very much for all your comments which was very helpful and inspirative. We answer to question raised in the detailed comments and suggestions in the pdf attached.

Comments and Suggestions for Authors

the MS aims to investigate the effect of turbidity on the behavior of bleak Alburnus alburnus under experimental conditions. Although the topic is really interesting, I found the MS very confusing, and control was missed in some experiments. Starting from the conclusions, they are forced and based on statements that are not connected with the real findings of the MS.

The introduction in general is well written and only lacks literature references in the description of the bleak biology (see pdf attached for details comments).

Materials and methods are really confusing, and details about the experiments are missed (for example the duration of the experiments). It is not clear why 8 groups of 5 individuals were selected (no details about the criteria used) and if all the groups were tested at the same time. It is not clear which other parameters were measured together with turbidity although temperature is one of the parameters that was considered in the model (line 165). Oxygen demand is a crucial parameter for fish, especially under experimental conditions. It is not clear if this parameter was measured and considered during the experiment but the effect of a potential oxygen decrease (due to the addition of clay in a low level of water 10 cm) could have affected the behavior of the fish bringing them to an increasing of vertical movements (of all the individuals together with a consequent decrease of the inter-individual distance) that could have been wrongly registered as a ''shoaling'' behaviors. 

The number of individuals that have been examined is also quite low and it is not clear the criterion used to set this minimum sample size.

We followed the 3R rule, i. e. reducing the number of fish per trial to a minimum, while maintaining sufficient number of data for statistical analysis. For each group each level of turbidity was tested.

Morever it is not clear if the sex of the fish was also recorded (given that the capacity of create group with individual sof the same sex is one of the main characterists of cyprinids. About this, it is not clear in which season the fish were collected.
The sex of the fish was not recorded. The fish were collected at the end of winter.

The lack of fish SL in the model is a big problem because the difference between the fish is affected also by the fish size itself. the average size of the individuals is reported but the descriptive statistic of the SL of the sample is not clear (minimum, maximum, and standard deviation are not given). So the fish size should be considered in the analyses. The same lack is also reported for the temperature that was measured but only the mean was given in the MS. 

Both the temperature and SL were measured during each experimental trial, although unfortunately not specifically provided in the Methods section. We added this into the text. These were further used for statistical analysis.

Figure 1 reports the difference between the groups but it is not specified what the error bars are (SD, SE, variance?) These are variances.

Discussions are not well supported by literature and the findings are not appropriately discussed.One of the main concerns is that it should be considered that the experiment was carried on in 10 cm of water, and the addition of clay could have also affected the quantity of oxygen in the aquarium putting the fish under stress. This stress could be the cause of their change in behavior. For example, if the movement of the fish after the addition of clay was vertical it is clear that the fish were all 'looking'' for fresh and oxygenated water given that bleak is a cyprinid that has a preference for oxygenated water. Thus, also the direction of the movement (together with the fish size) after the addition of clay should be considered as a variable in the model.Responded in the pdf.

     In general, it is not clear whether the protocols used were designed on purpose or where protocols are known from the literature (and tested for other species) but modified. 

The protocols were mostly designed on purpose, after consultation with a statistician and person experienced with ethological protocols, but partially they were based also on protocols found in literature.

For all these reasons the MS can not be accepted and should be reconsidered only after major revision (control missing in some experiments). 

Please find all the detailed comments and suggestions in the pdf attached.

Thank you very much for all your comments. We answer to all question raised in the detailed comments and suggestions in the pdf attached.

Author Response File: Author Response.pdf

Reviewer 2 Report

See attached Reviewer Report

Comments for author File: Comments.pdf

None

Author Response

We thank the reviewer for his comments and suggestion. 

 Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The MS was revised following the suggestions and comments. The revised MS is clearer and the quality  was improved.

The MS can be considered for publication

Reviewer 2 Report

The edits and recommended changes have been addresses.