Miniaturisation of the Daphnia magna immobilisation assay for the reliabletesting of low volume samples

Review
Thanks for the update and answering the comments.

Additional comment I have is for the authors to be consistent in format of EC50.

The paper adds to the knowledge of ecotoxicological tests

Note:
This review refers to round 2 of peer review.

Review
Aims of the journal:
The article fits into he aims of the journal. It has a new knowledge with a new approach for conducting experiments in ecotoxicology.

Scope of the journal:
The article describes a methodology that will be useful in carrying out ecotoxicological assays for water samples from surface water, ground water and leachate with availability of low volumes. This will be beneficial in environmental monitoring protocols for assessment of toxicity of environmental samples.

General comments:
This work is useful in conducting ecotoxicological assays where the sample volume is low and can attain the same level of sensitivity to the toxicant as the conventional approach with large volumes of samples. This method will prove an useful approach in regulatory aspects of toxicity tests. While the method is beneficial to Daphnia tests, it will be useful to extrapolate the toxicity to other organisms such as fish embryos.

Abstract
Provides a concise information of the content of the article. Comparison of approximate volume size from conventional approach and the present study will provide the purpose of the article.

Introduction
Line 91, should be …..µL to mL

Materials and Methods
Metal salts are not tested while nickel and cadmium toxicity are discussed later in the article.
Line 130 replace …….singly with individually
Line 137 destilled, should be distilled
Line 147 to 149 – Sentence too long, split into two sentences

Results
Line 232 specify the data type, EC50?

Discussion
Line 329 Compare EC50 values
Line 369 PDMS – provide full form
Line 370 PAH – provide full form

Overall comment
Please check for the representation of species name in italics.

Note:
This review refers to round 1 of peer review and may pertain to an earlier version of the document.

Review
Article title: Miniaturisation of the Daphnia magna immobilisation assay for the reliable testing of low volume samples
Authors: Eberhard Küster[1], George Gyan Addo[1], Silke Aulhorn[1], Dana Kühnel[1
Reviewer: Melanie Trenfield

On reading the above listed manuscript I would recommend it be accepted following minor revision. The study investigates an innovative concept of optimising the volume of sample water required for toxicity testing with Daphnia magna. There are a handful of other miniaturisation bioassays published for daphnids that have incorporated such a study design, where the chemicals have been tested both in miniaturised assays versus traditional assays. This study assesses a range of chemicals different to those previously investigated in this way. While I am not familiar with the challenges of having limited availability of test water, I appreciate there would be situations for certain contaminants and investigations where this would be the case.
The study has been thoroughly researched and designed well with a comprehensive range of recent literature cited. The introduction satisfactorily describes the need for the study and the conclusion suitably summarises the outcomes. The toxicity data has been rigorously compared with toxicity data from the USEPA Ecotox database to assess whether the EC50s generated from the miniaturised design are comparable with the sensitivity observed in conventional cladoceran testing based on the OECD method. The outcome was that the test design resulted in similar sensitivity to traditional testing and data published from other studies. While there were some differences in sensitivity (for some chemicals increased sensitivity was observed and for others reduced sensitivity) this is reasonable considering inter-lab differences and that in some cases comparison was being made with daphnid species other than D. magna.

My general comments (by section) are below:
Abstract
It is mentioned that standardised toxicity tests require high volumes of testing water. It would be more informative to provide actual values of volumes along with this statement. Likewise for the following statement where it is mentioned that volumes of leachates or microplastic aging experiments are limited - what volumes are considered ‘limited’? Not all readers would be familiar with the challenges of volumes generated from leachates or microplastic aging experiments. Reading further I note that you include this in the introduction, but it would be helpful in the abstract to provide a brief indication of the extent of test volume optimisation that the study achieves.
Introduction
An impressive list of organic and inorganic chemicals has been assessed, encompassing fungicides, herbicides, insecticides, pharmaceuticals, and a surfactant. I think it would be helpful to include in the introduction a brief description as such of the types of chemicals you have tested. Perhaps even to include a description of chemical use for each contaminant in Table 3 alongside where the CAS number is listed.
 
Methods
Culturing & biotesting
• Was there any consideration as to whether the volumes used for culturing could also be reduced? If so, it would be good to acknowledge or cite that it was considered and trialled elsewhere. In some cases, culture water can also be expensive to collect or produce. 80 mL per individual adult cladoceran seems a lot although that is for a 48-h period. We use a 30 mL volume for individual cladoceran culture, but with daily water exchange and a different clad species. (Trenfield, M. A., Pease, C. J., Walker, S. L., Mooney, T., Tybell, L., Humphrey, C., ... & Harford, A. J. (2020). Standardised chronic toxicity test protocols and culturing methods for a suite of tropical freshwater species. Australasian Bulletin of Ecotoxicology and Environmental Chemistry, 6, 1-80)
• Given the small test volume, have you looked at measured concentrations of the contaminants in the well plates following the 48-h test period to quantify any loss over the test period? Also how do you know if water parameters are being maintained and other stress factors are not being introduced i.e. how do you measure dissolved oxygen to ensure this remains optimal in the well over the 48 h?
• Would be good to explicitly mention that the daphnids aren’t fed during the exposure.
• What is the residence time of these chemicals in the environment? Is a 48-h exposure long enough to represent what the cladocerans would be exposed to during an environmental incident? If so, it would be good to justify the exposure period.
• Given the emphasis of using chronic exposure endpoints when deriving guideline values for contaminants, it would be useful to know if you anticipate this design could be adapted to a longer-term exposure if larger volume well plates were used, food was also incorporated, and water exchanges performed.
Substance selection
• I note that the published literature on miniaturisation studies with daphnids included silver, nickel and cadmium studies. Is there any reason why a metal contaminant was not included in this study? It would have been interesting to confirm if the well plate approach could be successfully applied to metal contaminants, or if there would be a substantial loss of metal to the surface of the wells. Would be good to include the reasoning for the selection of contaminants upfront here (in addition to the lipophilicity and availability of data that you mention).

Specific formatting/grammatical errors:
Line 85: ‘Usually’ should be ‘usual
Line 158: ‘build’ should be ‘built’
Line 163 & 264: need space between the two words in ‘potassiumdichromate’
Line 163: First mention of SDS – it should be spelled out in full
Line 268: for a ‘few substances’ – replace with the actual number of chemicals (3?) for which it was not possible to make a comparison.
Line 365: ‘disturb’ could be replaced with ‘interfere’
Line 373: ‘disturb’ could be replaced with ‘interfere with’
Table 1: Specify in the title that the miniaturisation is for daphnids only
Is there a reason why the first row is highlighted a different colour?

Note:
This review refers to round 1 of peer review and may pertain to an earlier version of the document.