Toxicity of Landfill Leachate to Stream-Dwelling Benthic Macroinvertebrates

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript assesses the toxicity of landfill leachate from the Winona County landfill to stream-dwelling benthic macroinvertebrates. The study combines field bioassessment of macroinvertebrate communities in nearby trout streams with laboratory toxicity bioassays using locally collected taxa, specifically focusing on the pollution-sensitive caddisfly Brachycentrus. The research question is practically relevant for local environmental management, and the approach of using indigenous species for toxicity testing adds ecological validity to the standard protocols.

Recommendations：

1. Framing of the ammonia-related hypothesis

The authors attribute the observed toxicity primarily to un-ionized ammonia based on literature links and high initial concentrations. At present, the evidence is largely associative; there are high ammonia levels consistent with published effects, but no specific manipulation (e.g., pH adjustment to alter un-ionized ammonia ratios without removing other contaminants) was performed to isolate ammonia as the primary toxicant. The manuscript should therefore present this mechanism explicitly as a strong inference rather than a proven fact, or discuss other potential toxicants (e.g., heavy metals like Chromium which was also high) in more detail.

2. Interpretation of aeration effects and statistical support

The authors suggest that aeration "may be a simple means of reducing the toxicity". However, the results section notes that the reduction in mortality was not significantly different (P=0.174) due to wide variability. Given that the statistical tests did not support the efficacy of pre-aeration in the 24-h tests, it will benefit readers if the discussion and conclusion more explicitly distinguish between the trend suggested by the data and the lack of statistical significance. The claim that aeration is effective should be softened to avoid overstatement of the experimental results.

3. Methodological details on control groups

In the laboratory testing section, while stream water was used, the specific survival rates of control groups (0% leachate) for all experiments should be explicitly tabulated or mentioned to ensure the validity of the mortality observed in treatment groups. For instance, in Figure 1, the 0% mortality is clear, but confirming that controls in the aeration experiments also had low mortality is important for interpreting the high variability.

4. Discrepancy between biotic indices (Hilsenhoff and B-IBI)

The manuscript reports a divergence between the Hilsenhoff biotic index (indicating "good to excellent" conditions) and the B-IBI (indicating "fair/good to poor"). While the authors discuss that multi-metric indices are more responsive, further elaboration on which specific metrics within the B-IBI drove the lower scores at the sites nearest the landfill would provide valuable insight. This distinction will help clarify whether the impairment is related to potential leachate exposure or other habitat factors not captured by the tolerance-based Hilsenhoff index.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study employed a combined approach of laboratory toxicity testing and field community investigation to assess the toxic effects of leachate from a closed sanitary landfill on benthic macroinvertebrates in coldwater trout streams in southeastern Minnesota, USA. The research addresses a clear and environmentally relevant issue, with an overall sound experimental design that effectively integrates field and laboratory work. The main conclusion is that the leachate currently shows no significant impact on benthic invertebrate communities in nearby streams, but exhibits considerable toxicity at higher concentrations. This work offers valuable insights for regional environmental risk assessment and management.

The following points require attention:

1. The color scheme used in the legend of Figure 1 includes several hues that are very similar, making them difficult to distinguish. It is recommended that the author enhance the color contrast to improve clarity and readability.
2. The reference list is notably outdated and lacks citations of recent studies in the field. The author should update the references to include more current and relevant literature.
3. The composition and concentration of landfill leachate can vary significantly with seasonal changes. The author should specify the time of year during which the leachate samples were collected.
4. The author should provide a clear specification of the mesh size (or aperture) of the D-frame aquatic dip net used for sampling.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors and Editors,

Thank you for the opportunity to review the manuscript: "Toxicity of Landfill Leachate to Benthic Macroinvertebrates in Streams," which is an interesting toxicological study. As the authors emphasize, the toxicity of landfill leachate is likely to remain a significant problem in the future. The manuscript, however, suffers from some flaws that I recommend be reviewed and corrected before acceptance for publication.

Unfortunately, due to the lack of line numbering, the communication may be slightly difficult; however, I will do my best to present my point of view.

Introduction

In my opinion, this section should also refer to water quality parameters. These analyses appear no earlier than in the Results section, without prior mention in the hypotheses. Therefore, it appears as an additional test, without a planned intention. Please consider incorporating it into the hypotheses.

Section 2.1.

I suggest adding a map with the landfill, the streams, and sampling sites marked.

Section 2.2.

Please add the info when the samples were collected.

Section 2.3.

- Sentence: „Macroinvertebrates and stream water were collected just prior to each test.” Prior to each test? With no acclimation to the laboratory conditions? Were the test temperatures the same as in the fields? Even if so, the transfer stress should be eliminated through acclimation.

- description of the tests: The rationale for selecting Brachycentrus larvae as one of the most sensitive is unclear. Is it based on literature data or the authors' own research (initial tests)?

- description of the tests: Were the dilutions prepared using distilled water or stream water collected in the field?

The section describing statistical procedures is missing. References to statistical analyses are included in the results. A proper section should be included within the Materials and Methods section. It also should contain the methodology for LC50 calculation, as well as the number of individuals used in the experiments

Additionally, in the M&M section, the authors should refer to the analyses of water quality parameters with more detailed description of methodology, sampling, transport etc.

Results

Figure 1 should be reconsidered. Should the concentrations invisible in the figure listed in the legend? Even if they overlap, the legend is confusing.

Discussion and conclusions

Please consider the reformulation of the Conclusion section. It does not refer directly to the hypotheses.

Namely, the one referring to the toxicity towards sensitive vs resistant taxa and the question of sampling point localisation (near or downstream of the landfill). What is the conclusion?

 

Moreover, the sentence: „Although several taxa present in the stream can suffer complete mortality when exposed to raw, undiluted leachate, sensitive taxa such as Brachycentrus caddisfly larvae typically were among the most abundant organisms within various sites in Burns Valley Creek, suggesting no current problems from leachate pollution.” Require more profound discussion. In my opinion, leachate pollution may still impact the environment; however, the population composition might have changed due to the pollution pressure, resulting in the selection of the most resilient individuals. The aspect of developing tolerance has not been discussed in the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has revised the manuscript in accordance with the reviewers' comments; however, several issues remain to be addressed. For example, it is recommended that all tables be reformatted as standard three-line tables, and the formatting of figures should be standardized, as Figure 2 and Figure 3 currently exhibit inconsistencies. Additionally, it is unclear whether a statistical significance analysis has been performed for the data presented in Figure 4.

Author Response

Reviewer: The author has revised the manuscript in accordance with the reviewers' comments; however, several issues remain to be addressed. For example, it is recommended that all tables be reformatted as standard three-line tables,

Response: All tables have been reformatted.  Production staff will need to adjust all tables to fit the page.

 

Reviewer: .....and the formatting of figures should be standardized, as Figure 2 and Figure 3 currently exhibit inconsistencies.

Response: Figures 2 and 3 have been standardized.

 

Reviewer: Additionally, it is unclear whether a statistical significance analysis has been performed for the data presented in Figure 4.

Response: The statistical test results are presented in the text when Figure 4 is referenced. That text appears immediately above the figure.