Phenol, Cyanide, and Thiocyanate in Aquatic Media: The Ecotoxicity of Individual Substances and Their Mixtures

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents the results of studies on the toxicity of phenols, cyanides and thiocyanates, which were assessed on the basis of a series of tests of the effect of these compounds on the growth and changes in chlorophyll of microorganisms (Aliivibrio fischeri, Pseudomonas putida) and aquatic plants (Chlorella sp., Lemna minor), as well as tests of plant growth inhibition, root growth and chlorophyll formation, and finally a test of seedling growth in Allium cepa. In addition, an attempt was made to determine their combined toxicity using binary combinations of these substances and ternary mixtures (of different concentrations) for the studies. The experimental part was preceded by a literature review, which revealed significant gaps in our knowledge of the ecotoxicity of the substances studied. I consider the manuscript interesting and worth publishing, but reading it raised several questions and doubts that should be resolved before its publication.

The most important question is the issue of measuring changes in chlorophyll content. In section "2.1.4. Lemna minor test" in lines 226-231 it is stated that the measurement of chlorophyll content in Lemna minor leaves was carried out spectrophotometrically after its extraction in 95% ethanol and the calculations were made using the equation given by Lichtenthaler (1987). However, according to the title, this publication concerns "Chlorophyll Fluorescence Signatures of Leaves during the Autumnal Chlorophyll Breakdown" and therefore it is probably not a good source for the equation describing the relationship between absorbance and chlorophyll concentration. In this case, one can at most expect that it cites some equation by another author (I do not have access to this publication). Moreover, the “Results and Discussion” section discusses and presents in detail the results of tests of chlorophyll changes in Lemna minor (compare: Fig. 1 and paragraphs in lines 339–367), whereas the “Conclusions” section highlights that a “luminescence inhibition test in Aliivibrio fischeri” was performed (line 501), about which the reader learns little in the text.

I also propose considering the formulation of the phrase in which the authors write "...phenols have a predominantly positive effect on Lemna minor, as it grows in its presence and the negative effect is only seen in the chlorophyll content ...". However, does not leaf chlorosis indicate that phenols also have a negative effect on Lemna minor in the long term, and the stimulation of more intensive growth is rather a temporary effect and should not be treated as a positive effect? ​​

I would also like to draw attention to the inconsistency in the formulation "... was accompanied by a slight promotion of root growth (up to 14.68%), although no trend was observed in this case ...) - either it promoted or it did not.

Please also specify the units in Table 2.

Author Response

For the research article environments-3573411

Responses to the comments of Reviewer 1

 

Dear Reviewer,

Thank you for your valuable comments, which have helped us to improve this manuscript. Below you will find the detailed responses to the comments, while the corresponding revisions are marked in red in the manuscript file.

The manuscript presents the results of studies on the toxicity of phenols, cyanides and thiocyanates, which were assessed on the basis of a series of tests of the effect of these compounds on the growth and changes in chlorophyll of microorganisms (Aliivibrio fischeri, Pseudomonas putida) and aquatic plants (Chlorella sp., Lemna minor), as well as tests of plant growth inhibition, root growth and chlorophyll formation, and finally a test of seedling growth in Allium cepa. In addition, an attempt was made to determine their combined toxicity using binary combinations of these substances and ternary mixtures (of different concentrations) for the studies. The experimental part was preceded by a literature review, which revealed significant gaps in our knowledge of the ecotoxicity of the substances studied. I consider the manuscript interesting and worth publishing, but reading it raised several questions and doubts that should be resolved before its publication.

• “The most important question is the issue of measuring changes in chlorophyll content. In section "2.1.4. Lemna minor test" in lines 226-231 it is stated that the measurement of chlorophyll content in Lemna minor leaves was carried out spectrophotometrically after its extraction in 95% ethanol and the calculations were made using the equation given by Lichtenthaler (1987). However, according to the title, this publication concerns "Chlorophyll Fluorescence Signatures of Leaves during the Autumnal Chlorophyll Breakdown" and therefore it is probably not a good source for the equation describing the relationship between absorbance and chlorophyll concentration. In this case, one can at most expect that it cites some equation by another author (I do not have access to this publication).”

Response to comment:

Thank you for your comment. We acknowledge that the title of the cited reference was listed incorrectly. The reference has been corrected, and a new, accurate citation has been added. Instead of: „Lichtenthaler, H.K. Chlorophyll Fluorescence Signatures of Leaves during the Autumnal Chlorophyll Breakdown. J. Plant. Physiol. 1987, 131, 101–110. https://doi.org/10.1016/S0176-1617(87)80271-7” it should be: „Lichtenthaler, H.K. Chlorophylls and Carotenoids: Pigments of Photosynthetic Biomembranes. Methods Enzymol. 1987, 148, 350–382. https://doi.org/10.1016/0076-6879(87)48036-1“

 

• “Moreover, the “Results and Discussion” section discusses and presents in detail the results of tests of chlorophyll changes in Lemna minor (compare: Fig. 1 and paragraphs in lines 339–367), whereas the “Conclusions” section highlights that a “luminescence inhibition test in Aliivibrio fischeri” was performed (line 501), about which the reader learns little in the text.”

Response to comment:

Thank you for your comment. In the conclusion section, emphasis was placed on all test organisms used in the toxicity assessment. Aliivibrio fischeri was specifically highlighted towards the end of the conclusion because it was the only organism used to evaluate the toxicity of the multicomponent system, while the other organisms were not included in this part of the study. Detailed results and interpretation of the Aliivibrio fischeri assay related to multicomponent toxicity are presented in Section 3.4.

In line with your suggestion, we have revised the conclusion and added a sentence referring specifically to Lemna minor to improve clarity and balance, lines 551-571.

“The ecotoxicity of phenols, cyanides, and thiocyanates was assessed using a diverse set of bioassays. These included luminescence inhibition with Aliivibrio fischeri, growth inhibition with Pseudomonas putida and Chlorella sp., a series of assays with Lemna minor targeting frond and root growth as well as chlorophyll a formation, and a seedling growth test using Allium cepa.

In the Lemna minor assays, phenol induced visible chlorosis and significantly re-duced chlorophyll a content, yet simultaneously stimulated both frond and root de-velopment, pointing to a potential metabolic adaptation. On the other hand, cyanide and thiocyanate demonstrated consistent toxicity across all measured parameters, with cyanide showing the strongest inhibitory effects, especially on root and frond growth.

Among the applied assays, Aliivibrio fischeri and Allium cepa exhibited the highest sensitivity to the tested compounds. However, the test with Allium cepa had serious drawbacks as it was time consuming and suffered from a high classification of inhibi-tion values due to a relatively low number of germinated individuals in the blank sample. The tests involving Pseudomonas putida and Chlorella sp., conducted over the same exposure period, revealed comparable sensitivity levels.

When comparing the toxicants, cyanide was generally the most harmful across all tests. Phenol exhibited either similar or slightly higher toxicity than thiocyanate, de-pending on the assay. Overall, most of these tests showed a clear negative effect of the selected toxicants, which underlines the importance of assessing the risk of the presence of these substances in the aquatic environment.”

 

• “I also propose considering the formulation of the phrase in which the authors write "...phenols have a predominantly positive effect on Lemna minor, as it grows in its presence and the negative effect is only seen in the chlorophyll content ...". However, does not leaf chlorosis indicate that phenols also have a negative effect on Lemna minor in the long term, and the stimulation of more intensive growth is rather a temporary effect and should not be treated as a positive effect?”

Response to comment:

Thank you for your insightful comment. We agree that the observed chlorosis of Lemna minor fronds in the presence of phenol indicates a potential negative effect, particularly in the context of long-term exposure. While our results demonstrated a stimulation of frond and root growth, we acknowledge that this response may represent a short-term adaptive mechanism rather than a truly beneficial effect.

In light of your suggestion, we have revised the wording in the manuscript to avoid describing the effect of phenol as "predominantly positive" and instead emphasize the complexity of the response, which includes both stimulation of growth and inhibition of chlorophyll formation. This change more accurately reflects the dual nature of the observed effects and avoids potential misinterpretation regarding the overall impact of phenol on Lemna minor, lines 416-423.

“These findings suggest that the response of Lemna minor to phenol is complex, involving both stimulation of frond and root growth and inhibition of chlorophyll formation, likely indicating a short-term adaptive mechanism rather than a genuinely beneficial effect. In contrast, cyanide and thiocyanate clearly impair overall growth and physiological function. The observed differences in toxicity are most likely related to the distinct structural properties of the tested compounds, although a general difference in the physiological response of Lemna minor to organic versus inorganic pollutants cannot be excluded.”

 

• “I would also like to draw attention to the inconsistency in the formulation "... was accompanied by a slight promotion of root growth (up to 14.68%), although no trend was observed in this case ...) - either it promoted or it did not.”

Response to comment:

Thank you for your valuable observation. We agree that the original formulation may appear inconsistent. To clarify, although a slight increase in root growth (up to 14.68%) was recorded, the effect was not consistent across all concentrations, and no clear dose-dependent trend could be established. We have therefore revised the sentence to more accurately reflect the results, emphasizing the variability in the data rather than implying a definitive stimulatory effect, lines 396-398.

“The promotion of frond growth observed in our study was accompanied by a slight increase in root growth (up to 14.68%). However, due to variability in the data, no consistent dose-dependent trend could be established in this case (grey circles in Figure 1A).”

           

• “Please also specify the units in Table 2.”

Response to comment:

The comment is accepted, and the units are added in Table 2.

Yours sincerely,

Dajana Kučić Grgić

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript provides an interesting discussion on the ecotoxicity of the pollutants Phenol, cyanide and thiocyanate as individual component and in mixtures.

The topic has relevance in the field of sustainability and environmental protection. The manuscript may have potential for consideration for publication.

There are, however, a few points to address and that may require revision to further strengthen the manuscript.

 

1)

Section 1. Introduction.

This is an important section to establish objective of the study.

As is, the current manuscript may be lacking in novelty and impact, as it is already known that phenol, cyanide and thiocyanate are eco-toxic. It is therefore also not surprising that their mixtures will also be eco-toxic.

The authors should revise the introduction to justify the novelty and significance of the research.

 

2)

Section 1. Table 2.

Provide units to the concentrations – are they in ppm or ppb range.?

What are the standards for such pollutants according to the references in Table 2?

Also to describe and provide schematics to explain what may be existing treatment processes (biological activated sludge or chemical methods?) to treat such industrial wastewaters? What are the capacity of the treatment plants?

 

3)

Section 4.

Line 523. “The analysis revealed a synergistic behavior of phenol in combination with cyanide or thiocyanate…”

Please justify with more details on the synergistic behaviour.

 

4)

Section 4. Conclusion.

The authors may wish to provide a table to summarise key findings from the study.

And after knowing the ecotoxicity of the pollutants individually as well as in binary or ternary, what could be the recommended treatment processes to treat and manage such pollutants? Please also list down what are the research priorities and roadmap in treating and managing such pollutants.

 

5)

References

Please review and list only relevant references. Typically, the number of references should not exceed 50 for a research paper.

 

 

Author Response

For the research article environments-3573411

Responses to the comments of Reviewer 2

 

Dear Reviewer,

Thank you for your valuable comments, which have helped us to improve this manuscript. Below you will find the detailed responses to the comments, while the corresponding revisions are marked in red in the manuscript file.

 

The manuscript provides an interesting discussion on the ecotoxicity of the pollutants Phenol, cyanide and thiocyanate as individual component and in mixtures. The topic has relevance in the field of sustainability and environmental protection. The manuscript may have potential for consideration for publication. There are, however, a few points to address and that may require revision to further strengthen the manuscript.

 

• “Section 1. Introduction. This is an important section to establish objective of the study. As is, the current manuscript may be lacking in novelty and impact, as it is already known that phenol, cyanide and thiocyanate are eco-toxic. It is therefore also not surprising that their mixtures will also be eco-toxic. The authors should revise the introduction to justify the novelty and significance of the research.”

Response to comment:

Thank you for your valuable comment. We agree that the ecotoxicity of phenol, cyanide, and thiocyanate is generally known; however, the objective of this study was not merely to confirm their toxicity, but to provide a detailed and comparative assessment of their effects on multiple aquatic species from different trophic levels, as well as to evaluate their combined effects in binary and ternary mixtures. To the best of our knowledge, no published studies currently exist that address the joint toxicity of these three compounds in aqueous mixtures, particularly using the selected range of bioassays. Although these substances are assumed to be toxic, precise documentation of their toxic thresholds is essential—especially to determine concentration levels that pose a risk to aquatic organisms, with reference to the most sensitive species. Moreover, phenol, cyanide, and thiocyanate commonly co-occur in industrial effluents, particularly in wastewater from the coking industry. Understanding their combined toxic action is crucial for accurate ecological risk assessment and for informing the design of effective wastewater treatment strategies.

In response to your suggestion, we have revised the Introduction section to better emphasize the novelty and scientific relevance of our research, highlighting the knowledge gaps it addresses and the methodological advancements it introduces, lines 120 – 135 and 160-167.

 

• ” Section 1. Table 2. Provide units to the concentrations – are they in ppm or ppb range? What are the standards for such pollutants according to the references in Table 2? Also to describe and provide schematics to explain what may be existing treatment processes (biological activated sludge or chemical methods?) to treat such industrial wastewaters? What are the capacity of the treatment plants?”

Response to comment:

Thank you for your constructive comment. We have carefully addressed all the points raised and incorporated the necessary clarifications and additions into the manuscript. The units of concentration are added in Table 2. The new content has been added in lines 46 – 63 of the revised manuscript.

“Wastewaters containing phenols, cyanides and thiocyanates are regarded as one of the most difficult industrial effluents to treat due to their complex composition. Consequently, modern treatment strategies usually demand a combination of physical, chemical, and biological processes to meet increasingly stringent environmental regu-lations [2]. Figure 1 provides an overview of treatment methods for wastewaters con-taining phenols, cyanides, and thiocyanates. According to the EU Industrial Emissions Directive and the corresponding Best Available Technique (BAT) Reference Document for Common Waste Water and Waste Gas Treatment/Management Systems in the Chemical Sector (CWW BREF), the maximum allowable concentrations for phenol, free cyanide, and thiocyanate in treated industrial effluents discharged into natural water bodies are typically set at 0.1 mg/L for each compound [12]. While treatment technologies are well established, the design capacity of industrial wastewater treatment plants varies significantly depending on the specific production scale and local regulations. For example, Bargiel et al. [13] report a treatment plant capacity of 600 m³/day for coking industry wastewater, while the authors are familiar with two facilities in the region with capacities of approximately 1200 m³/day and 700 m³/day, respectively. This is not surprising given that such industries typically generate enormous volumes of highly polluted effluents that require complex and large-scale treatment systems [2].”

Figure 1. Overview of treatment methods for wastewaters containing phenols, cyanides, and thiocyanates.

 

• “Section 4. Line 523. “The analysis revealed a synergistic behavior of phenol in combination with cyanide or thiocyanate…” Please justify with more details on the synergistic behaviour.

Response to comment: Thank you for your comment. In order to justify our statement about the observed synergistic behavior, we have changed the sentence in lines 523-524 of the old manuscript: „The analysis revealed a synergistic behavior of phenol in combination with cyanide or thiocyanate.“

The new sentences are added, lines 575-583:

„Isobolographic analysis showed a synergistic deviation from additivity for solutions in which phenol was combined with cyanide. A higher content of cyanide (as a significantly more toxic component) masked the synergism by bringing the toxicity of the mixtures closer to that of the cyanide solution, thus positioning two of the mixtures (i.e. two TU points) within the predefined additivity interval of ±20%. However, when all three mixtures are considered together, a deviation from the additivity interval with a clear tendency towards synergistic behavior can be seen. The synergistic deviation from additivity was more pronounced in the solutions where phenol was combined with thiocyanate, as all three TU points were below the additivity interval.“

 

• “Section 4. Conclusion. The authors may wish to provide a table to summarise key findings from the study. And after knowing the ecotoxicity of the pollutants individually as well as in binary or ternary, what could be the recommended treatment processes to treat and manage such pollutants? Please also list down what are the research priorities and roadmap in treating and managing such pollutants.

Response to comment:

Thank you for your valuable comments and suggestions. In response, we have revised the Conclusion section to provide a more structured overview of the key findings from the study. Although we did not include a separate table, the text has been reformulated to clearly summarize the results of the toxicity assessments and their implications.

We addressed both individual and combined toxic effects of phenols, cyanides, and thiocyanates. The study highlighted cyanide as the most toxic compound across all assays, while phenol demonstrated complex behavior—stimulating growth in Lemna minor despite reducing chlorophyll content, indicating a potential short-term adaptive response. The ecotoxicity tests involving Aliivibrio fischeri and Allium cepa proved to be the most sensitive, although the Allium cepa test had limitations due to low germination rates in the control group.

Importantly, we analyzed the combined toxicity of binary and ternary mixtures using isobolographic analysis. The observed synergistic deviations—particularly in phenol-thiocyanate and phenol-cyanide combinations—underline the need to consider mixture effects in environmental risk assessments, as they may lead to underestimations of actual toxicity in complex aquatic environments.

Regarding treatment and management strategies, the revised text briefly discusses commonly used approaches. Biological methods (e.g. activated sludge) are effective for phenols, while combined chemical-biological systems may be more suitable for cyanides and thiocyanates. Additionally, the observed synergistic effects point to the importance of developing integrated monitoring and treatment systems capable of responding to the complexity of multicomponent industrial wastewater.

Finally, based on the findings, research priorities have been outlined, including the optimization of treatment strategies for mixed pollutant systems, the development of more sensitive bioassays, and further exploration of synergistic toxicity effects in diverse organisms. These additions aim to provide a clearer roadmap for future investigations and environmental management efforts.

The new content has been added in lines 551 – 610 of the revised manuscript.

“The ecotoxicity of phenols, cyanides, and thiocyanates was assessed using a di-verse set of bioassays. These included luminescence inhibition with Aliivibrio fischeri, growth inhibition with Pseudomonas putida and Chlorella sp., a series of assays with Lemna minor targeting frond and root growth as well as chlorophyll a formation, and a seedling growth test using Allium cepa.

In the Lemna minor assays, phenol induced visible chlorosis and significantly reduced chlorophyll a content, yet simultaneously stimulated both frond and root development, pointing to a potential metabolic adaptation. On the other hand, cyanide and thiocyanate demonstrated consistent toxicity across all measured parameters, with cyanide showing the strongest inhibitory effects, especially on root and frond growth.

Among the applied assays, Aliivibrio fischeri and Allium cepa exhibited the highest sensitivity to the tested compounds. However, the test with Allium cepa had serious drawbacks as it was time consuming and suffered from a high classification of inhibition values due to a relatively low number of germinated individuals in the blank sample. The tests involving Pseudomonas putida and Chlorella sp., conducted over the same exposure period, revealed comparable sensitivity levels.

When comparing the toxicants, cyanide was generally the most harmful across all tests. Phenol exhibited either similar or slightly higher toxicity than thiocyanate, depending on the assay. Overall, most of these tests showed a clear negative effect of the selected toxicants, which underlines the importance of assessing the risk of the presence of these substances in the aquatic environment.

In addition to the toxicity of the individual toxicants, their joint toxic action was also analyzed. For this purpose, the experimentally determined toxicities of binary and ternary mixtures (according to the Aliivibrio fischeri test) were compared with the toxicities predicted by the additive model. Isobolographic analysis showed a synergistic deviation from additivity for solutions in which phenol was combined with cyanide. A higher content of cyanide (as a significantly more toxic component) masked the synergism by bringing the toxicity of the mixtures closer to that of the cyanide solution, thus positioning two of the mixtures (i.e. two TU points) within the predefined additivity interval of ±20%. However, when all three mixtures are considered together, a deviation from the additivity interval with a clear tendency towards synergistic behavior can be seen. The synergistic deviation from additivity was more pronounced in the solutions where phenol was combined with thiocyanate, as all three TU points were below the additivity interval. In addition, cyanide and thiocyanate showed additive behavior, indicating that they have a very similar toxic mode of action. The ternary mixtures did not differ significantly from the behavior of the binary mixtures. The presence of a synergistic deviation from additivity observed in the case of Aliivibrio fischeri increases the likelihood of such a scenario in the case of other organisms. Therefore, this should be seriously considered when assessing ecological risks in re-al-life scenarios.

Based on the ecotoxicological data obtained, the selection of appropriate treatment strategies should be tailored to the specific physicochemical properties of the pollutants and the nature of their interactions. For phenolic compounds, biological treatment technologies—such as activated sludge systems and membrane bioreactors—have demonstrated high efficacy due to the biodegradability of these substances. In contrast, cyanides and thiocyanates, owing to their pronounced toxicity and limited biodegradability, often necessitate advanced chemical oxidation techniques, including alkaline chlorination, ozonation, or other advanced oxidation processes (AOPs). In many cases, the integration of biological and chemical treatments within hybrid systems may offer the most robust and efficient approach. Special consideration should be given to pollutant mixtures, where synergistic interactions may enhance overall toxicity and si-ultaneously compromise treatment performance.

Future research should prioritize the development of integrated, multi-barrier treatment solutions capable of addressing complex, multi-component industrial effluents. This includes advancing early-warning monitoring systems for the detection of combined effects, as well as improving mechanistic understanding of sublethal and chronic impacts on aquatic organisms. A comprehensive roadmap for pollutant management should encompass (i) the design of cost-effective and scalable treatment technologies, (ii) refinement of bioassays with improved ecological sensitivity and predictive value, and (iii) implementation of risk assessment frameworks that explicitly consider mixture toxicity and non-additive effects.”

 

 

• ”References: Please review and list only relevant references. Typically, the number of references should not exceed 50 for a research paper.

Response to comment:

The comment is accepted, and only relevant references are added.

Yours sincerely,

Dajana Kučić Grgić

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has been strengthened. Nice work by the authors.

However there could be still minor error(s) – for example, Table 2 with the message [Error! Reference source not found.]. Currently, the methods used are essentially lab-based. It will be good to carry out further practical studies involving real wastewaters and site results in the future.

 

Author Response

We sincerely thank the reviewer for the positive feedback and constructive suggestions.

The referencing error in Table 2 ([Error! Reference source not found.]) has been corrected in the revised version of the manuscript. The references in the text are now all linked to the reference list. We appreciate your attention to detail in pointing this out.

We agree that future research involving real wastewater samples would enhance the practical relevance of the study. While the ecotoxicity tests applied in this research are currently performed under controlled laboratory conditions, it is important to note that these standardized bioassays are designed to be applicable to real wastewater samples as well. However, due to the sensitivity and variability of test organisms, all tests must still be conducted in controlled laboratory environments, as no reliable on-site (in situ) ecotoxicity tests are currently available.

In parallel, research on the treatment of real coking wastewater is being conducted as part of an applied research project in collaboration with industry. We plan to publish the results of that study in the near future to complement the findings presented in this manuscript.