Intergrading Water Quality Parameters, Benthic Fauna and Acute Toxicity Test for Risk Assessment on an Urban-Rural River

Round 1

Reviewer 1 Report

Brief summary

This article discusses on the intergrading several tests for risk assessment in polluted areas. Water quality had been the main approach in assessing river health. Then, this was improved by a more recent assessment based on benthic organisms such as Index of Biological Integrity (IBI). The latest that seems to further improve the assessment is toxicity test. The findings are rather expected where the rural reaches (goose farm areas) of river appeared to be more polluted than the urban reaches. However, no detailed discussion provided to support the findings.

 

General concept comments:

1.       The title of this does not clearly describes what this study about. The term ‘intergrading’ gives the impression that this study combines all approaches and compares how the combined-approach performed in assessing the river pollution. Or, at least compare how each approach performed. However, it does not.  

2.       It is suggested the authors to get this article for English proofreading. There are many parts of this article that need a substantial improvement in terms of English.

3.       This article lacks a sound problem statement and justification as to why this work is important. It describes the shortcomings of previous approaches i.e water quality and biotic index, but does not offer a solution of how this study can improve.

4.       Method section describes the study area; whilst it is not fairly well written, it also lacks of justification as to why those areas / stations were selected, other than due to different human activities. The characteristics of Site 3 (which is far from river flow) is not described. Similarly, the inclusion of tributary sites is also not justified. Samples collection was briefly described for Site 1 -4, but no information for tributary sites.

5.       Sampling strategy is also not clear, especially on how many samples altogether were collected. The methods also do not provide detailed description. For example, how the sample was divided into two parts  for different analyses.

6.       For ZET test, please provide info where did zebrafish embryos were collected from.

7.       In Results section, authors use the term ‘linkages’ referring to the group of worms. I would suggest to classify them under the phylum i.e. Annelida. If there is any specific reason of using the ‘linkage’, then it needs to be described beforehand.

8.       Table 1 is confusing for the use of ‘N’ as it describes as ‘number of benthic species’. N normally refers to number of individuals while S normally refers to number of species.

9.       I would suggest to be consistent in terms of structure for e.g. the Figure 6 should appear after paragraph, not in the beginning of the sub-section. It is the same case with Figure 7.

10.   Figure 7 of the PCA should not only simply stating the obvious about the cluster, but also should also describe what is the meaning of cluster. For example the closer the plot means they are more similar to those far away.

11.   Discussion section lacks arguments and discussions. For example Section 4.1 mostly re-describe the results. Only a small part of this section provide the arguments and discuss as to why such findings were recorded. The same comment on the section 4.2 where it lacks of discussion and argument. If any, the discussion is mainly to compare the findings of this study with the previous studies, without really providing the reason behind the findings.

12.   Conclusion is not convincing and does not really in-line with the tittle.

Specific comments

Comments as in the document

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Brief summary:

This article discusses on the intergrading several tests for risk assessment in polluted areas. Water quality had been the main approach in assessing river health. Then, this was improved by a more recent assessment based on benthic organisms such as Index of Biological Integrity (IBI). The latest that seems to further improve the assessment is toxicity test. The findings are rather expected where the rural reaches (goose farm areas) of river appeared to be more polluted than the urban reaches. However, no detailed discussion provided to support the findings.

 

1. Comment:The title of this does not clearly describes what this study about. The term ‘intergrading’ gives the impression that this study combines all approaches and compares how the combined-approach performed in assessing the river pollution. Or, at least compare how each approach performed. However, it does not.  

Reply: We gratefully appreciate for your comment. Our intention was to apply several methods that are currently widely used by scholars to conduct a comprehensive study of water bodies in terms of water quality index conditions, aquatic fauna community structure, and toxic effects on organisms. The study does not involve the combination of all approaches. Based on your suggestion, we have changed the title to "Intergrading water quality parameters, benthic fauna and acute toxicity test for risk assessment on an urban-rural river". Thanks again for your valuable comment.

 

2. Comment:It is suggested the authors to get this article for English proofreading. There are many parts of this article that need a substantial improvement in terms of English.

Reply: Thank you so much for your careful check. We have followed your suggestion to proofread the article in English and have already made changes to the problems found.

 

3. Comment:This article lacks a sound problem statement and justification as to why this work is important. It describes the shortcomings of previous approaches i.e water quality and biotic index, but does not offer a solution of how this study can improve.

Reply: Thank you for your rigorous comment. The purpose and significance of the study have been added to the revised abstract and introduction.

 

Abstract: Climate change, river pollution and loss of biodiversity are increasing and becoming global environmental concerns. The Yellow River is China's mother river, providing water for about 114 million residents in towns and cities along its route. Yet in 2012, the Yellow River received 4.474 × 10⁹ tons of sewage containing a large number of exogenous pollutants, posing a huge ecological and public health threat. Water quality safety is not only a matter of ecosystem health, but also of human survival and social development. Therefore, the effects of pollutants on water quality safety should be carefully studied, which is important to ensure the sustainability of the Yellow River and the surrounding cities and towns.

Introduction: Direct discharges of wastewaters from industrial and personal use are contributing large amounts of exogenous pollutants to rivers [3]. It has been reported that 90% of rivers flowing through towns and cities are heavily polluted [4], posing significant ecological and public health threats, including loss of biodiversity and the induction of cancer [5,6]. A statistically significant correlation has been found between the incidence of biological tumors and the concentration of chemical contaminants in water [7,8]. In addition, for rivers in urban-rural areas, there are often significant differences in the pollution status of rivers in different sections, such as urban and rural areas, due to the type and intensity of human activities [9], and therefore the main pollutants and toxic effects on ecosystems [10], with rivers around cities with high population density often having poorer water quality than areas far from cities [11].

Before the 1990s, river health assessment mainly relied on water quality indicator tests, i.e., quantitative testing of certain indicators, a method that allows visual assessment of whether and how much pollutant levels are exceeded [12]. However, water quality indicator analysis alone cannot achieve a comprehensive assessment of a water body because pollutants are always present as a mixture [13]. Whereas, it has been found that the use of appropriate bioanalysis can effectively compensate for this shortcoming. Bioanalysis can reflect the extent of water pollution and reveal the potential adverse effects of pollutants on organisms and humans [14,15]. Jeppesen et al. [16] applied indices related to aquatic community structure to assess the health of aquatic ecosystems [17,18]. Brack et al. [19] monitored water quality and thus revealed its potential health risk to humans by analyzing the short-term toxicity of pollutants to organisms, which is beyond the analytical capacity of water quality indicators. Thus, biologically oriented approaches were integrated into water quality monitoring.

Reference:

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5.Burdon, F.J.; Munz, N.A.; Reyes, M.; Focks, A.; Joss, A.; Räsänen, K.; Altermatt, F.; Eggen, R.I.L.; Stamm, C. Agriculture versus Wastewater Pollution as Drivers of Macroinvertebrate Community Structure in Streams. Sci. Total Environ. 2019, 659, 1256–1265, doi:10.1016/j.scitotenv.2018.12.372.

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7.Liu, Y.; Liu, G.; Yuan, Z.; Liu, H.; Lam, P.K.S. Presence of Arsenic, Mercury and Vanadium in Aquatic Organisms of Laizhou Bay and Their Potential Health Risk. MAR POLLUT BULL 2017, 125, 334–340, doi:10.1016/j.marpolbul.2017.09.045.

8.Ogbeide, O.; Uhunamure, G.; Okundaye, F.; Ejeomo, C. First Report on Probabilistic Risk Assessment of Pesticide Residues in a Riverine Ecosystem in South-South Nigeria. CHEMOPHERE 2019, 231, 546–561, doi:10.1016/j.chemosphere.2019.05.105.

9.Zhao, H.; Li, X. Risk Assessment of Metals in Road-Deposited Sediment along an Urban–Rural Gradient. Environ. Pollut. 2013, 174, 297–304. https://doi.org/10.1016/j.envpol.2012.12.009.

10.Chetty, S.; Pillay, L. Assessing the Influence of Human Activities on River Health: A Case for Two South African Rivers with Differing Pollutant Sources. Environ Monit Assess 2019, 191, 168. https://doi.org/10.1007/s10661-019-7308-4.

11.Liu, J.-S.; Guo, L.-C.; Luo, X.-L.; Chen, F.-R.; Zeng, E.Y. Impact of Anthropogenic Activities on Urban Stream Water Quality: A Case Study in Guangzhou, China. Environ Sci Pollut Res 2014, 21, 13412–13419. https://doi.org/10.1007/s11356-014-3237-5.

12.Sinche, F.; Cabrera, M.; Vaca, L.; Segura, E.; Carrera, P. Determination of the Ecological Water Quality in the Orienco Stream Using Benthic Macroinvertebrates in the Northern Ecuadorian Amazon. Integr Envir Assess & Manag 2022, ieam.4666. https://doi.org/10.1002/ieam.4666.

13.Gerber, R.; Smit, N.J.; van Vuren, J.H.J.; Ikenaka, Y.; Wepener, V. Biomarkers in Tigerfish (Hydrocynus Vittatus) as Indicators of Metal and Organic Pollution in Ecologically Sensitive Subtropical Rivers. Ecotoxicol. 2018, 157, 307–317, doi:10.1016/j.ecoenv.2018.03.091.

14.Dalzochio, T.; Ressel Simões, L.A.; Santos de Souza, M.; Prado Rodrigues, G.Z.; Petry, I.E.; Andriguetti, N.B.; Herbert Silva, G.J.; Gehlen, G.; Basso da Silva, L. Water Quality Parameters, Biomarkers and Metal Bioaccumulation in Native Fish Captured in the Ilha River, Southern Brazil. CHEMOSPHERE 2017, 189, 609–618, doi:10.1016/j.chemosphere.2017.09.089.

15.Bradley, P.M.; Journey, C.A.; Berninger, J.P.; Button, D.T.; Clark, J.M.; Corsi, S.R.; DeCicco, L.A.; Hopkins, K.G.; Huffman, B.J.; Nakagaki, N.; et al. Mixed-Chemical Exposure and Predicted Effects Potential in Wadeable Southeastern USA Streams. Sci. Total Environ. 2019, 655, 70–83, doi:10.1016/j.scitotenv.2018.11.186.

16.Jeppesen, E.; Nõges, P.; Davidson, T.A.; Haberman, J.; Nõges, T.; Blank, K.; Lauridsen, T.L.; Søndergaard, M.; Sayer, C.; Laugaste, R.; et al. Zooplankton as Indicators in Lakes: A Scientific-Based Plea for Including Zooplankton in the Ecological Quality Assessment of Lakes According to the European Water Framework Directive (WFD). HYDROBIOLOGIA 2011, 676, 279–297. https://doi.org/10.1007/s10750-011-0831-0.

17.Kaboré, I.; Ouéda, A.; Moog, O.; Meulenbroek, P.; Tampo, L.; Bancé, V.; Melcher, A.H. A Benthic Invertebrates-Based Biotic Index to Assess the Ecological Status of West African Sahel Rivers, Burkina Faso. J ENVIRON MANAGE 2022, 307, 114503. https://doi.org/10.1016/j.jenvman.2022.114503.

18.Wu, J.; Mao, R.; Li, M.; Xia, J.; Song, J.; Cheng, D.; Sun, H. Assessment of Aquatic Ecological Health Based on Determination of Biological Community Variability of Fish and Macroinvertebrates in the Weihe River Basin, China. J ENVIRON MANAGE 2020, 267, 110651. https://doi.org/10.1016/j.jenvman.2020.110651.

19.Brack, W.; Aissa, S.A.; Backhaus, T.; Dulio, V.; Escher, B.I.; Faust, M.; Hilscherova, K.; Hollender, J.; Hollert, H.; Müller, C.; et al. Effect-Based Methods Are Key. The European Collaborative Project SOLUTIONS Recommends Integrating Effect-Based Methods for Diagnosis and Monitoring of Water Quality. Environ Sci Eur 2019, 31, 10. https://doi.org/10.1186/s12302-019-0192-2.

 

4. Comment:Method section describes the study area; whilst it is not fairly well written, it also lacks of justification as to why those areas / stations were selected, other than due to different human activities. The characteristics of Site 3 (which is far from river flow) is not described. Similarly, the inclusion of tributary sites is also not justified. Samples collection was briefly described for Site 1-4, but no information for tributary sites.

Reply: Thank you very much for pointing out this problem. The selection of sampling sites in our study was based on the different sources of pollution along the Jishan River, and the main sites where potential pollution exists were selected. Considering that the wastewater discharge from the goose farm would have an impact on water quality, sample points were set up in the tributaries of the Jishan River. We have made changes to the sampling point selection and sample collection in the method section, and the revised version is attached below for your review.

Study area: The sampling sites are located in the Juancheng County of Shandong Province, where it is on the lower reaches of the Yellow River. The water of the Yellow River flows through the water intake site into the Jishan River of the County. Based on the different sources of pollution along the Jishan River, the sampling sites included the primary locations of potential pollution. Of these, 4 sampling sites were selected on the main stream, i.e., site 1 (35°30′4.56″N, 115°21′18.28″E) was in water intake point of the Yellow River. The site 2 (35°26′45.12″N, 115°32′44.08″E) was in Shaheqiao, where is the drinking water source reservoir for the County. There is a city sewage treatment plant next to the river at site 3 (35°34′36.96″N, 115°31′26.06″E), and the sampling point will be located at its outlet, because the effluent from the plant is discharged into the river. The site 4 (35°39′18.53″N, 115°40′35.48″E) was located downstream of the rural area. In addition, considering that the wastewater discharge from goose farms will have an impact on the water quality, 6 sites were set up in a tributary where the goose farm lies on nearby. Tributary site 1 (TS1, 35°67′96.38″N, 115°57′07.23″E) was 10 km away from the estuary, TS2 was inside the goose farm (35°68′00.49″N, 115°58′07.93″E), TS3 (35°67′08.37″N, 115°59′01.68″E) and TS4 (35°67′71.39″N, 115°59′91.72″E) were 8.7 and 7.4 km away from the estuary, respectively. TS6 was in the estuary (35°66′18.11″N, 115°68′00.29″E), and TS5 (35°65′15.46″N, 115°61′01.47″E) was in another tributary as a reference, where no goose farm within the basin of this tributary.

 

5. Comment:Sampling strategy is also not clear, especially on how many samples altogether were collected. The methods also do not provide detailed description. For example, how the sample was divided into two parts for different analyse.

Reply:  We gratefully appreciate for your comment. Sample collection methods and the number of samples have been added to the revised methods section. We feel sorry for our carelessness.

Sampling and sample treatments: During field investigation, 100 mL glass vials, which were pre-cleaned with pure water and dried at high temperature in an oven (GW-024E, Juwei, China) at 120°C, were used to collect surface water samples at S1-4. A total of 12 surface water samples (4 sites × 3 replicates) were finally collected. Sediment samples were collected using a 1/40 Peterson grab sampler (surface area: 15 × 30 cm²) in triplicate from shallow to deep at the bottom of the river and then combined into one sample. Surface soil samples (0-25 cm) were collected at TS2 inside the goose farm using a shovel. Each sediment or soil sample consisted of three sub-samples, hence a total of 27 sediment samples and 3 surface soil samples were collected during the study. Each sediment and soil sample was then divided into two parts of equal weight, one prepared for toxicity testing and material identification and stored in tin foil bags at 4°C, and the other for benthic identification and stored in formalin solution.

 

6. Comment:For ZET test, please provide info where did zebrafish embryos were collected from.

Reply: Thank you for your valuable comment. We have added information about the collection of zebrafish embryos in the ZET test.

Adult zebrafish were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China) and maintained in the laboratory at a constant temperature of 26°C and 14 h of light/10 h of darkness. Adult females and males were placed in a 1:2 ratio in spawning boxes one hour before the start of the dark cycle the night before the experiment, and eggs were collected on the day of the experiment.

 

7. Comment:In Results section, authors use the term ‘linkages’ referring to the group of worms. I would suggest to classify them under the phylum i.e. Annelida. If there is any specific reason of using the ‘linkage’, then it needs to be described beforehand.

Reply: Thank you so much for your valuable suggestion. We have replaced "linkages" with "Annelids" in the text.

    A total of 21 benthic species were identified (Table 1), belonging to molluscs, arthropods and annelids. Among which, arthropods are the most common species, accounting for 67% of total species. The annelids (3 species) accounted for 14%, and molluscs (4 species) amounted to 19%.

 

8. Comment:Table 1 is confusing for the use of ‘N’ as it describes as ‘number of benthic species’. N normally refers to number of individuals while S normally refers to number of species.

Reply: We apologize for the confusion caused by the unclear presentation of terms. N in Table 1 refers to the total number of benthic individuals rather than the number of species. We have made the correction in Table 1. Thank you for your careful check.

 

9. Comment:I would suggest to be consistent in terms of structure for e.g. the Figure 6 should appear after paragraph, not in the beginning of the sub-section. It is the same case with Figure 7.

Reply: Thank you so much for your valuable suggestion. We have corrected the correlation analysis section by shifting the position of Figures 6 and 7 back to maintain a consistent structure throughout the text.

 

10. Comment:Figure 7 of the PCA should not only simply stating the obvious about the cluster, but also should also describe what is the meaning of cluster. For example, the closer the plot means they are more similar to those far away.

Reply: Thank you for your rigorous comment. We have added the results of the PCA analysis to the article and attached it below for your review.

Teratogenic rate of zebrafish embryos and Species diversity index were positively correlated with DO and pH since their intersection angles in the PCA plot were not greater than 90 degrees. However, their correlation with P, TP, ORP, TN, SPC is reversed.

 

11. Comment:Discussion section lacks arguments and discussions. For example, Section 4.1 mostly re-describe the results. Only a small part of this section provides the arguments and discuss as to why such findings were recorded. The same comment on the section 4.2 where it lacks of discussion and argument. If any, the discussion is mainly to compare the findings of this study with the previous studies, without really providing the reason behind the findings.

Reply: We feel sorry for the inconvenience brought to the reviewer. We have made changes to the discussion section. In sections 4.1 and 4.2, we have removed the parts that are repetitive with the results and added the reasons behind the results as arguments.

4.1 Water quality and benthic fauna: It has been known that benthic community reflect ecological quality and integrate the effects of different stressors providing a broad measure of their impact [34]. Therefore, benthic fauna is widely used as water quality indicators [12,35]. Its species diversity is a reflection of biome composition and structure [36]. Biological communities have their own natural evolutionary characteristics, while external disturbances such as changes in natural conditions and human activities may cause changes in the species composition of the community, and in the long run, the diversity of ecosystem structure will be destroyed [37,38]. In our study, we found that the biodiversity of freshwater ecosystems varied greatly among sample sites. The most abundant species taxa were found at sample site S2, and decreasing downstream to S3 and S4. This is consistent with previous studies by Huh et al [39] and Nestlerode, J.A. [40]. Species diversity is richer in flood risk areas and these results can be attributed to the natural vertical distribution of riverine fauna and the influence of artificial flood control systems. Many aquatic animals spend all or part of their life cycle in floodplains [41], and therefore species richness of freshwater fauna is usually higher in these areas. The sample sites located downstream had significant sediment deposition, reduced flow velocity, reduced habitat heterogeneity, and a decreasing trend in benthic faunal diversity. Moreover, due to the poor stability of the sandy riverbed, which is easily disturbed by water flow, as well as the lack of apoplankton input and limited organic matter input from upstream, the benthic fauna was dominated by shaker mosquito larvae with strong migratory ability and adapted to poor nutrition [42,43]. The lowest number of species was found at S1, which may be due to the sharp narrowing of the river channel at this point after the inflow of the Yellow River water into the territory of Yancheng, resulting in a high scouring force [44]. Also pollution is highly correlated with human activities in the watershed, which manifests itself as differences in species diversity in biological distribution [9]. Among the different types of anthropogenic disturbance intensity selected in this study, the analysis revealed that benthic organisms were more abundant and more uniformly distributed at the sample sites of drinking water reservoirs with less human activities, and more indicator organisms of clean water bodies were present, while the crowded areas such as urban sewage treatment plants and rural farms had less benthic organisms due to intensive human activities, and the input of domestic sewage and farming wastewater disturbed the original system of the water ecosystem.

4.2 Toxic effects to zebrafish embryos and ecosystem risk: Zebrafish is an important model organism for toxicological studies [45]. In this study, we assessed the ecological risk through zebrafish embryos and found that raw water presented teratogenic effects that were not significantly different from the control, and the effects were more pronounced when the embryos were exposed to sediment. This is due to the enrichment of the sediment with low-dose，hard-to-detect contaminants in the treatment. Multiple malformed embryos were found in the sediment samples at S2. A possible reason is the hydrological conditions of the sampling site. Frequent floods occur here and the river has been in operation for a long time, resulting in severe siltation, with a large average siltation depth [46]. The water level is raised thus creating an anoxic environment where organic matter is reduced under these conditions, producing alcohols and aldehydes as harmful substances and accumulating here [47].The high lethality at S3 and S4 may come from treated reclaimed water from sewage plants and from farm wastewater. Further analysis of samples collected from goose farms revealed that samples from within the farms were more toxic to zebrafish embryos [48], which may be due to the high level of eutrophication of water bodies caused by excessive levels of organic substances such as N and P in farm wastewater [49]. Total coliforms are another contamination factor in the surface water near the farm [50], resulting in poor overall water quality at this sampling site. This can be explained by the fact that poultry farms are usually located near densely populated residential areas, where domestic wastewater and livestock wastewater are discharged together into a common sewer system causing an integrated impact on water quality. This suggests that the source of pollutants is mainly from human activities, including urbanization and farming development. This is consistent with previous findings on endocrine disruptors in the area [51], which pose a challenge to the protection and preservation of drinking water resources.

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51.Tan, J., Liu, L., Li, F., Chen, Z., Chen, G.Y., Fang, F., Guo, J., He, M., Zhou, X., 2022. Screening of Endocrine Disrupting Potential of Surface Waters via an Affinity-Based Biosensor in a Rural Community in the Yellow River Basin, China. Environ. Sci. Technol. 56, 14350–14360. https://doi.org/10.1021/acs.est.2c01323

 

12. Comment:Conclusion is not convincing and does not really in-line with the tittle.

Reply: We appreciate that you have provided us with important comment. The conclusion is an important part of the paper and should echo the purpose of the study. Unfortunately, we were not able to do this. We have made changes in this section as you suggested and have attached the revised version below for your review. Thank you again for your comment.

The tests on zebrafish embryos indicated that sediments inside rural farm were more toxic than others, similar results were obtained from aquatic organism community. Therefore, it is of importance to protect the environment of farmed river sections in rural areas. This study can provide scientific basis and regulatory reference for the sustainable management of urban-rural rivers.

Reviewer 2 Report

Shao et al has carried out a comprehensive study and assessed water quality and healthy risk in a urban-rural river in the lower reaches of the Yellow River. The intent of the manuscript is really good as the authors analyzed the ecological healthy risk in an urban-rural river according to some indexes, and the results highlight the importance to protect watershed environment in rural reaches of the river in the watershed of the Yellow River.

 However, there are several issues which are of concern to me and I would like to draw the attention of the authors to this sectors. I recommend Reconsider after major revision. My specific comments are enumerated below:

1.   I do not know why you designed the Zebrafish embryotoxicity test, do you know the main pollutant in this river?

2.   In the statistical analysis part, do you conduct the normal test of the data? Because it is very important for the next analysis such as ANOVA and correlation analysis.

3.   The result part should be carefully checked. For example, in Figure 2B, it sounds no difference of P among four sites, but the authors added abcd above the column. In addition, why you set different colors in all sub-figures? I did not find the illustrations in the Figure caption.

4.   The Discussion part has been written in a very loose manner. The authors should discuss their results in depth.

Author Response

Reviewer 2

Shao et al has carried out a comprehensive study and assessed water quality and healthy risk in a urban-rural river in the lower reaches of the Yellow River. The intent of the manuscript is really good as the authors analyzed the ecological healthy risk in an urban-rural river according to some indexes, and the results highlight the importance to protect watershed environment in rural reaches of the river in the watershed of the Yellow River.

Reply: We are truly thanks for your comments and suggestions, which can help us improve the quality of the paper significantly.

 

However, there are several issues which are of concern to me and I would like to draw the attention of the authors to this sectors. I recommend Reconsider after major revision. My specific comments are enumerated below:

Reply: Thank you very much for your comments. We will modify our manuscript according to the reviewers’ suggestions.

 

1. Comment: I do not know why you designed the Zebrafish embryotoxicity test, do you know the main pollutant in this river?

Reply: Thank you for your comment. Zebrafish embryotoxicity is recognized as acute toxicity around the world, which has been frequently used for risk assessment in environments. We would like to investigate the water physio-chemical parameters, aquatic biota and the toxicity of the water body to illustrate the health status of this river.

 

2. Comment: In the statistical analysis part, do you conduct the normal test of the data? Because it is very important for the next analysis such as ANOVA and correlation analysis.

Reply: We performed normal distribution and variance homogeneity test in advance, when the assumptions of homogeneity of variance and normal distribution were not met, a Kruskal–Wallis H test with Dunn post hoc tests was performed to conduct multiple comparisons. We added “The normal distribution and variance homogeneity test in advance, when the assump-tions of homogeneity of variance and normal distribution were not met, a Kruskal–Wallis H test with Dunn post hoc tests was performed to conduct multiple comparisons.” into the data analysis section.

 

3. Comment: The result part should be carefully checked. For example, in Figure 2B, it sounds no difference of P among four sites, but the authors added abcd above the column. In addition, why you set different colors in all sub-figures? I did not find the illustrations in the Figure caption.

Reply: Thank you for your comments. We are sorry that we made a typing error for the letters above the column. We have re-corrected and changed the colors for sub-figures in revised manuscript.

 

4. Comment: The Discussion part has been written in a very loose manner. The authors should discuss their results in depth.

Reply: Thanks you for your comments. We made improved distinctly in discussion part according to comments from reviewers.

Author Response File: Author Response.pdf

Reviewer 3 Report

Figure 1: It would be good to indicate/show the activates around the sampling sites on the map.

Line105: How were they cleaned?

Line 106: Do you mean triplicates I do not understand what you mean.

Fig 6: Some of the figures are not clear.

Line 303: In your view what accounted for the high biodiversity at S2. can the surrounding contribute to this.

Line 315-317: at S2 Where are the toxins coming from? at S3 and 4 they are from the Goose wastewater. is the wastewater from the Goose farm discarded directly into the river without treatment?

Line 322-324: The E.coli from human activity is this again due to untreated discharge wastewater?

Conclusion: Why were heavy metals and nutrient (nitrate and phosphates) level of the river not tested? these could have a negative effect on the water quality especially from human activity.

 

Author Response

Reviewer 3

1. Comment:It would be good to indicate/show the activates around the sampling sites on the map of Fig.1.

Reply: Thank you for your valuable suggestions. We have re-described the criteria for setting sampling points in the revision process. The type of human activity has been changed to the different sources of pollution along the river and the type of pollution source at the sample point has been briefly described in the sample point setting.

Study area: The sampling sites are located in the Juancheng County of Shandong Province, where it is on the lower reaches of the Yellow River. The water of the Yellow River flows through the water intake site into the Jishan River of the County. Based on the different sources of pollution along the Jishan River, the sampling sites included the primary locations of potential pollution. Of these, 4 sampling sites were selected on the main stream, i.e., site 1 (35°30′4.56″N, 115°21′18.28″E) was in water intake point of the Yellow River. The site 2 (35°26′45.12″N, 115°32′44.08″E) was in Shaheqiao, where is the drinking water source reservoir for the County. There is a city sewage treatment plant next to the river at site 3 (35°34′36.96″N, 115°31′26.06″E), and the sampling point will be located at its outlet, because the effluent from the plant is discharged into the river. The site 4 (35°39′18.53″N, 115°40′35.48″E) was located downstream of the rural area. In addition, considering that the wastewater discharge from goose farms will have an impact on the water quality, 6 sites were set up in a tributary where the goose farm lies on nearby. Tributary site 1 (TS1, 35°67′96.38″N, 115°57′07.23″E) was 10 km away from the estuary, TS2 was inside the goose farm (35°68′00.49″N, 115°58′07.93″E), TS3 (35°67′08.37″N, 115°59′01.68″E) and TS4 (35°67′71.39″N, 115°59′91.72″E) were 8.7 and 7.4 km away from the estuary, respectively. TS6 was in the estuary (35°66′18.11″N, 115°68′00.29″E), and TS5 (35°65′15.46″N, 115°61′01.47″E) was in another tributary as a reference, where no goose farm within the basin of this tributary.

 

2. Comment: How were they cleaned in Line 105?

Reply: The 100 mL glass vials were used to collect surface water samples after prior washing with pure water and drying in an oven at 120°C. We have made changes in the article. Thank you very much for your careful check.

During field investigation, 100 mL glass vials, which were pre-cleaned with pure water and dried at high temperature in an oven (GW-024E, Juwei, China) at 120°C, were used to collect surface water samples at S1-4.

 

3. Comment: Do you mean triplicates in Line 106? I don’t understand what you mean.

Reply: We took three replicate samples at each sampling site and then mixed them into one sample mixture, meaning that each sample contains three sub-samples. We have also reorganized the language in the methods section and have attached the revised version here for your review.

Sediment samples were collected using a 1/40 Peterson grab sampler (surface area: 15 × 30 cm²) in triplicate from shallow to deep at the bottom of the river and then combined into one sample. Surface soil samples (0-25 cm) were collected at TS2 inside the goose farm using a shovel. Each sediment or soil sample consisted of three sub-samples, resulting in 27 sediment samples and 3 surface soil samples collected during the study.

 

4. Comment:Some of the figures are not clear in Fig.6.

Reply:  We have re-adjusted the layout of Fig.6 to make it look clearer.

 

5. Comment:In you view what accounted for the high biodiversity at S2. Can the surrounding contribute to this?

Reply: High biodiversity was associated with surroundings of the sample site. Floods are frequent at this site, and many aquatic animals spend all or part of their life cycle on the floodplain, resulting in higher species diversity. The low level of anthropogenic disturbance and low disruption of aquatic community structure is another reason for the richness of biodiversity at this site.

 

6. Comment:Where are the toxins coming from at S2? They are coming from the Goose wastewater at 3 and 4. Is the wastewater from the Goose farm discarded directly into the river without treatment?

Reply: The river is silted up at S2 thus creating an anoxic environment where organic matter is reduced and harmful substances such as alcohols and aldehydes are produced and accumulated. The farm wastewater is treated, but the treatment is not effective enough so that the effluent still has a potential impact on organisms.

 

7. Comment: The E.coli from human activity is this again due to untreated discharge water?

Reply: Thank you very much for your question! We have added this to the revised discussion section. The results of the analysis of the samples collected inside the farms showed that they were more toxic to zebrafish embryos, so it was assumed that an important factor in the contamination of surface water inside the farms was E. coli from livestock manure.

Discussion: Further analysis of samples collected from goose farms revealed that samples from within the farms were more toxic to zebrafish embryos [48], which may be due to the high level of eutrophication of water bodies caused by excessive levels of organic substances such as N and P in farm wastewater [49]. Total coliforms are another contamination factor in the surface water near the farm [50], resulting in poor overall water quality at this sampling site. This can be explained by the fact that poultry farms are usually located near densely populated residential areas, where domestic wastewater and livestock wastewater are discharged together into a common sewer system causing an integrated impact on water quality. This suggests that the source of pollutants is mainly from human activities, including urbanization and farming development. This is consistent with previous findings on endocrine disruptors in the area [51], which pose a challenge to the protection and preservation of drinking water resources.

 

8. Comment:Why were heavy metals and nutrient (nitrate and phosphates) level of the river not tested? These could have a negative effect on the water quality especially from human activity.

Reply: That is a really good suggest! We will do it in our further study.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have addressed my comments in satisfaction.