Point by Point Response to the Reviewers Comments for the Manuscript
Point by point response to the reviewers’ comments for the manuscript:
AECT-D-12-3447
Assessing the Chronic Toxicity of Atrazine, Permethrin and Chlorothalonil to the Cladoceran Ceriodaphnia cf. dubia in Laboratory and Natural River Water
Comments from Reviewer 1
General Comment
1. The main argument that the authors use to explain the similarity in toxicity found using the standard exposure medium versus ecologically relevant natural river water was the lack of organic matter the latter. However it is not clear why the TOC levels of in ? 7 mg/l was regarded as low? It would be useful to provide an indication of what concentration will be regarded as high DOC in freshwaters. Also in the introduction the role of suspended matter in reducing the toxicity of organic pollutants is mentioned and also used as supporting argument for the absence of differences in toxicity in conclusions section, but the SPM concentrations are not reported in the paper. If available then these should be provided.
Response 1
We compared the toxicities of three pesticides between Laboratory water and River water. Since river water contained natural water constituents such as DOC and SPM, we assumed that these natural water characteristics may modify the resulting toxicities of the chemical tested. The Total Organic Carbon (TOC) content of the Colo river water was in the range of 6-8 mg/L, which is the average range for most rivers. Gundersen and Steinnes (2003) mentioned that TOC level greater than 8 mg/L was regarded as high.
We measured and reported the TOC level in our study as TOC consists of both dissolved organic carbon (DOC) and particulate organic carbon. Hence, we did not provide either DOC or SPM concentrations individually.
2.Avoid one sentence paragraphs.
Response 2
The one sentence paragraph has been amended as suggested. Please see (Li 82-86) in the revised MS.
3. The authors mention that percentage recoveries (li. 256) of what I would assume are the nominal concentrations. Were there any other measures taken for quality assurance, e.g. procedural blanks? To me the random verification of the administered concentrations would be sufficient but I am not sure what editorial policy is.
Specific comments:
Response 3
The percent recoveries mentioned in (li 253-254 in revised MS) for atrazine, chlorothalonil and permethrin (95, 85 and 80%, respectively) were the measured concentrations of spiked solutions of atrazine, chlorothalonil and permethrin prepared in Colo river water. The percent recoveries were used as correction factors for the corresponding pesticides.
Nominal treatment concentrations were used in this experiment; however, the treatment concentrations were measured in a number of randomly selected treatments. We have not included the actual measured concentrations of the treatment solutions in the manuscript as we did not measure them for every treatment. Instead, we measured the treatment concentrations in a number of randomly selected treatments and used these analyses as a verification of the nominal concentrations used. Furthermore, we renewed the treatment solutions every 24-hours over the 7-day test period (it is mentioned in Experimental Design Li 219-220 revised MS) to ensure pesticide concentrations were maintained close to nominal values. The budget also did not allow us to do measurements for all the toxicity tests as they are very costly and labour intensive.
4. Li. 149 - space between value and unit for conductivity.
Response 4
This has been changed as suggested.
5. Li. 152 - degree sign.
Response 5
This has been corrected (Li 150 revised MS)
6. Li. 153 - what was analysed in the Colo River samples? The parameters mentioned in the previous paragraph? If so then switch the two paragraphs.
Response 6
The Colo river water samples were extracted and analysed to measure the concentrations of atrazine, permethrin and chlorothalonil. Hence we do not wish to switch the two paragraphs. However, Li 152 (revised MS) has been amended to provide more information.
7. Li. 162 - dubia should be lower case.
Response 7
This has been amended (Li 160 revised MS).
8. Li. 170 - what is the reason for including Perrier water into the exposure medium?
Response 8
5% Perrier® mineral water was added to generate moderately hard (USEPA 1994) reconstituted laboratory water that is optimal for culturing cladocerans as well as providing sufficient essential trace elements (USEPA 1994).
9. Li. 196 and 198 - first reference to the terms "LW" and "RW" so provide full term and abbreviation in brackets.
Response 9
The above comment has been addressed (Li. 193 and 196 Revised MS).
10. Li. 206 - why and what solvent control was used in this study?
Response 10
Solvent control was used to make sure that the test animals were not affected by the carrier solvent. A carrier solvent was used for the preparation of pesticide stock solutions. In our study, acetone was used as a carrier solvent and the maximum volume used was not more than 0.5% of the total volume of treatment solution.
Solvent and blank control treatments were run in conjunction with all pesticide tests in order to determine if the amount of solvent used in the highest concentration caused any lethality. In no case did the solvent cause lethality that was significantly different to that of the control indicating that the solvent had no toxic effect.
11. Li. 212 - space between unit and "for".
Response 11 This has been amended (Li 211revised MS)
12. Li. 256 - Rather provide range or average and RSD of recoveries than only the average.
Response 12
We provided the percent recovery results for atrazine, permethrin and chlorothalonil. We used the term average as we extracted and analysed three replicates for each chemical and used the average of them. However, we have now added the recovery range for each pesticide. Please see Li 253- 254 in the revised MS.
13. Li. 280 - consistency in the use of lower case p. From this point onwards the capital P is used.
Response 13. All capital Ps have been replaced with lower case p.
14. Li. 326 - data are
Response 14.
This has been amended (Li. 324 revised MS)
15. Li. 333 - is there any possible reason why chlorothalonil was more toxic (albeit non-significant) in RW when compared to the other pesticides that had greater toxicity in LW?
Response 15
We have checked the concentrations of all three pesticides in the Colo River water prior to use in the toxicity tests as diluent and none of the three pesticides was detected in the river water. Given the log Kow value of chlorothalonil, we expect that its EC50 value in LW would have been lower than that in RW, but this was not the case. Hence, we do not suggest any reason why the EC50 value of chlorothalonil in RW was lower than that in LW.
16. Li. 342 - insert full stops after et al. as well as after sentence "?(1990)."
Response 16.
Li. 340 (revised MS) has been amended.
17. Li. 344 - why is there referral to DOM here and then to TOC in li. 346? Are there values for the DOM and SPM to support these arguments?,
Response 17.
Li. 342 (revised MS) we were just referring to the water constituents such as DOM or suspended sediment individually instead of using the general term TOC. As we have already mentioned above, TOC consists of dissolved, colloidal and particulate fractions of organic carbons in river water. However, in Li. 342 (revised MS), we refer to TOC as a general term. We measured TOC of the river water used in our study, but we did not measure either DOC or SPM individually.
18. Li. 355 and 356 - use the abbreviated genus names for those genera that have been provided earlier in the paper, e.g. Daphnia carinata, Paratya, etc.
Response 18.
The genus name for daphnia was provided earlier in the paper hence the abbreviated genus name is subsequently used (Li. 353 revised MS), however, the genus name for other species are provided as they have not been mentioned earlier in the paper.
19. Li. 364 - remove comma before data of reference
Response 19.
Li. 362 (revised MS) it has been amended.
20. Li. 365 - use abbreviation for DOM and provide full term where it is mentioned the first time in li. 344.
Response 20
Li. 342 (revised MS) and Li. 363 (revised MS)- have been amended.
201. Li. 367 - ".. there are no data?"
Response21.
This has been amended (Li. 365 revised MS)
22.Li. 371 - wording e.g. "Work by Yang et al. (2006) indicates that?"
Response 22
We would like to leave it as it is (Li. 366 revised MS).
23. Li. 391 - once again how much DOM and SPM and are these regarded as high or low?
Response 23
This comment has been addressed in the earlier (See Response 1) .
We stated in Li. 388-391(revised MS) that “Despite having moderate organic carbon-water partition coefficients, testing in river water, containing dissolved organic matter and suspended particles, did not significantly (p > 0.05) alter the toxicity and bioavailability of atrazine, chlorothalonil and permethrin to C. cf. dubia compared to testing in laboratory water”. We refer to Table 1 which provides the physico-chemical properties of the three pesticides including the Log Kow values (organic carbon-water partition coefficients). All three pesticides have moderate Log Kow values as given in the Table. We just suggested that since we used natural river water as the diluent, it would definitely contain DOM and SPM; however, since we measured only the TOC of the river water, we are unable to provide the concentrations of DOM or SPM.
4. Li. 547 - remove comma before date.
Response 24
The above mentioned comment was not found.
25. Table 2 - the fiducial range provided for the molar LC25 does not make sense.
Response 25.
We are providing a complete set of results including LC25 values and their fiducial ranges as mole/L values. Therefore, we would like to keep them as they are.
Comments from Reviewer 2
Abstract
Page numbers used refer to the revised MS.
1. P2, line 31: change "amount" to "amounts"
Response 1
This has been amended.
2. P2, line 40: change "classed" to "classified"
Response 2
.This has been amended
3. P2, line 44: change "tested pesticides" to "pesticides tested"
Response 3
We prefer to leave it as it is.
4. P2, line 44: change "provides" to "provided"
Response 4
This has been amended
Introduction
5. P3, line 64-65: change "environment that omit" to "environment, omitting"
Response 5
P3, Li. 65 This has been amended
6. P5, line 115: add space after "membranes"
Response 6
P5, line 115 This has been amended.
Materials and Methods
7. P6, line 138: delete return before "Table 1."
Response 7
P6, line 136 This has been amended
8. P6, line 149: check DO value, should it be "100.2%"?
Response 8
P6, line 147: DO value has been corrected to "102%"
9. P6, line 149: need to explain the relationship between the TOC values presented and DOM. Also, the study is based on comparing toxicity with and without DOM. Need to explain whether the TOC values in the Colo River are considered low, high?
Response 9
This comment has been addressed in the earlier comments made by the Reviewer1 (See Response 1).
10. P6, line 154: change "schott" to "Schott"
Response 10
P6, line 153 This has been amended
11. P6, line 155: change "25 C" to "25°C"
Response 11
P6, line 155 This has been amended
12. P7, line 163: change "US" to "U.S."
Response 12
P7, line 160 This has been amended
13. P7, line 163: change "Dubia" to "dubia"
Response 13
This has been amended
14. P8, line 193: insert "water (LW)" after "laboratory"
Response 14
P8, line 193 This has been amended
15. P8, line 193: change "river waters" to "river water (RW) with"
Response 15
P8, line 193 This has been amended
16. P8, line 195-198: The experimental design is not ideal for testing the difference in toxicity of a chemical under different conditions. The individual chemicals should have been tested with both water types at the same time.
Response 16
P8, line 190-196 We agree with the reviewer’s comment that the individual chemicals should have been tested with both water types at the same time. Although we conducted the toxicity tests in laboratory and river waters separately, a LW control (with ten replicates) was added in the toxicity tests conducted in RW. If the LW controls from LW and RW tests were not significantly (P > 0.05) different, then the results of both sets of experiments could validly be compared. This is explained in Li. 198-204 Pg 8.
17. P8, line 200-201: this sentence is unclear. The second set of experiments is stated as being in RW, but this states that LW was used
Response 17.
This sentence has been deleted.
Discussion
P 14, line 310-313: Suggest re-word and include reference to chlorothalonil's effect on glutathione. Could also include toxicity values for D. magna as comparison.
P 14, line 345-347: Why was this river water used if it did not contain sufficient DOM to bind the pesticides? Why not use other river water or add DOM to make a valid comparison?
Response14.
We did not state the effect of chlorothalonil on glutathione in our MS. We would like to keep the discussion as it is since we have done this part with all the available results from literature.
Colo river water was used as it is one of the most pristine rivers in NSW and contains an average concentration range of TOC. We wanted to find out if the water constituents such as DOC and SPM in natural water (within the average range) would modify the toxicity of the pesticides. We did not want to add DOM to the river water to modify the pesticide toxicity, which may have caused a significant difference in toxicity. We just wanted to investigate the effect of natural river water on the toxicity of pesticides tested. Hence, we are reporting a very valid comparison of two different water types with and without natural constituents at a level, which is environmentally relevant.