# External Peer Review of EPA’s Draft Reassessment of the

1999 Ambient Water Quality Criteria for Ammonia

– Freshwater

Peer Reviewer Comments Final Report

EPA Contract No. EP

- C

- 07

- 059 Work Assignment No. 1

- 04

Submitted to:

Lisa Huff Health and Ecological Criteria Division Office of Science and Technology U.S. Environmental Protection Agency 1200 Pennsylvania Avenue, NW Washington, DC 20460

Submitted by:

Eastern Research Group, Inc. 110 Hartwell Avenue Lexington, MA 02421

- 3136

Septembe r 21 , 2009 Printed on Recycled PaperQUALITY NARRATIVE STATEMENT

ERG selected reviewers according to selection criteria provided by EPA. EPA confirmed that the scientific credentials of the reviewers proposed by ERG fulfilled EPA’s selection criteria. Reviewers conducted the review according to a charge prepared by EPA and instructions prepared by ERG. ERG checked the reviewers’ written comments to ensure that ea ch reviewer had provided a substantial response to each charge question (or that the reviewer had indicated that any question[s] not responded to was outside the reviewer’s area of expertise). Since this is an independent external review, ERG did not edit the reviewers’ comments in any way, but rather transmitted them unaltered to EPA.i



## Table of Contents

SECTION A: Technical Charge to Peer Reviewers

................................ ................................ ............. A

- 1

SECTION B : Peer Review Comments Organized by Charge Question

................................ ............ B

- 1

Acute criteria in fresh waters

................................ ................................ ................................ .................... B

- 3 1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?

................................ ................................ .. B

- 3

2

. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?

................................ .......... B

- 7

3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe there is an alternative approach to the use of this d ata that would be more scientifically sound?

................................ B

- 11

4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scientifically defensible principle for structuring the population exposure duration problem and designing further research to quantify such a duration?

................................ ................................ ......................... B

- 15
Hyalella azteca position statement and proposed rationale

................................ ................................ ..... B

- 19 5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientificall y sound recommendations?

................................ ................................ ................................ ..................... B

- 19

Chronic Freshwater Criterion

................................ ................................ ................................ ................. B

- 21 6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such use? Are you aware of other relevant data that were not used?

................................ .. B

- 21

7. Is the freshwater chronic criterion scientifically defensible with mussels present and mussels absent?

................................ ................................ ................................ ................................ ....... B

- 25

Use of 28

- day Juvenile Test Data

................................ ................................ ................................ ........... B

- 29 8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defe nsible as "chronic" test data for criteria development?

................................ ................................ ................................ ................. B

- 29

9. Should toxicity studies on the growth rates of mussel shells dur ing 28

- day tests be considered quantitatively when developing water quality criteria?

................................ ........... B

- 33

10. Regarding the position statement and rationale on use of juvenile mussel growth data

– ........ B

- 35

11. The values of the acute and chr onic ammonia criteria have a strong dependence on pH. Juvenile and adult mussels, as sediment

- dwelling organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have suggestions on how states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?

................................ .................... B

- 39ii

12. In general, should the criteria include a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically def ensible way to account for these differences when deriving protective water quality criteria?

......................... B

- 43

13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?

................................ ................................ ................................ ................................ .... B

- 45

Additional Comments

................................ ................................ ................................ ............................. B

- 49



### SECTION C: Individual Peer Reviewer Comments

................................ ................................ ........ C

- 1 Alex Barron

................................ ................................ ................................ ................................ .. C

- 3 Steven P. Canton. MS

................................ ................................ ................................ ................ C

- 17 Jerome M. Diamond, Ph.D.

................................ ................................ ................................ ....... C

- 31 George Dixon, Ph.D.

................................ ................................ ................................ .................. C

- 47 Jerry L. Farris, Ph.D.

................................ ................................ ................................ .................. C

- 53 Will iam L. Goodfellow Jr., MS

................................ ................................ ................................ . C

- 63 James J. Pletl, Ph.D.

................................ ................................ ................................ ................... C

- 71A

- 1 

## Charge to Peer Reviewers
EPA’s Draft Reassessment of the 1999 Ambient Water Quality

Criteria for Ammonia

– Freshwater

July 24, 2009

The document to be reviewed is a draft revision of EPA’s aquatic life criterion for ammonia. The new criterion is intended to replace the current aquatic life criterion previously updated in 1999.

BACKGROUND: The aquatic life criterion for ammonia is used for determining the level of pollution control necessary to attain the aquatic life uses of water bodies, and in particular to allow the propagation of fish and shellfish, per Section 302 of the Clean Water Ac t.
In a 2004 interagency memo EPA stated that it would conduct a criteria re

- evaluation of the 1999 ammonia aquatic life criteria in light of new studies indicating that freshwater mussels may be more sensitive to ammonia than the organisms considered in the 1999 Update of Ambient Water Quality Criteria for Ammonia. In addition, a Federal Register Notice (FRN) was published in 2004 notifying the public of the criteria re

- evaluation and requesting any pertinent toxicity data. Responding to comments from t he FRN regarding concerns about uncertainty in the new test protocol and data, in 2005 EPA hosted the Mussel Toxicity Testing Protocol workshop to convene experts to discuss the new test protocol for glochidia and juvenile mussels.

The current draft cri teria reassessment of the 1999 Update incorporates new toxicity data on freshwater mussels as well as snails and a couple of ESA listed fish species. The focus of the 1999 Update was on the chronic criteria whereas this reassessment updates both the acute and chronic criteria. The current draft recommendation includes criteria for waters with mussels present and another applied to waters with mussels absent.

DOCUMENT TO BE REVIEWED

The document is titled “Reassessment of the 1999 Ambient Water Quality Criteria for Ammonia

- Freshwater”. The document presents acute and chronic ammonia criteria for fresh waters with mussels present and a separate criterion for waters with mussels absent.A

- 2



### CHARGE QUESTIONS
Acute criteria in fresh waters:

1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?



### 2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?

3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe there is an alternative approach to the use of this data that would be more scientifically sound?



### 4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scie ntifically defensible principle for structuring the population exposure duration problem and designing further research to quantify such a duration?

Hyalella azteca position statement and proposed rationale (see Appendix B):
The EPA workgroup developed a position statement and proposed supporting rationale describing the concerns over using Hyalella azteca toxicity test data in criteria development due to the uncertain health of the test organisms in different test water composition. The rationale defin es the specific concerns and uncertainties supporting the recommended exclusion of the Hyalella data from use in criteria derivation, at this time; the position statement is based on the workgroup’s review of a number of toxicity tests on Hyalella, referen ced in the rationale.

5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?

Chronic Freshwater Criterion:

6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such use? Are you aware of other relevant data that were not used?

7. Is the freshwater chronic criterion scientifically defensible with mussels present and mussels absent?A

- 3



### Use of 28

- day Juvenile Test Data (see Appendix C):
Water quality criteria for the protection of aquatic life are derived using toxicity endpoints that relate to population level impacts. In general, these end points relate to survival, growth and/or reproduction. The 28

- day test with juvenile mussels, while similar in duration to a standard chronic test, is not technically an early

- life stage test according to the 1985 Guidelines for Aquatic Life Criteria as m uch of the early development will have already occurred.

8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defensible as "chronic" test data for criter ia development?

9. Should toxicity studies on the growth rates of mussel shells during 28

- day tests be considered quantitatively when developing water quality criteria?

10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chanc e they could recover or stabilize is one potential outcome only)?

11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Juvenile and adult mussels, as sediment

- dwelling organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have suggestions on how states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?

12. In general, should the criteria include a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically defensible way to account for the se differences when deriving protective water quality criteria?

13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?B

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## SECTION B

Peer Review Comments Organized by Charge QuestionB

- 3



## Acute criteria in fresh waters

1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?

Alex Barron Yes, all the toxicity tests that influence the derivation of the new criteria for ammonia appear to be scientifically defensible. All relevant data that I am aware of has been considered in this reassessment document.

Steven Canton Yes, in general the toxicity tests used to derive the water column criteria , as cited in

the draft update, are scientifically defensible . Plus, it appears they provide sufficient data to evaluate inter and intra

- species comparisons using the guidelines established for de riving water quality criteria.
Additionally, a few years ago, we conducted a review of a number of ambient water quality criteria (AWQC) as part of the Arid West Water Quality Research Project (AWWQRP 2006). One aspect of that project included a review and update to the 1999 EPA Ammonia criteria document, similar to this draft. Checking those updates side

- by

- side, it appears there are a few more studies that could be included in this re

evaluation:
Augspurger, T., A.E. Keller, M.C. Black, W.C. Cope, a nd F.J. Dwyer. 2003. Water quality guidance for protection of freshwater mussels (Unionidae) from ammonia exposure. Environmental Toxicology and Chemistry 22:2569

- 2575.
Khatami, S. H., D. Pascoe, M. A. Learner. 1998. The acute toxicity of phenol and uni onized ammonia, separately and together, to the ephemeropteran Baetis rhodani (Pictet). Environmental Pollution 99:379

- 387.
Rubin, A.J., and M.A. Elmaraghy. 1977. Studies on the toxicity of ammonia, nitrate and their mixtures to guppy fry. Water Research 11:927

- 935.
Russo, R.C., D.J. Randall, and R.V. Thurston. 1988. Ammonia toxicity and metabolism in fishes. In R.C. Ryans, ed. Protection of River Basins, Lakes, and Estuaries . pp. 159

- 173. American Fisheries Society, Bethesda, Maryland.
Sangli, A. B. and V. V. Kanabur. 2001. Toxicity of ammonia to a freshwater fish, Gambusia affinis and its effect on oxygen consumption. Geobios 28:56

- 58.
Tomasso, J.R., C.A. Goudie, B.A. Simco, and K.B. Davis. 1980. Effects of environmental pH and calcium on ammonia toxic ity in channel catfish. Transactions of the American Fisheries Society 109:229

- 234.
I should also note that in that prior review of the 1999 Ammonia document (AWWQRP 2006), we raised concerns regarding the appropriateness of some studies/data used in the 1999 EPA document (see Table 1 below). These studies may need an additional evaluati on to determine if they provide toxicity data meeting EPA data quality guidelines (Stephan et al. 1985).B

- 4 Table 1 Deletions of Inappropriate Data Presented in the EPA 1999 AWQC Ammonia

Document (from AWWQRP 2006)

Species References Comments Acute Simocephalus vetulus Mount 1982 Insufficient data to validate results Oncorhynchus mykiss Calamari et al. 1977 Unable to validate citation or data

- other data available by same author with this species Oncorhynchus mykiss Thurston et al. 1981 Unable to validate citation or data Oncorhynchus mykiss Reinbold & Pescitelli 1982 Unable to validate citation or data

Pimephales promelas Thurston et al. 1981 Unable to validate citation or data Pimephales promelas Reinbold & Pescitelli 1982 Unable to validate citation or data

Gambusia affinis Wallen et al . 1957 Insufficient data to validate results Lepomis macrochirus Reinbold & Pescitelli 1982 Unable to validate citation or data

Sander vitreum Reinbold & Pescitelli 1982 Unable to validate citation or data

Chronic Ictalurus punctatus Colt & Tchobanoglous 1978 Insufficient data to validate results Source: ( AWWQRP 2006)

Jerome Diamond In general, the toxicity tests used to derive ammonia criteria in the draft Reassessment Document (RD) are scientifically defensible given EPA’s criteria development guidelines (Stephan et al. 1985). However, I have reservations regarding the use of snail data derived from field

- collected organisms as explained below under Chronic Freshwater Criterion . I am not aware of other relevant freshwater toxicity test data that were not either used or considered in the RD.

George Dixon I am familiar with the majo rity of the literature cited and, in my opinion, the tests used

to develop the acute criteria are scientifically defensible. I am, however, relying on the fact that the Draft Reassessment states that a process to identify data meeting the minimum requireme nts for inclusion was followed. I have not obtained all of the cited literature and reviewed each paper for suitability of inclusion of the findings in the data sets; this type of detailed audit is beyond the scope of this review. I follow the ammonia lit erature closely, and I am unaware of any additional data that could be of

use.

Jerry Farris With greater recent attention dedicated to standardization of test techniques suitably addressing the feasibility of repeatability and precision of results from ju venile mussel responses (Augspurger, 2007), data available at the time of this reassessment meet a higher standard of acceptability for guidance modified from the US EPA (Stephan et al., 1985) and follow consensus test protocols and quality assurance recom mendations sufficient to support water quality criteria development. The majority of tests used to derive the criteria seem scientifically defensible for such use. Upon review of those included and others not specifically included in the calculation, bu t cited in Table 1 of the draft addendum, there are tests that present concern for acclimation procedures ofB

- 5 test organisms and culturing (either reported or unreported) and lack of reference testing that can insure level of expertise or consistency associ ated with such field collection, organism transfer, and culturing that can significantly influence test responses. Specifically, sufficient acclimation information for water quality and temperature was not included in Mummert et al., 2003 and Scheller, 19 97.
I am not aware of other data from standardized toxicity tests with ammonia that could be considered relevant to this reassessment.

Bill Goodfellow From my review, I believe that the acute toxicity testing data is relevant. My only concern is that this document was careful to use data that was from published references which I believe is a strength of this document as compared to earlier water quality criteria documents. Since the only unpublished data used that demonstrates a somewhat low toxicity response to ammonia was from Keller 1999 for the Pondshell Mussel, maybe putting a synopsis as an attachment would be warranted. This would help make the document transparent as the other Attachments help do.

The Table A in text of the GMAV for the Moun tain Whitefish does not match Table 1. The Text table reports 12.11 and Table 1 reports 12.09. I am expecting that the Table 1 is correct and is what was used for the FAV. Please verify.

Jim Pletl The question requires that the term “scientifically defensible” be defined within the context of the use or the consequences of the use. The use is to support development of water quality criteria that will be promulgated by states as legally enforceable water quality standards. Failure to meet water quality standards instream and/or at the edge of respective mixing zones will result in TMDLs, implementation plans, or NPDES permit limits for dischargers to the respective stream. Failure to meet water qual ity standards also infers that designated uses are not being supported. In the case of ammonia criteria, the designated use is aquatic life. The Clean Water Act also states that violations of such permit limits can result in significant monetary fines an d/or jail time for responsible individuals. Therefore the use, in this case, is quite demanding of the data.
The level of scientific defensibility set by EPA for this use is limited to that defined by EPA’s 1985 “Guidelines For Deriving Numerical Natio nal Water Quality Criteria For The Protection of Aquatic Organisms And Their Uses”, hereafter referred to as the Guidelines, and the published, peer reviewed toxicity test methods used in each case. The tests in question appear to meet these standards; ho wever the data quality objectives set by these documents may not be sufficient for the data use. The toxicity test methods and the Guidelines do not require what are considered to be critical elements of scientifically defensible toxicity tests. Acute te sts typically only have one quality objective: minimum survival in controls. Other than control survival there are no other quality objectives that must be met for the results of these tests to be considered reliable. For example, 1) the methods and Guid elines do not require a concentration

- response curve for data to be deemed acceptable for any use. There can be little certainty in a toxicity test result where response does not change predictably with the concentration exposure. If demonstration of thi s curve is not required of the test or EPA, data of questionable quality may be used to derive water quality criteria. 2) Analysis of acute toxicity test data typically also does not address the intra

treatment variability that commonly occurs in these te sts; providing results with biased estimates on uncertainty. 3) The Guidelines also do not require replication of any test result for the most sensitive species. This would allow a single test result toB

- 6 significantly bias the calculation of water quality criteria. ASTM E2455

- 06 for testing freshwater mussels recommends that “different batches of the same species and the same life stage should be collected and tested over time in order to obtain a measure of the variability associated with testing the par ticular species”. This goal was not accomplished for the pink mucket, the third most acutely sensitive genus of the database.
Aside from universal issues for acute tests 4) there is also concern that the acute juvenile mussel tests may be biased toward s findings of more sensitivity and toxicity. The tests proposed to be used for this reassessment are conducted without sediment in the test vessels. Newton and Bartsch (2007) found that without sediment juvenile organisms did not grow in 96 hour toxicity tests. This clearly shows that the organisms are stressed by the lack of sediment in the test vessels. This also strongly suggests that the survival endpoints from acute tests may be biased low (falsely indicating greater toxicity) due to the added stre ss of failing to provide sediment in the test vessels. All of these issues characterize a significant level of uncertainty which is indirectly proportional to scientific defensibility. EPA should address these uncertainties before using new data to updat e this criteria document.

I am not aware of other relevant data that were not used for this reassessment.B

- 7

2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?

Alex Barron Table B compares Final Acute Value (FAV) and Criterion Maximum Concentration (CMC) values calculated with the entire dataset including data for bivalve mollusks and also with the dataset with the data for bivalve mollusks removed. The Asiatic clam ( Corbicula fluminea ) is in a different Family ( Cobiculidae ) and Order ( Veneroida ), and is not a Unionid mussel. Corbicula has shown very similar sensitivity to ammonia compared to Unionid mussels howev er. Table B really represents a non

- bivalve mollusk dataset, which would be appropriate where no bivalve mollusks of any kind are present. The FAV and CMC calculated without the data for bivalve mollusks is less than a factor of two different than the FA V using the entire dataset, which is 62.75 % higher. The 1999 ammonia criteria also provided separate recommended acute criteria based on observed sensitivity of salmonid species. The 1999 criteria document also presented separate acute criteria that ar e approximately 50 % different between the salmonids present or absent acute criteria. The recommendations in the 2009 reassessment document providing separate criteria , calculated with and without a group of sensitive species is in keeping with precedent s set in the earlier ammonia criteria for calculating separate criteria when one group of species appears to be especially sensitive to ammonia. This can be considered appropriate for ammonia criteria because of the relatively large database and the fac t that ammonia toxicity is usually caused by mg/L concentrations as compared to ug/L concentrations as seen with many other toxic pollutants. The implementation of criteria based on the dataset without freshwater mussels will require some way of determining whether or not a water body is not likely to contain freshwater mussels, before such criteria can be considered appropriate. The presence or absence of freshwater mussels in a water body may be difficult to ascertain without a survey that spec ifically targets this issue. Most monitoring techniques for collecting benthic macroinverte brates such as the widely used EPA Rapid Bioassessment Protocol (RBP) are not designed or intended to regularly collect mussels that are often tightly imbedded in t he sediment. Thus a typical RBP survey that did not collect freshwater mussels cannot be accepted as proof of absence of mussels in that waterbody. I would recommend a default of assuming the presence of freshwater mussels unless several surveys that spe cifically targeted the identification of mussels have been conducted on that water body. A brief review of biological survey data in Virginia showed that the presence of freshwater mussels was detected in 83 % of water bodies that had been surveyed with m ethods capable of finding mussels. However, in some waterbodies the presence of mussels was confirmed only after several surveys. Because the distribution of freshwater mussels is often dependent on fish for dispersal, it might be suspected that mussels m ay not be present in some headwater or intermittent steams where it can be demonstrated that fish are not present due to physical barriers preventing fish migration.

Steven Canton Interestingly, we made a similar recommendation for this particular situa tion (i.e., the

concept of with and without mussels

- based criteria for ammonia) in that prior report ( AWWQRP 2006, Chapter 4). I have attached a copy of the relevant chapter from that analysis, which may be useful, as least in reference to how this has b een looked at by other parties.
In addition to a with and without mussel approach, the document could also include other “implementation” issues, such as evaluating additional site

- specific information needed to provide in

- depth technical recommendations . For example:B

- 8  Is there a definitive cause and effect relationship between ammonia sources and the diversity and abundance of mussels downstream of the source?

 How have the manageable sources (i.e., Agriculture, WWTP) of nitrogen changed over the last decade within the study waters in comparison to measured changes in mussel communities?

 Is there evidence of anthropogenic nitrogen loading that is equal to or gr eater than the natural occurrence of decomposition due to heterotrophic bacteria?

 What is the within

- site spatial and temporal variability in NH 3 of the pore water and is this variability linked to season, storm events, temperature, or flow conditions.

In addition it would be advisable to include a statement to recognize the importance of developing “site

- specific standards”, since mussels now play an important role and are the most sensitive species in the list. Site

- specific standards reflect the reality of which species are needed to be protected in a particular waterbody, particularly with mussels that may be more abundant in some parts of the country, whereas in some other regions their presence can be very limited. In this case, some guidance on when and how to do mussel surveys to determine when to apply a “with mussels” or “without mussels” criterion would be a useful addition to the criteria document.

Jerome Diamond Attempting to differentiate freshwater ammonia criteria on the basis of mussels bei ng present or absent is in my opinion a very tenuous and probably unsupportable idea scientifically. First, the acute data indicate that Corbicula (Asiatic Clam) is nearly as acutely sensitive as the most sensitive unionids. Corbicula is not a unionid an d has a totally different life cycle than unionids. Furthermore, they are now widespread in the U.S. so it may prove fairly inefficient to separate criteria on the basis of presence of bivalves in general. As an implementation issue, distinguishing between surface waters with mussels and those without is not nearly as straightforward as it is for salmonids or “cold water” species presence/absence because the latter are dependent on a fairly known, measurable water characteristic (water temperature regime). Also, while most states have cold water as well as warm water aquatic life uses in their water quality standards, I’m not aware of many (perhaps any) that have specifically a mussels use. The only example of which I’m aware is Virginia DEQ, which distinguishes different application of the chlorine standard based on whether federally listed mussels are known to be present in a given stream segment. They apply a “halogen ban” (no chlorine or other halogenate d disinfection chemicals are allowed to be used for wastewater treatment) in those segments for those permittees that do not have intermittent dischargers and have discharges < 20,000 gallons per day. However, in this case the water quality standard was not changed but rather a risk management approach was taken to reduce the major point sources of chlorine or other halogenated disinfection chemicals to the stream. Mussels can and do occur (or have occurred) in a wide variety of habitat types and water q uality regimes and so their presence is not often known without specifically sampling for them. Such sampling may need to be fairly intensive to “prove” the absence of mussels. In addition, mussels have been extirpated from a number of streams in the U.S . due to historic land use and habitat changes, toxic spills, and poorly treated wastewater and nonpoint runoff (e.g., see EPA 2002). If today, a state finds that mussels are not present but historically they were, what then should be the ammonia criteria ? I believe such differentiations in criteria suggested in the RD beB

- 9 relegated to EPA’s recalculation of site

- specific criteria (USEPA 1994). That procedure was designed to identify whether certain types of species are present or could occur in a given s ite or waterbody. This would be a more efficient and scientifically defensible way to handle the apparent sensitivity of freshwater mussels to ammonia.

George Dixon The document is silent on the appropriate methods for determining whether mussels are pre sent or absent in a given receiving environment, and therefore which criteria should be applied. I expect that a guidance document will have to be developed to assist those applying the revised criteria. My expertise is in aquatic toxicology, while the que stion of presence or absence is based more in field

- based aquatic ecology, particularly mussel ecology. As such I do not have detailed recommendations for the determination. I would, however, expect that some consideration would be given to a method tha t was able to deal with environments where mussels were once present, but have now been expatriated.

Jerry Farris With mussels presenting such an extreme sensitivity and complex life history in comparison to other groups, not only will their presence establish consideration for site variance demonstrations related to specific media, habitat or host fish interactions, but their spatial positions and ecological requirements against the backdrop of a stream’s ability to meet water quality standards

- will require some unique approaches to:

 Short

- term authorizations to discharge

 Compliance with permits that account for ammonia levels, but in streams or those segments that fail to meet water quality standards

 Designated mixing zones and how “shock effect” as sociated with ammonia will be considered

 The applicability of biocriteria and use of in

- stream community assessments related to the concept of mussels present and absent that would incorporate differences between degree of colonization and distribution ra tes for fish, other benthic organisms of conventional use in stream surveys, and freshwater mussels. These considerations coupled with only a recent attempt to describe how certain stream characteristics may serve to structure mussel communities, will ne ed to be carefully approached when and if such recommendation is made.
Since many of the ‘mussel present’ instances may involve listed species, the ability to qualify broad testing and culturing of suitable surrogate species for the time and seasonal l imits related to any testing related to permit or enforcement actions, will likely present an additional set of laboratory qualifying or certification considerations related to accessing suitably sited mussel species for testing. Laboratories listed in

re ference to the range of studies cited with this reassessment and that have implemented the new test standards (ASTM, 2006) have certainly advanced the methodologies needed for risk assessments and management decisions. However, this relatively limited num

ber of research and agency laboratories will likely be challenged to fulfill the rigorous requirements of consistency with organism culturing and quality assurance that may be expected of more conventional test demands. While consideration of these outcom es may reach beyond the scope of the question for mussel presence, increased demand as a technical consideration would be expected to impact population availability.B

- 10 Relative use of reference sites in determining relationships between areas of reduced f reshwater mussel diversity and abundance and known significant ammonia sources will also likely become a technical consideration. Definitive cause

- and

- effect relationships have not been documented in such instances (Bartsch et al., 2003) and such consider ations would require more detailed attention between the field exposure and effects data.

Bill Goodfellow I believe that EPA has adequately defended their position for when mussels were used or not used for the generation of the two phased acute criterion.
I do feel that EPA should include guidance that the lower mussel criterion should only be used when mus sels are currently present in the water system and not historic or wished to be in the system. I can see States using the lower mussel numbers without a strong technical reason to use them because someone wished them to be in the system, etc. When States choose to go this way arbitrarily, it will weaken the acceptability of the criterion for everyone.

Jim Pletl Generally, water quality criteria are designed to protect populations. The first step in determining whether mussels are present or absent is to define a standard for the conditions describing that a population exists at a site. This is not a straight

- forward exercise and can be quite difficult as seen in various site

- specific criteria projects around the country. There must be guidance as to wha t parameters and measures of those parameters are to be used to define presence/absence of a population. Quantitative evidence of reproduction would be required at a minimum, as well as the presence of various developmental stages. The normal abiotic con ditions documented for a species should also be compared to that of a particular surface water to determine whether a conclusion of presence or absence is supported. Certainly the availability of host species could influence the determination of absence/p resence. Once absence/presence is established EPA should only use mussel data to develop water quality criteria for waters where mussels are deemed present. EPA should ensure that conditions of the site where absence has been concluded have not been anthropogenica lly changed to a state where mussels cannot reproduce. Historic records of water quality and biological surveys would help support this determination. It is clear that EPA and the states will share the burden of the presence/absence determination and tha t there will be differences of opinion as to whether, when absence seems apparent, conditions changed due to human activity that could have been reasonably precluded or the species simply never successfully established a population at the site due to speci es

- specific factors.
EPA should also determine whether simply the presence of mussels is important or the presence of specific species is important. Given that the sensitivity of mussel species in acute tests spans a six

- fold concentration range the pre sence of only certain species could have a similar effect on the resulting water quality criteria; which then could have significant impacts on water quality management decisions. Further, different species have different life cycle durations. If individ ual species were used to develop criteria rather than the presence/absence of a taxonomic group the differences in life cycles may also impact criteria implementation.B

- 11

3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe there is an alternative approach to the use of this data that would be more scientifically sound?

Alex Barron The position statement in Appendix B of the reassessment document acknowledges several concerns about the use of toxicity data for glochidia and recommends that these data should not be used for criteria development at the present time. This position see ms reasonable given the uncertainties regarding glochidia testing as outlined in Appendix B. Glochidia, with such short

- lived life stages and with variable lengths of natural viability in the water column, ranging from a few hours to several days, it is di fficult to know how to use some of these data for developing criteria. For some species where the natural viability of glochidia, or where the actual exposure to the water column is short, the result of 48 hour tests may not provide a realistic exposure scenario, while a shorter exposure may be more important..

There are some additional short

- term (6 and 24

- hour) exposure data available that may provide some additional information on this issue. Table 2 of the Reassessment Document provides acute value s based on 48

- hour exposures for tests conducted with glochidia in several publications. For some of these same tests, additional information on 6 hour and 24

- hour exposures are also contained in Wang et al 2007b, in their table 3. These 6 and 24

- hour d ata indicate that glochidia are less sensitive to ammonia during 6 and 24

- hour exposures than during a 48 hour exposure. For the species where glochidia may only be viable or exposed to the water column for a few hours, the results of 6 hour and 24 hours t ests may provide useful information and these data could be normalized to the proper standard conditions and included in Table 2, to provide additional information for comparison. As reported in Wang et al 2007b; the data suggest that at 6 to 24 hour test durations, the sensitivity of glochidia for most species of freshwater mussels is less than or equal to the sensitivity of the juveniles of the same species at 96 hours. This provides some support that the revised ammonia acute criteria (based on 96 ho ur data for juveniles) may also provide protection to glochidia at exposure durations of 24 hours or less. The fact the acute criterion is applied as a one

- hour average should also provide some additional level of protection.

Steven Canton Given the rat ionale provided, in combination with EPA criteria development guidance,

general ecotoxicological principles, and my own views on general invertebrate biology, I agree it is scientifically defensible to exclude glochidia data in ammonia criteria development for the following reasons.

 First, the document notes that despite having four acute toxicity studies that consider five different mussel species (with the glochidia stage) ,with the majority of test having a duration of 2 days, there are still several questions to be answered when testing these particular life stages:

o How long do they stay attached to the host?

o How long do they stay in conglomerates and does this jelly (for those species) protect them from exposure to contaminants?

o If they sink to sedim ent how long do they stay there in the conglomerate before transforming to the juvenile mussel?

o I agree with the authors that these valid questions and also note that the questions will probably have different answers for different species of mussels, wh ich makes the data of even more uncertain general ecological relevant and, therefore, use of glochidia data even more problematic and difficult to apply generally to this group forB

- 12 criteria development purposes.

 Second, concerning the mechanisms of ammoni a toxicity in this particular stage of development there is also a long list of unanswered questions: would glochidia be able to take up ammonia if available in the host?, what is the mechanism of glodichia exposure to ammonia when they are released in

con glomerates?

- Again

– great uncertainty with unknown ecotoxicological significance.

 Third, based on the results from those four studies it seems that if the glochidia acute data were included in the calculations, they would dramatically reduce the acute c riteria and probably be over conservative for other fresh water species without actually knowing if the information has any ecological relevance.

Given these uncertainties

– which are well articulated in the draft document, there appear to be two scient ifically defensible approaches: 1) Do not use the glochidia acute toxicity data 2) Do a final acute criteria calculation “with and without glochidia”. The first approach would be the easiest to avoid confusion and possible errors in development of safe lev els for ammonia. The second approach would give flexibility

– although it would be necessary to include a strong caveat to a “with glochidia” value, given the questions noted in the criteria document and my thoughts above.

Jerome Diamond I agree with the RD that glochidia data should not be included in criteria derivation at this time but not entirely for the reasons given by EPA. First, a female mussel often produces hundreds or even thousands of glochidia in a single season. This helps counteract t he fact that a high percentage of the glochidia will not survive because they have not encysted on a host fish within a reasonable amount of time. As many researchers have documented, glochidia are incapable of surviving and maturing on their own

— they req uire nourishment from a host organism typically within days of being produced. It could be argues that basing criteria on a 50% reduction in glochidia survival would be similar to basing criteria on a 50% reduction in egg survival of other species that br oadcast an abundance of eggs (e.g., marine barnacles) in which only a small fraction of the larvae are likely to survive and become established under natural, unstressed conditions. I am not aware that EPA has ever based acute criteria on egg survival (ev en fertilized egg survival) and I believe this might contradict EPA’s criteria development guidelines for establishing acute criteria. A second reason I don’t think glochidia should be included at this time is that the majority of mussel species at risk to day (i.e., threatened, endangered, or species of concern) appear to have relatively narrow specificity in terms of a vertebrate host and have evolved structures (e.g., lures) or behaviors that enable very rapid encysting in the host (e.g., see Rogers

- Lower y and Dimock 2006). Of the mussel species actually used in glochidia testing that are discussed in the RD, most of them have evolved adaptations to enable rapid infestation of a host (often < 6 hours), resulting in a very short free

- living glochidia stage . Thus, for the majority of mussel species at risk, exposure periods > 24h are probably inappropriate, regardless of whether control survival is > 90%. A third reason is that while the ASTM glochidia test procedure appears to be reasonably robust in terms of intra and inter

- laboratory testing, the procedure is still a “Guide”, not a “Method”, which indicates that there are still many aspects requiring expert judgment, trial and error, and research, especially in terms of culturing andB

- 13 maintaining glochidia under laboratory conditions. Also the inter

- lab study reported by Wang et al. (2007) used the same water source and glochidia stocks for all labs. This would tend to underestimate the true interlab variability, where each lab uses their own source of g lochidia (and techniques for obtaining glochidia) as well as water source. In addition, at the present time there are still relatively few laboratories that have performed this test (e.g., compare with daphnia or amphipod tests). Much of the glochidia da ta under consideration were produced by a few laboratories. This may be acceptable for tests that use species closely resembling those for which a standard EPA or ASTM method has been developed and used extensively; e.g., an acute test with a different sp ecies of minnow or Daphnia but the identical test design and organism life stage as used for the standard fathead minnow or Daphnia acute test. However, such is not the case with the ASTM glochidia test, which uses a fairly different test design and the e ndpoint is dependent on sensitivity to a sudden stress (i.e., rapid valve closure to NaCl exposure). While the ASTM procedure appears to address these concerns for the most part, there is still some uncertainty in my mind regarding defensibility of the te st endpoint. As noted in the ASTM method, exposures beyond 24h may not achieve 90% control survival, indicating the precarious nature of keeping this lifestage alive without a host. Given this, is the apparent increase in ammonia sensitivity between 6 an d 48h a true difference in sensitivity or is it an artifact of the method (i.e., glochidia are under increased stress). I also question the ecological importance of having valve closure within one minute or less to the introduction of a NaCl solution. I’ m not altogether sure this endpoint is similar to other acute endpoints relied upon by EPA in their water quality criteria. In addition, the ASTM Guide points out that live or dead mussels could be open or closed at a given time and some may respond more slowly to the NaCl shock than others (Kernaghan et al. 2005). If there was a more direct way to determine mortality of glochidia to ammonia exposure, for example, that would be preferable in terms of using such data for criteria development.

I wonder if b etter use of glochidia survival data couldn’t be made by considering the level of juvenile recruitment necessary to maintain a viable mussel population, similar to the EPA fish larval recruitment model for dissolved oxygen in coastal waters in the Virginia n Province (USEPA 2000). Perhaps a similar type of framework could be used based on the mussel species (or, if feasible and defensible, genus or even family), when glochidia are typically produced in a given region, and a function specifying ammonia toxic ity to glochidia given ambient temperatures and pH at that time. The modeling framework could be made general so that a user could input necessary temperature and pH data, recruitment timing (e.g., season, month) and certain assumptions regarding host abu ndance. This would not be unlike the larval recruitment model EPA has developed for dissolved oxygen with the exception that the abundance of another species (i.e., a host) is not needed or relevant for the dissolved oxygen model. However, if such a mode l was considered (albeit simplistic but using conservative assumptions based on expert malacologist judgment), it could put glochidia toxicity testing results into an ecological context in a more useful way.

George Dixon Exclusion of the glochidia data, a s detailed in Appendix A, is fully appropriate and

well explained. Until tests are completed to determine (on a species by species basis) the duration of the period of longevity (and by inference viability) for glochidia, LC50s obtained with those life st ages will be suspect. I am unaware of any other approach which would be more scientifically valid

Jerry Farris Information from tests with glochidia can be supportive of the consideration for siteB

- 14 variance information, but should not be used to provide stand alone estimates in the absence of supportive acute juvenile estimates. Multiple lines of evidence comprised of testing with more than one mussel life stage that include comparisons with glochidia responses, would seem more scientifically sound, and may remove much of the seasonal dependence upon culturing and provision of test organisms with less invasive techniques in instances mentioned in the above mentioned technical considerations

Bill Goodfellow Not only is it scientifically defensible, I believe that it is the right way to go. Too much is unknown with regards to the biology of glochidia and the associated toxicology or appropriate methods to evaluate the toxicity of various chemicals for this life stage.

Jim Pletl The alternative question would be: Is it scientifically defensible to include glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Again, EPA has not defined standards in the Guidelines for this determination other than requir ing acute tests be at least 48 hours in duration and test organisms not be fed (except for mysids). These requirements were not developed to address the duration of the lifestage unique to glochidia. Toxicity data for glochidia appear to show that durati on of tests with this lifestage significantly affect the test results when tests are 24 hours in duration or longer. Therefore it seems that the glochidia are stressed in some fashion when tests meet the 48 hour requirement of the Guidelines. The test de sign for water quality criteria development is intended to measure stress due to the treatment (ammonia) independent of all other stressors. Tests of 48 hour duration and more fail to meet this goal. Since tests of duration less than 48 hours do not meet the Guidelines’ requirements but tests of duration equal to or greater than 48 hours provide biased results it seems that a new standard for test duration unique to these mussels must be first developed. However the literature indicates a great deal of d iversity among freshwater mussels in terms of their glochidia ecology. The extent of this issue demands either more detail in this criteria document or more opportunities for site

- specific ammonia criteria implemented by the States for only the mussel spe cies present. The States will also require guidance on how to develop site

- specific ammonia criteria based on the issues unique to glochidia. To support the scientific defensibility of EPA’s decision it would be appropriate to first develop guidance for the appropriate test duration to be used for developing water quality criteria relative to the duration of the life stage. Currently acute test duration commonly is within an order of magnitude of life stage duration. To accomplish this goal for glochidi a a test duration of 12 hours or less may be necessary. Such a guideline would help defend the scientific defensibility of using glochidia data for developing water quality criteria.
One should conclude that EPA’s decision to not use glochidia data is l ogical and correct without more information. There is a great deal of uncertainty associated with test results using glochidia. The question that must be answered is whether the level of uncertainty realized by using glochidia data is so great that it com promises the integrity of the resulting water quality criteria. This, in fact, seems the case; however it should be stressed that scientific defensibility should not be based on judgment but minimum standards of quality. Without these standards of qualit y the magnitude of uncertainty is unknown and probability of erroneous actions is elevated.B

- 15

4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scientifically defensible principle for structuring the population exposure duration problem a nd designing further research to quantify such a duration?

Alex Barron I believe that the approach outlined in appendix B is defensible. Given the significant uncertainties concerning the appropriate exposure durations for glochidia as discussed in the a ppendix B, attempting to use toxicity data currently available for glochidia to derive defensible criteria at the present time appears to be untenable. This would be especially important if the final criteria were significantly influenced by glochidia dat a with the recognized uncertainties about practical ecological importance. Water quality criteria should be based on the best information available in order to gain acceptance and avoid continued uncertainty. Additional information as discussed in append ix B would be needed to provide sufficient confidence in utilizing glochidia data to derive water quality criteria.

Steven Canton The proposed approach will give researchers and scientific community time to come

up with some answers and provide relevant information concerning the appropriate time of duration for glochidia exposure testing

– if such testing is determined to be

necessa ry for this short

- lived, parasitic life stage. However, if this approach is proposed, it will also create a limbo period, in which this issue will have to be addressed while decisions are made for application of a criteria that some may fight as “non

- prot ective” since the criteria document has “acknowledged” that a sensitive life stage was not considered!

I’ll be the first to admit it is a difficult to task to structure a criterion that includes population exposure approach to criteria development when de aling with a wide variety of fresh water mussel species that have different life spans (few months to several years), not to mention a glochidia life

- stage that can be released individually or in conglomerates, and that can get attached to fish, rocks, sed iment or plants.
Such variability could mean that for the particular issue of including glochidia data or not, we perhaps should be treating these mussels individually, by genus or species, rather than thinking of them simply as the “mussel” group. Regardless, it will take a considerable amount of time and effort to develop some way to make “uniform” relevant exposure toxicity testing time for the glochidia life stage that will be relevant for all if not the majority of fresh water mussels.

Jerome D iamond I am not sure I concur entirely with the proposed approach for further research discussed in Appendix A of the RD. I do agree that it would be useful to have better information regarding the natural life expectancy of free

- living glochidia in nature for

s everal representative species covering the range of different reproduction strategies observed. Such information would help indicate whether a 6, 24, or 48h exposure is warranted. I do not, however, understand EPA’s proposal specifying the free

- living du ration based on 95% of the glochidia that attach to a host. This appears to be a fairly restrictive proposition and it is not clear to me how this would be determined. As Rogers

- Lowery and Dimock (2006) observed, the encapsulation process in fish varies with the species and organism history of exposure to mussel infestations. Many researchers have demonstrated poor encapsulation rates of glochidia of certain mussel species (including several listed as threatened or endangered) with several common fish sp ecies (e.g., Dodd et al. 2006). In other words, the number of glochidia thatB

- 16 encyst on a host has as much to do with the host as it does glochidia viability in and of itself. Somehow, there would need to be the presence of a known host with the glochidia to determine an answer to EPA’s proposal. However, viable host species are not known for many mussel species. Therefore, this evaluation would require testing with a few mussel species for which host species are known with certainty.

George Dixon I am not a mussel ecologist, but the approach taken, and its imp lications for further

research appear valid. Having said that, it is likely that modifications to th e approach will have to be made as such research proceeds. It is also apparent that there is suff icient species variability in the life habits of glochidia that a single approach will not fit all species.

Jerry Farris The support of life history information and exposure information can support this contention, but the pertinence of this endpoint wou ld continue to be challenged for its scientific defensibility as relates to what we currently understand of exposure durations related to this life stage and variety of adaptations.

Bill Goodfellow See comment for Question 3, the same comment is relevan t for Question 4.

Jim Pletl As discussed earlier the uncertainties of using juvenile mussel data for deriving water quality criteria are significant, but EPA has identified additional concerns associated with tests of glochidia. The sensitivity of glochidia as a function of exposure duration, within the constraints of the Guidelines, precludes the use of glochidia data to derive water quality criteria. This is primarily due to the relatively short duration of instream exposure that can occur with t his lifestage and the sensitivity of glochidia to that duration of exposure. The issue of exposure duration instream versus that of lab toxicity tests is critical to the use of all toxicity data in deriving water quality criteria and is not unique to fres hwater mussels and their glochidia lifestage. Decapod crustacean larvae are sensitive to duration of test exposure without food for durations required by the Guidelines. The literature refers to the “Point of No Return” or the PNR (Klaus Anger, various p apers), where larvae will not survive beyond a certain time period without food even if food is provided to the larvae. Toxicity tests of duration approaching the PNR are likely stressing the test organisms in addition to the toxicant in question. The sa me can be said of glochidia tests with durations of 48 hours and more and likely durations even greater than 24 hours.
Development of criteria must consider all test factors that might bias test results in comparison to responses that are expected instream , and exposure constitutes one of the most important factors. Exposure is a function of frequency, magnitude and duration. The default frequency assumption for water quality criteria toxicity tests is that exposure is continuous. Magnitude of exposure i s usually dependant on concentration, which may also be a function of other factors like pH, alkalinity, DOC, etc. The magnitude of exposure for glochidia will also be affected by the species

specific conditions unique to glochidia of different species. For example, some glochidia are released into the water column singly; some are released in groups encased in a gelatinous shell where exposure would be mitigated. Duration of test exposure should be related to the duration of the lifestage, unless it can be demonstrated that a different test duration is representative of that lifestage. This is a fundamental of the Guidelines although it is not clearly delineated. In the case of glochidia 48 hour or longer test durations have been shown to result in res ponses notB

- 17 expected instream. This is a defensible reason to reconsider use of the acute glochidia data to derive water quality criteria and defines well the research needs for this taxonomic group and lifestage. Shorter duration tests will be necessary to provide reliable test results for deriving water quality criteria.B

- 19



## Hyalella azteca position statement and proposed rationale

5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?

Alex Barron Yes. The EPA Guidelines for Diving Numerical National Water Quality Criteria for the Protection of Aquatic Organisms and Their Uses, in section IV. 5. H specifies that in order to be useful for calculating a Final Acute Value, the must be relatively good agreement of the data within a species. Generally “if the acute values differ by more than a factor of 10, some or all of the values probably should not be used in the calculation” of a Species Mean Acute Value (SMAV). This applies to the available dat a set for Hyalella azteca, where the normalized acute values listed in Table 2 range from 1.58 mg/L to 83.9 mg/L, differing by as much as a factor of 53. This wide range of acute values for H. azteca required more careful review before determining what if any of the data for this species is acceptable for criteria development. EPA has conducted an additional review of these data and has determined that there are significant uncertainties with data for this species, enough to preclude their use in derivin g criteria. The position statement provides a summary of the information known concerning possible factors that could cause the acute toxicity values for H. azteca to be affected by some water quality characteristics not previously recognized or considere d and not fully understood at this time. Based on the current information available, it appears reasonable that until these factors are better understood, the data for H

. azteca should not be used in the calculation of a SMAV or a FAV.

Steven Canton I support the position statement based on the workgroup’s review of a number of

toxicity tests on Hyalella, referenced in the rationale. I can’t help but wonder if this recommendation holds for Hyalella test data for other EPA criteria documents, as well

– for example, Hyalella is the most sensitive species in EPA’s chronic cadmium database (EPA 2001). Is this position statement generic to all EPA criteria databases, or simply this ammonia update?
I should note that our prior review of the study also r ecommended removal of Hyalella from the ammonia database, primarily because of the poor control performance (AWWQRP, 2006), which it now appears may be due to the ionic balance issue noted by the position statement. So, I support non

- use of Hyalella toxic ity data for this ammonia update.
Most of the recommendations provided have scientific basis. It appears that ongoing studies will provide quality data that will widen the knowledge for water quality requirements for H. azteca husbandry. However, the statement concerning the complications due to possible H. azteca genetic or taxonomic diversity is not relevant because the way this information is used in criteria development, which only considers appropriate data at genus level (i.e., GMAVs) not new species or subspecies. In addition, the issue of a wide range of surface waters inhabited by H. azteca could be easily addressed setting a site

- specific standard, which will consider only the species present at a particular site. Thu s, inclusion of this information may add confusion rather than help explain a decision on non

- use for criteria development.B

- 20

Jerome Diamond I have reviewed the Hyalella data in a previous outside peer review for EPA and agreed with EPA’s concerns regardi ng Hyalella test data for ammonia. I agree with the position statement in Appendix B that Hyalella toxicity data should not be used in water quality criteria development. There is a clear need to resolve water quality requirements and other methodologica l issues for this species in water

- only tests.

George Dixon The exclusion of Hyalella azteca data, as summarized on page 13, is fully defensible. I

would suggest that while all of the points are relevant, the pivotal consideration is the impact of chlorid e and bromide ion on the viability of the species and the potential interaction of the concentration of these ions with ammonia toxicity. The Hyalella data will be suspect until that information is obtained.

Jerry Farris Those points are very critical giv en the consideration for osmoregulation by this organism and the points cited in the workgroup’s review. This seems well illustrated in the background material furnished for this review.

Bill Goodfellow I believe that the position statement and supporting rationale is appropriate and

justified.

Jim Pletl The position statement is written to address the use of H. azteca data for the ammonia update; however the issues presented would likely be important for many other water quality criteria using data for this species. Therefore the position statement is no t reasonable and scientifically sound if H. azteca data are considered within the context of all water quality criteria calculations. There is a concern that decisions such as this do not have associated rules as normally defined in the Guidelines. EPA i s being reasonable and responsible in considering factors that may bias the development of water quality criteria in this case, but it is unclear whether EPA has a process to identify such factors as new information is collected over time. Perhaps there w as insufficient data to identify the H. azteca issues when data for this species was considered for use, but EPA needs to develop and implement a system to periodically review data for issues such as this and publish the system as part of the Guidelines.
Based on this comment the position statement may be reasonable but the scientific basis of the recommendation is questionable without a well defined system of review. Such a system should include review of data relative to standards of quantity and quali ty required to make such an assessment. EPA needs to determine how much information is necessary to make this type of recommendation as well as the qualitative nature of the data (measured vs nominal concentrations, static vs flow through tests, etc.) Wi thout a system it will become increasingly difficult to be consistent in making these types of decisions as more variances arise.B

- 21



## Chronic Freshwater Criterion

6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such use? Are you aware of other relevant data that were not used?

Alex Barron Yes. The toxicity studies referenced all appear to be good quality studies and provide useful information on the chronic t oxicity of ammonia to aquatic organisms. The use of EC 20 values calculated by regression analysis is excellent and provides a consistent estimation of low

- level effects under chronic conditions. All toxicity data relevant to ammonia toxicity that I am aware of are included in the references and were considered in the reassessment document.

Steven Canton While , the toxicity tests used to derive the criteria are generally scientifically

defensible (see discussion below) ; I am uncomfortable with the calculation because there is insufficient data to evaluate intra

- species comparisons using the guidelines established for deriving water quality criteria. More specifically, th e 2009 data set still does not fully meet the EPA guidelines for developing water quality criteria. A salmonid representative has been added to the data set (which was missing in the 1999 document) , but a representative from the Class Insecta is still mis sing. It appears that the option for using acute

- to

- chronic ratios for determining chronic criteria would be more relevant than simply using a “hypothetical GMAV for insects”.
One of the studies noted in Question 1 may provide additional relevant chronic data.
It is also important to point out that there are only two studies (Anderson et al. 1978, Sparks and Sandusky 1981) available for Musculium genus. Interestingly, those two studies provide substantially different estimates of what level of ammonia is protective to fingernail clams

– creating uncertainty in the true chronic value. A closer analysis of these two papers reveals that while the later Sparks and Sandusky study was designed to confirm Anderson et al’s findings, the researchers faced some se rious methodology deficiencies, as the authors recognize: “Although it would have been desirable to perfect the culture methods first and then employ them in the toxicity tests, methods were developed as testing proceed because of limited time” (Sparks and Sandusky 1981). As part of their study design, Sparks and Sandusky used clinoptilolite to remove ammonia, but note this compound could also have remove potassium and chlorinated hydrocarbons

– which raises the question that toxic effects observed in Muscu lium transversum in this particular study may be also attributed to other confounding factors in the test water and not exclusively to ammonia exposure. In addition, authors conclude that also biological processes (i.e., naturally occurring bacteria that c onverts ammonia into a relatively non

- toxic nitrate) may be involved in ammonia removal rather than physico

- chemical processes. Thus, it would be advisable to re

- analyze the relevance of these studies for this update. As it stands, I cannot fully support calculating a GMAV for this fingernail clam using these two studies.

Another issue that is relevant to mention is the continued inclusion of a temperature relationship for chronic ammonia criteria originally used by EPA (1999), which was derived from a si ngle study. It appears the EPA used the Arthur et al. (1987) study, which evaluated acute ammonia toxicity to 14 species (9 invertebrate and 5 fish species), to incorporate temperature dependence into the chronic equations. There are three problems with in corporating temperature in the chronic relationship, but notB

- 22 acute. First, there is lack of chronic ammonia toxicity studies for fish or invertebrate species to demonstrate the relationship between temperature and chronic ammonia toxicity. Second, the rati onale for deriving an invertebrate chronic temperature slope from acute data (i.e., fish and invertebrates) is unclear. Finally, the assumption that chronically exposed invertebrates will have similar temperature dependence compared to acutely exposed indi viduals is based on the Thurston et al. (1984) study and those authors indicated that a 96

- h period is insufficient to determine an acute toxic concentration of ammonia for insects. Consequently, it appears there are no appropriate data to support incorpor ation of a temperature component to the chronic ammonia standards, just as EPA concluded with the acute equations. The position statement notes that the temperature

- dependent toxicity model now only applies to invertebrates.
I have to admit I was hoping t he actual equations, inflection points, and pH and temperature relationships would be thoroughly re

- evaluated as part of this update, given the abundance of new data included not in the 1999 document. I would strongly recommend re

- evaluation of the chroni c temperature component of the equations rather than just accept the 1999 update evaluation. Although, I have to admit, the draft document doesn’t actually provide any equations to test

– just recalculations of acute and chronic endpoints. This is anothe r disappointment.

Jerome Diamond Most of the chronic toxicity tests relied on by EPA for chronic freshwater criterion development are consistent with EPA’s Guidelines and other water quality criteria developed. I am not convinced that the 28 day juvenile test data for the snails included in Table C should be used. Both of these tests used mixed

- aged organisms, and in the case of the O zark spring snail, organisms were also field

- collected adding uncertainty in terms of their condition and acclimation to laboratory test conditions. In general, EPA has not relied on such tests for chronic criteria development, and I believe for good reas on. I am not aware of any other relevant data that EPA did not already consider in this document.

George Dixon Please see the response to question 1 above. That statement is also applicable to the

chronic data base.

Jerry Farris The tests utilized for t he criterion are scientifically defensible for such use and I’m not aware of other relevant data that were not used.

Bill Goodfellow I believe that they are scientifically defensible for the use in the development of the chronic criterion. I am not awar e of other relevant data.

Jim Pletl Using the standards established by the Guidelines and the respective toxicity test methods the toxicity tests used to derive the chronic criterion are scientifically defensible. However, this does not mean that the data generated from these tests and the resulting test endpoints are scientifically defensible for the use. As stated before, there are standards that should be met to establish that the test results are of sufficient quality to be used in deriving water quality criteria. These standards inclu de an appropriate concentration response curve and limits on variability within and between test treatments, replication of tests and use of reference toxicant tests to gauge organism sensitivity, as well as defensible control response across tests. One o f theB

- 23 shortcomings of the chronic freshwater mussel method is that it does not include minimum organism size (weight, length) requirements that must be met at the end of the test in controls. It is unknown whether the growth expressed in the controls of t hese tests is acceptable in a natural environment where stress does not exist. The tests only can establish weights attainable in controls in the lab environment when organisms in the controls meet the minimum survival requirement. The organisms may be s tressed in all test vessels, resulting in a bias of the test endpoint. The combination of treatment stressors (ammonia and vessels without sediment, for example) may produce a synergistic effect resulting in a lower ammonia IC20 for growth and survival th an that resulting from a test which has only one stressor (ammonia). In this case, the control response cannot account for the synergism, even though the control is intended to account for all factors independent of the tested treatment.

I am not aware of any other relevant data that was not used.B

- 25

7. Is the freshwater chronic criterion scientifically defensible with mussels present and mussels absent?

Alex Barron Yes. Similar distinctions for the presence or absence of certain sensitive organisms showing different sensitivities to ammonia have been used before. The 1999 ammonia criteria recommended different chronic criteria based on the presence or absence of

ea rly life stages of fish in the water body, setting a precedent of allowing different criteria for different situations. These different criteria were based on a similar magnitude of difference in sensitivity between the early life stages and the older lif e stages of fish (in the 1999 reassessment criteria) as is seen between the sensitivities of freshwater mussels and other species (in the 2009 reassessment). This policy of providing these separate criteria based on the presence absence of freshwater muss els will provide some flexibility in implementing the criteria in some instances and is reasonable.
Given the wide spread nature of ammonia in natural environments and the difficulties and expense imposed on municipalities in treating for ammonia in sewa ge treatment facilities, a significant lowering of the ammonia criteria is likely to have important consequences for municipalities. Providing these two criteria may help alleviate concerns that any additional restrictions on ammonia discharges are target ed to providing the needed level of protection for the waterbody, based on whether or not the more sensitive mussels are present. Providing two EPA recommended criteria that already take these issues into account could help reduce the potential for reque sts for consideration of developing site

- specific criteria based on the recalculation procedure, which can require significant resources to pursue.
Table C shows the four most sensitive genera and the calculation of the CCC for the two different criteria (with and without Unionid mussel data). The data under “excluding freshwater mussel data (family Uniondale)” includes the GMCV for the fingernail clam, Musculium transversum as the third most sensitive genera and this GMCV is used in the calculation of the CCC for “non

- Unionid containing waters”. While it is true that M

. transversum species is not in the family Unionidae , it is a bivalve mollusk.

In the recommendations for developing separate acute criteria, the calculation of the FAV and CMC as shown in Table B, the Asiatic clam was removed from the dataset to produce a criteria based on a “non

- bivalve mollusk” dataset (as commented on under question # 2 above). There ma y be water bodies with physical habitats (such as some headwater streams) that are unsuitable for bivalve mollusks of any kind, not just Unionids. EPA should consider whether or not to base their recommendations regarding separate criteria based on remov ing data for Unionid genera only, or for all bivalve mollusks. The same approach should be used for both the acute and chronic criteria. (Note: there is a small typo in Table C; the far right hand column should be labeled GMCV, rather than GMAV)

Steve n Canton The freshwater chronic criterion based on a mussels present/absent approach maybe scientifically defensible

– but will potentially result in very different approaches, even in regions of the United States where mussels are known to be absent or rare. In fact, the primary question will become “what level of data is required” to determine whether the habitat is or is not suitable to support mussels and/or whether mussels have ever occurred within the site h istorically. Guidance on such data needs questions would be useful to include in the criteria document to support a with/without musselB

- 26 criterion.
Additionally, even when the “mussel present” option is applied, a whole other series of issues arise, bec ause there is often a poor correlation between the water column NH 3 concentration and the pore water NH 3 concentration where most juvenile mussels reside. Site

- specific sediment conditions can greatly influence the availability of NH 3 , and in the case of the upper Mississippi River, as much as 6

- 30 times greater than the surface waters (Bartsch et al. 2003).
In such cases, criteria developed without considering the complete affects of sediment on the accumulation of NH 3 , or other confounding affects on mu ssel communities (i.e., the fact that mussel beds can self

- produce nutrients, such as ammonia) (Dankers & Koelemaij 1989), may greatly underestimate, or even overestimate, a criterion that is protective of mussels. In addition, this also brings up the que stion of whether the use of sediment is appropriate in chronic toxicity tests designed to be protective of aquatic life in the water column, and what endpoint should be evaluated using such methodologies.

Jerome Diamond I don’t think it is. See answer to question 2. The chronic value used for L. fasciola in Table 4 (0.39 mg N/L), and which must have been used in the GMCV calculation in Table C, doesn’t appear to make sense given an IC25 at the same pH and temperature reported of 0.38 mg N/L on p. 20 and the IC25 based on the actual data (pH = 8.2 and 20  C) of 0.39 mg N/L. The IC or EC20 should be lower than the IC or EC25. The IC pin value of 0.23 mg N/L appears more in line with the data for this species. EPA should at least defend why 0.39 mg N/L is a more appropriate value than 0.23 mg N/L for this species. In addition, wouldn’t it be more consistent to use the ICp value for both Lampsilis species because the L. siliquoidea chronic value could only be calculated using the ICpin method?

George Dixon The chronic criterion is defensible, provided tha t suitable guidelines are developed to

differentiate environments where mussels are present from those where they are absent. Please see my response to question 2 above.

Jerry Farris The criterion does not seem defensible for locations where mussels are present as would relate to considerations under the Endangered Species Act, but would likely be defensible under considerations with the Clean Water Act. I pose this statement as an example of the difficulty in relying on scientific defensibility in the face of a very broad range of considerations in risk and decision management that may rely on these estimates.

Bill Goodfellow Similar to the acute criterion, I believe it is scientifically defensible to develop the criterion with mussels present and mu ssels absent. I do believe that the EPA should have a cautionary note as in the acute criterion as to when and how to determine if mussels are present or absent.

Jim Pletl As stated in question #2, EPA should determine whether simply the presence of mussels is important or the presence of specific species is important. The first step in determining whether mussels are present or absent is to define a standard for the condit ions describing that a population exists at a site. This is not a straight

- forwardB

- 27 exercise and can be quite difficult as seen in various site

- specific criteria projects around the country. Guidance must be provided as to what parameters and measures of those parameters are to be used to define presence/absence of a population. Quantitative evidence of reproduction would be required at a minimum, as well as the presence of various developmental stages. The normal abiotic conditions documented for a spec ies should also be compared to that of a particular surface water to determine whether a conclusion of presence or absence is supported. Certainly the availability of host species could influence the determination of absence/presence.
Once absence/pres ence is established EPA should only use mussel data to develop water quality criteria for waters where mussels are deemed present. EPA should ensure that conditions of the site where absence has been concluded have not been anthropogenically changed to a state where mussels cannot reproduce and biological records have documented the presence of previous populations. Historic records of water quality and biological surveys would help support this determination. It is clear that EPA and the states will sha re the burden of the presence/absence determination and that there will be differences of opinion as to whether, when absence seems apparent, conditions changed due to human activity that could have been reasonably precluded or the species simply never suc cessfully established a population at the site due to species

- specific factors. Given that the chronic sensitivity of mussel species drives the CCC approximately 44% lower than when mussel data is not used the management actions and costs based on criteri a without mussel data could be significantly different than when mussel data is used to develop the CCC.
The presence/absence of species is usually a question that is asked when criteria are recalculated or a site

- specific criterion is being developed. In these cases the issue of removing species from a database is addressed by determining whether data for other species with similar taxonomy is available. The taxonomy and reproductive cycle of the freshwater mussels is so unique that this will not possibl e. Therefore a decision must be made as to whether to apply a CCC with or without mussel data. Given that there is so little chronic data available for these mussels and that there are outstanding issues that must still be addressed (use of shell measure ments to represent growth, tests without sediment, irregular concentration response curves, etc.) a CCC with mussel data will result in water quality standards with uncertain consequences. This uncertainty demands that criteria without mussel data be avai lable for states to use when mussels are not resident (reproducing population found year to year). At this point in time a CCC without mussel data would be more scientifically defensible than one with mussel data.B

- 29



## Use of 28

- day Juvenile Test Data

8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defensible as "chronic" test data for criteria development?

Alex Barron I do not believe that growth measured during a 28

- day test can be considered a true measure of a chronic effect as intended by the requirements in the 1985 Guidelines for Aquatic Life Criteria, such that they can be related to significant population impacts. G iven the life span of the organism and the time needed to reach maturity, a 20% difference in growth observed over 28 days may be reversible and may not be relevant on a population level and should not be used to derive criteria.

Steven Canton Water quality criteria for the protection of aquatic life are derived using toxicity

endpoints that relate to population level impacts. In general, these endpoints relate to survival, growth and/or reproduction. I would agree that the 28

- day test with juvenile mussels, while similar in duration to a standard chronic test for many other groups of organisms, is not technically an early

- life stage test according to the 1985 Guidelines for Aquatic Life Criteria, and as such, should perhaps not be included in the ch ronic database.
If this is carried forward, I would support the approach of the 28

- day exposure tests being considered as “other data’

– if the test are acceptable in all other respects. Reasons for not including in chronic criteria calculations could fo llow and build on the arguments presented in the position paper:

 First, it is not representative. If the duration of the life stage is 2

- 6 years for juvenile life stage of fresh mussels, 10% of this amount of time would be 73

- 219 days. Thus, a longer e xposure (i.e., 90

- day) would be more representative for a “chronic” study for juvenile fresh water mussels than 28

- day tests.

 Second, extending the exposure time would allow the tested animals to adapt better to laboratory conditions, thus reducing the st ress and decrease the risk of having test condition

- biased results.

 Third, although it will be more challenging to keep all the water quality parameters under control in a longer exposure it could also provide other avenues for future research on juvenile mussels (i.e., if captivity conditions and pollutant exposure would enhance growth over longer periods of time, if they may need additional food source, among others)
Once possible conflicting issue is that other species used in EPA criteria development h ave “allowable” chronic test conditions that also represent a small portion of their total life span

– e.g., 60

- day post hatch ELS for salmonids, which can live for years. The difference, conceptually, is that the 28

- day test for mussels does not include the earliest life stage prior to juvenile testing commences

– i.e., it does not start with egg, then hatch, then juvenile exposure more typical of an ELS for fish. If we can conceptually overcome this “weakness” for a 28

- day juvenile mussel test, then I

s uppose growth

- effects data in a 28

- day juvenile mussel test could be at least evaluated in the context of other reported data

– and if comparable to effects data from longer

- duration testing, then perhaps it could be used in some form for criteria calculat ions.B

- 30

Jerome Diamond The question posed here by EPA implies that a 28

- d test may not be sufficiently long for freshwater mussels that live 2

- 6 years (actually, many unionid species live far longer than that

— 20 years is not uncommon). If this is the case, I fail to see how EPA justifies use of 28

- d tests for fish such as salmonids that live 3

- 5 years or sturgeon that live > 30 years. I realize there’s McKim’s 1977 paper but that was based on outdated test methods and has questionable relevance to data col lected in more recent years. The discussion concerning the usability of 28

- d mollusk survival and growth tests in chronic criteria development (pages 15

- 16 of the draft document) appears to have contradicting statements and questionable rationale in my vie w. The discussion begins with the statement that 28

- d tests do not qualify as life

- cycle, partial life cycle, or early life stage tests as defined in EPA’s Guidelines. According to those guidelines, 28

- d tests can only be used for fish because it has not been demonstrated that a 28

- d exposure is a reasonable predictor of invertebrate sublethal chronic effects (defined as at least 90d for vertebrates and presumably for invertebrates such as mussels which live for several years). However, the draft documen t goes on to say that 28

- d survival information could be used to inform the chronic criterion because obviously, lethality is not reversible. Yet in footnote #2 on page 15 of the document, as well as in Appendix C, uncertainties regarding the 28

- d mussel test method are discussed which include optimal quality and quantity of food, exposure apparatus, etc. needed to sustain juvenile mussels in good health and maintain adequate growth throughout the test. Based on my limited experience testing bivalves, I b elieve it may not be as challenging as one might expect to keep control juvenile mussels alive for 28

- d given adequate oxygen and clean water but it may be quite another thing to document that organism condition (or alternatively, susceptibility to stress) is truly adequate. Therefore, the fact that juvenile mussel survival is lower with exposure to certain levels of ammonia may be due to having an already stressed population due to inadequate food or other factors. A similar finding is now being made by E PA after reviewing chronic Hyalella chronic test data (reported to be one of the most sensitive species in the 1999 ammonia criteria document). In that case, control survival was apparently satisfactory but test organisms were in fact ultrasensitive to the chemical due to suboptimal test c onditions. A second point I would raise regarding the chronic mussel tests is that they were only conducted once (based on published information) for a given species in one laboratory. While this in itself is not a reason to exclude test data, it is clear that there is considerable variability in acute endpoints among juvenile mussel tests using the same species and conditions (even within the same lab as mentioned previously), which should apply to chronic survival with ammonia as well. Given that certai n mollusks appear to be far more sensitive to ammonia than any species tested thus far, there should be some replication of these results to be sure we’re not going to find out that EPA needs to do yet another reassessment because of issues similar to the ones they are dealing with now for Hyalella

. It is important to note that while interlaboratory testing was conducted for acute juvenile tests (Wang et al. 2007), similar interlab testing has not been conducted (or at least published) to my knowledge for 2 8

- d mussel chronic tests. In addition, one should view the acute interlaboratory results cautiously because, as mentioned previously, a single culture and water source was used by all laboratories in that study. As Wang et al. (2007) point out: “However , the present study used the same batch of test organisms and the same dilution water, which were different from the referenced inter

- laboratory studies and might have contributed to the lower variability in test results. The present study was designed to determine the inherent variability in theB

- 31 test. Higher variability would be likely if the inter

- laboratory tests were conducted with different sources of dilution water and organisms (e.g., from different populations or watersheds). Therefore, additional s tudy is needed to further characterize potential variability associated with the newly developed ASTM standard methods for conducting acute toxicity tests with early life stages of freshwater mussels.” Therefore, the CVs reported by Wang et al. (2007) are likely to be underestimates of true test method variability. This is borne out by the acute data generated for the same species by different labs as noted previously in my comments.

George Dixon This is well covered in Appendix C, and the arguments are convincing. I agree that the

28

- d test does not qualify as a chronic test, since it is neither a life

- cycle test nor a partial life

- cycle test. I also agree that a concentration causing a greater th an 20% reduction in survival could be used as an upper limit for the Species Mean Chronic Value, assuming the test met the requirement for good lab practice.

Jerry Farris Research that validates effective endpoints related to the range of pollutant effec ts in freshwater mussels have shown that 28

- 30

- day exposure tests in both field and laboratory settings can be included to predict long term impacts related to biomarker responses, bioaccumulation, and even comparisons to conventional “chronic” test data. The 28

- day test exposure period will continue to be temporal as relates to the life cycle, but can be relational to long

- term considerations and offer critical information when combined with multiple lines of evidence. Inclusion of more than one life s tage response also advances mussel toxicity assessments, rather than providing semantic arguments over longevity for a life stage and whether the exposure period qualifies as “chronic”. Data from the 28

- day test can be defensible when supported by “other ” data. Other data should also be scientifically defensible.

Bill Goodfellow I do not believe that 28 day mussel studies should be considered as chronic test data for the national database as described in the position statement and supporting documen tation. I believe this is very well thought out and applaud the developers.

Jim Pletl This question is not only a function of the duration of exposure in the test but whether the test meets the requirements of the Guidelines. Clearly the test does not me et the Guidelines requirements because it does not address either the entire life cycle or, for partial life

- cycle tests, the appropriate life stages and test endpoints defined by the Guidelines. The reliability of this test is also clouded by the apparen t impact of testing juveniles in the absence of sediment and the use of shell length as a chronic test endpoint for freshwater mussels (see comments below). Perhaps the Guidelines require updating (has not been formally revised since its first release in 1985), but until this is done the 28

- day juvenile test does not meet the standard for scientific defensibility that EPA has established.
One might suggest using the data from these tests as an estimate of freshwater mussel chronic toxicity because the tests did not include the glochidia life stage and it is more appropriate than not using the data for criteria development. Unfortunately there is enough concern regarding the reliability of this test even to accurately represent the sensitivity of juveni les that it would not be appropriate to use the data in any water quality criteria capacity.
Independent of the issues raised above, it would seem that a 28

- day exposure test withB

- 3 2 juvenile mussels would be representative of the sensitivity of juveniles. H owever, chronic tests used for developing a CCC are intended to evaluate or extrapolate the sensitivity of a population to a stressor. This requires reliable knowledge of the sensitivity of the most sensitive life stages of that population. It is unknown how the sensitivity of glochidia compares to juveniles because of the issues associated with both acute and chronic test methods. Without this knowledge and reasonable certainty it cannot be determined that the 28

- day juvenile test with freshwater mussel s is scientifically defensible relative to development of a CCC.B

- 33

9. Should toxicity studies on the growth rates of mussel shells during 28

- day tests be considered quantitatively when developing water quality criteria?

Alex Barron No. I do not believe that differences in growth rates measured over a 28

- day period should be considered to provide acceptable quantitative chronic endpoint data for freshwater mussels that should be used to calculate chronic criteria for ammonia. Chronic criteria for ammonia must be based on clearly recognized adverse effects in order to be acceptable. Reductions in survival are irreversible and are clearly adverse effects, while slight reductions in growth may not be significant to the population, or th e organisms may be able to compensate over time and reverse the difference in growth. These type tests do not meet the requirements of the EPA Guidelines for use as a quantitative value in driving a chronic criterion. Using such data to derive chronic criteria, especially if it significantly influences the final criteria would likely increase uncertainty about the final criteria.

Steven Canton As described in the previous question, the studies on growth rates of mussels shells

during 28

- day tests shou ld only be used as information to better evaluate appropriate longer

- term chronic studies (i.e., 90

- day studies). The results from longer chronic tests could certainly be used quantitatively for the developing the water quality criteria. Unless 28

- day test data provide similar endpoints to long

- term testing, it would be necessary to keep the results separate and potentially have two conflicting sets of data (i.e., 28

- day and 90

- day data)

– which will only make the calculations of the new criteria more compl icated, and can lead to confusion when trying to implement. And I have to say that ammonia criteria development is confusing enough as is.

Jerome Diamond I don’t believe there is sufficient information or adequate test method development and standardizat ion at this time to be able to use growth rates of mussel shells in 28

- d tests for criteria development. Growth rates, based on shell measurements, are not sufficiently large over a 28

- d period to have much confidence in the results. Based on the studies reported in this draft, as well as others of which I’m aware, shell length is too coarse a measurement to be used as an indicator of mussel growth over such a short time period. Furthermore, it is generally not known what the expected growth rate should be for a given species under these test conditions. As I indicated earlier, without having such information, I don’t believe that survival data from such tests can be used with confidence as well.

George Dixon The approach taken in the document (Appendix C), that of not using data for growth

of organisms in non

- life

- cycle tests with durations of less than 90 days in the derivation of a Species Mean Chronic Value (SMCV), is in my opinion perfectly appropriate. The justification given in paragraph 5 of Appe ndix C centers on two points: lack of direct comparisons of growth between shorter tests and life

- cycle tests and uncertainties around the relationship between short

- term growth reductions and growth effects over a full life cycle. Both are valid reasons t o exclude the data from calculations of SMCV.B

- 34

Jerry Farris Growth rates of mussels during the 28

- day tests should not be considered quantitatively until more information is available on the validity of this test. Variability is still attributable to diet and holding conditions and has been cited broadly.

Bill Goodfellow The problem with using shell growth rates, it doesn’t necessarily link to a smaller organism being any less viable or having less reproductive benefits. Since mussels are not a commercially or recreational important organism, I believe that the use of growth rates are not valid as compared to a fish species or commercially important bivalve.

Jim Pletl Review of Wang et al. (2007) for both the copper and ammonia 28

- day tests shows that there is a very small difference between the length of the s hells at the beginning and at the end of the test based on control responses. In fact it appears that some of the tests show no statistical difference in shell length between the beginning and end of a test at alpha=0.05 and assuming a normal distribution . This does not support the use of shell length to represent growth in the 28

- day mussel tests. Further this paper shows that using shell length to represent growth does not provide reliable dose response curves in most cases. Although the authors only conducted 21

- day tests both Bringolf et al. (2007a) and Bringolf et al. (2007b) found that the concentration response curves for different chemicals were not what one would expect for a toxicant. Although there may be issues with organisms benefitting fro m certain concentrations of chemicals, this fact only complicates the interpretation of this data and increases the uncertainty of using toxicity test results based on shell length.B

- 35

10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chanc e they could recover or stabilize is one potential outcome only)?

Alex Barron  Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?
Yes. Although too many questions surround the use of growth effects during a 28 day test in relation to long lived species such as freshwater mussels to consider using differences in growth as data that directly influence the calculation of the final chronic criteria, these data still deserve consideration and inclusion in the EPA criteria document. These data do provide useful information to help judge the potential for “other” effects that have a potential for concern. Including such data under the category of “other data” can provide additional information that can lend confidence that the criteria based on survival can provide an adequate amount of protection. The magnitude of difference in growth may make a difference; a 20 to 25 % reduction in growth may not be of much ecological significance, especially if there is a thought that the effect may be short term and reversible, while a > 75% reduction in growth may be considered more of a concern. The magnitu de of difference between the effects on survival and the effect on growth can also provide useful information. Altogether, I see value in presenting these growth data under the classification of “other data” in the criteria document.

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?

I woul d agree that this statement could also be true. We simply do not know whether or not an effect on growth observed over a 28

- day exposure can be considered reversible or indicative of a permanent effect. As discussed above, I believe the magnitude in diff

erence in the growth and the magnitude of difference between the survival end point verses the growth endpoint could influence the level of concern regarding such data.

Steven Canton  Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?
My understanding of data included as “other data” in the criteria document is that these data are provided just as a reference to make the interested parties know that these data exist. They can certainly be presented that way and not considered in the calculations of the chronic criteria fo r the reasons outlined in the previous two questions.

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the ch ance they could recoverB

- 36 or stabilize is one potential outcome only)?
As noted in the position paper, it is not possible with the available data to conclude that impaired growth after 28

- day exposure will necessarily lead to continued reductions in growth over time. The position paper rightly points out the possibility of organisms to “recover” from potentially short term stress and show no further decline. In fact, these organisms may exhibit normal growth during a more relevant long

- term test. However, given the lack of information available to compare short

- term and long

- term toxicity studies in fresh water mussels, it would be advisable to note, as appropriate, that any of the three outcomes: continued decline, stabilize, or recovery is possible. In ad dition, the propensity for any of these three outcomes will most likely depend upon the particular mussel species and the stage of development used in the toxicity tests.

Jerome Diamond I agree with EPA’s position that mussel growth data should be categor ized as “other data” until it is demonstrated that there is a repeatable, defensible methodology for generating adequate growth data and that the growth endpoint calculated is ecologically meaningful. For this reason, I don’t think it matters whether impa ired growth observed in a 28

- d test is indicative of effects at longer exposures or not. However, I would recommend that EPA not consider 28

- d mussel growth as a valid endpoint based on methodological and perhaps biological uncertainties, as I mentioned

u nder (9) above, rather than because reduced growth rate is theoretically a reversible effect. I see no reason why reduced growth would not be an even bigger effect with increased exposure duration to a toxicant.

George Dixon With respect to the first poi nt above, inclusion of the data as “other data” is perfectly appropriate. This is consistent with the practice for short

- term growth results with other species, and I see no reason why mussel data should be treated differently. With respect to the second q uestion, this is a statistically valid point, but it does not reduce our level of uncertainty around the actual meaning of the data. The issue will only be resolved when someone undertakes 90

- day tests.

Jerry Farris  Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?

Growth should not be considered as de fensible for the above mentioned reasons relating to lack of reliable test conditions, but can be used as supportive information that might influence the criteria. Studies that include recovery and or stability in growth or even degrowth with freshwater b ivalves, have produced more reliable estimates of pollutant effects and could therefore be used to influence the criteria when included with broader scale studies.

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?
As stated, the above consideration seems very confusing and should be cl arified to relate to the defensibility as considered with “other data”.B

- 37

Bill Goodfellow  Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?

I believe that it should remain in the other data section so the relevant data can be used in site specific evaluations.

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?
I don’t believe that data supports this and such a statement should not be made.

Jim Pletl  Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?
Based on the uncertainty associated with testing juveniles without sediment and the lack of reliability in the shell length endpoint it is not appropriate to use the chronic data in any way associated with development of a CCC.

 Should the statement also c onsider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?

The comments provided above on the reliability of the shell length endpoint for chronic tests of freshwater mussels indicate that it is not a reliable endpoint to represent the chronic sensitivity of these organisms. Without more data, it is not defensible to specula te on the outcome of tests with longer duration when the reliability of the endpoint being used is in question.B

- 39

11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Juvenile and adult mussels, as sediment

- dwelling organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have suggestions on h ow states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?

Alex Barron The toxicity data currently available for freshwater mussels are for glochidia and

juveniles. The exposure to unency sted glochidia will be primarily via water exposure, and once encysted on a host fish they will be in a less exposed condition. Juvenile mussels will also be initially exposed to ammonia in the water column following dropping off (excystment) from the hos t fish. As the juvenile mussels are deposited onto the substrate they may remain on the surface or may become situated within a few centimeters of the surface. They will need to maintain a position on or near the surface in order to ensure access to well

- oxygenated water as well as filterable food. In all cases, the exposure to ammonia will be over the gills via water; either surface water or interstitial water and the water quality criteria can be adjusted to account for any differences in pH between the water column, the surface

- sediment interface or pore water at a specific depth in the sediment. For normal use in establishing permit limits, or assessment of waterbodies; the criteria should be applied to the water column. If there is a special concer n at a site, the option of monitoring the pH of interstitial water can be investigated, and the pH characteristic of the interstitial water can be used to assess the criteria. Care should be taken to determine the pH in the interstitial water at depths wh ere mussels are likely to be found at the site.

Steven Canton Given that juvenile mussels of most species reside completely in the sediment, filter

pore water, and feed on the sediment, I would suggest one approach might be determining a mean burial depth for the juveniles of the selected mussel species within the 20 09 data set. In laboratory tests, juvenile Villosa iris are reported to bury <1cm, yet were not exposed to the overlying water (Yeager et al. 1994). Perhaps it would be useful to use this burial depth and evaluate the average pH conditions within a buria l zone, per se. In addition, it is likely any measures of pH in the mussel micro

environment (i.e., taking measures of water quality over a mussel bed) may also show elevated ammonia levels simply from the metabolic activity of those organisms, themselves . I can’t help but wonder how to determine attainment of “safe” ammonia levels in future field monitoring, given the potential for organism

- generated ammonia values.

Jerome Diamond I am not so sure that applying ammonia criteria to juvenile mussels at le ast, and possibly adults as well, should consider sediment pH at all for several reasons. First, larger juvenile and adult mussels are reported to be exposed to waterborne contaminants via siphoning the overlying water and not as much via sediment ingesti on or pore water exposure during their growth season (Strayer et al. 2005). This is particularly true during times when water temperature exceeds 50  F (Watters et al. 2001; Schwal and Pusch 2007). In fact, many mussel sampling protocols specify a minimu m water temperature of 50

- 60  F because mussels are near or on the surface under those conditions and more readily seen (e.g., Pennsylvania DEP and Army Corps sampling protocol in the Allegheny River, PA). During this time (typically spring, summer, and f

all in most waterbodies of the temperate U.S.), mussels are actively feeding via siphoning the water column and sediment pH is probably not aB

- 40 factor affecting water column ammonia toxicity. Rather it is the overlying water pH that is probably more importa nt. Second, many researchers report that sediment pH and ammonia can vary substantially from one location to another within even small areas and may be dependent on very complex redox gradients that are difficult to measure much less predict (Burton 1992; Stemmer et al. 1990; Sarda and Burton 1995). It is no easy matter to identify an appropriate sediment pH that should be used to apply an ambient water ammonia criterion. Third, many of the mussel species tested thus far, or that are species of concern, in habit well

- aerated sediments that often have either fairly coarse particle size (gravels

– cobbles) or sand, both of which have interstitial water closely resembling the water quality conditions of the overlying water (Salmon and Green 1983; Neves and Widl ak 1988; Way et al. 1990). In these cases one would not expect a large difference between sediment and overlying water pH. Fourth, it seems premature to consider complex pH adjustments based on sediments when it hasn’t been demonstrated that juvenile or adult mussels are as sensitive to sediment ammonia as they appear to be to water column ammonia in lab tests. If indeed sediment pH needs to be considered, then it seems likely that mussels are not being exposed to surface water (including surface water a mmonia) but rather sediment and interstitial ammonia. If this is the case, there is no need to adjust water column ammonia criteria based on sediment pH.
Finally, basing a water column criterion on sediment water characteristics represents a major departu re for EPA in terms of criteria development. While I would encourage the Agency to consider more holistic criteria that take into account multiple media (e.g., sediment and water), I don’t believe there is sufficient information at this time to warrant al tering a water quality criterion, which was derived using water exposure test methods exclusively, due to sediment characteristics.

George Dixon While mussels live in sediment, the pH of importance from an ammonia criteria

perspective is the pH of the res piratory environment, which in this case is the pH at the sediment

- water interface. The sediment pH is of importance only to the extent that it influences the pH of the water at the interface. Remember these are water quality criteria, not sediment quality criteria. I have no suggestion as to the appropriate method for determining the pH at the interface. The methodology, more in the realm of environmental chemistry, falls outside of my area of expertise.

Jerry Farris Even if an adequate amount of informa tion was available for any benthic invertebrates for chronic toxicity related more specifically to the generic relationship of ammonia toxicity to pH, larger issues challenge this consideration with the reassessment for changes related to pH shifts, which may increase when phase waters are isolated for testing. These relationships have only recently been examined for juvenile freshwater mussels, without sufficient detail furnished as to the pH adjustment used with diluter systems in those tests or their ef fect upon alkalinity changes measured during the assays (Wang et al., 2007). Although these tests seem suitably performed to establish the influence of pH on the acute toxicity of ammonia and even the pertinence of normalizing ammonia toxicity data for mu ssels to a common pH end point (total ammonia nitrogen at pH 8.0), information is still lacking for the sediment relations specific to mussel responses. As relates to this consideration, the bulk of the studiesB

- 41 that have examined sediment and phase water relationships to date rely heavily on marine field monitoring, and not standardized laboratory testing or field monitoring with freshwaters (Salazar and Salazar, 2007). Any scientifically defensible way to account for these differences would greatly bene fit from studies with comparable methods examining habitat preferences, feeding, and mobility of a sensitive mussel life stage (juvenile) among sediment compartments, since the synthesis of ammonia toxicity data confirmed its status as a sensitive species. Data from these studies would also be needed to generate pertinent modeling of the ammonia fluxes from sediment and the concentration profiles in the phases that would have consequence to mussel impact.

Bill Goodfellow It is much easier to do this for water column organisms. I believe that the agency will open themselves up for tougher battle if they attempt to make this issue even more complex than it already is. I believe that noting that this is the case and stating t hat pH dependence vs. the criteria as it relates to mussels was not developed at this time given the uncertainty about what the appropriate pH assignment should be.
I also argue whether pH in sediment is a relevant question with regards to criteria since the management tool for water quality is going to be primarily the NPDES permit. How will anyone be able to control sediment pH to manage it? I am not sure what this does for development of a defensible criterion.

Jim Pletl It will be necessary for stat es and EPA to directly measure the pH of the environment where each life stage exists. For juvenile and adult mussels this will require measurements of the sediment in each location where the criteria are applied as water quality standards. Guidance from EPA on how to appropriately represent the pH of sediments where ammonia criteria are applied to protect freshwater mussels will be necessary. Measurements in the field will also be necessary because the pH of a discharge regulated by a NPDES permit canno t be used to estimate the pH of the sediment. The pH of the sediment can only be assessed through direct measurement. This may be done in situ or by rapidly measuring the pH of relevant sediments collected with sediment grab or core equipment.B

- 43

12. In general, should the criteria include a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically defensible way to account for these differences when deriving protective water quality criteria?

Alex Barron The potential differences in pH between water and sediment is not so much directly

related to the criteria development, as it is an issue related to how and where the criteria may be applied. The water quali ty criteria should provide recommendations for protective criteria based on ammonia concentrations in water and adjusted for pH and temperature. On a site

- specific basis, if in sediment there are significant differences between pH in the interstitial pore water in sediment and the overlying surface water, this can be assessed by site

- specific measurement of pH levels in the sediment (at depths where juvenile mussels are likely to be found) and then adjusting the criteria to those pH conditions. Another c oncern is whether or not the pH at the appropriate depth is relatively constant. These issues will likely vary considerably depending on the characteristics of the sediment, and perhaps seasonally. The criteria can be adjusted to the pH in the interstiti al water to assess the potential for adverse effects at the site.

Steven Canton Because adult mussels siphon directly from the water column, there is no need for a

pH differential calculation. However, to be protective of the juvenile life stage which ty pically filters pore water, there may be a need for differential pH measurements. But how does one account for all of the biotic and abiotic factors that affect pH in the sediment? Because site

- specific sediment conditions greatly influence the pH levels
, adjusting the pH should only be an option for site

- specific calculations (or considered as “other data”) rather than within the national water quality criteria.

Jerome Diamond I don’t think criteria should consider sediment pH or the difference between sediment and water pH in deriving water quality criteria. Rather, EPA should establish sediment ammonia criteria, using toxicity test data for mussels and other benthic species ( e.g., Hyalella
, Hexagenia , etc.) exposed to sediments with ammonia.

George Dixon My opinion here is partially covered in the response to question 11. The pH of the sediment is only of consequence to the degree that it influences the pH in the water column at the sediment

- water interface. I am not aware of any methodologies, other than direct measurement, that would allow estimation of the interface pH if the only data available were the sediment pH and the water

- column pH.

Jerry Farris In addition to the aforementioned comments regarding the need for additional studies involving mussel responses before including a consideration for this difference, site

specific variance procedures inclusive of sediment characteristics for a “mussels present “ site might b e especially applicable in this instance. Should these site criteria account for such differences in testing of site waters with and without sediment, they would be expected to be applicable for the species (or suitable surrogate) and sediment of interest . Use of a site

- specific criterion for copper that incorporated such broad test considerations has been utilized to insure protection of mussel species for almost two decades within the Clinch River, Virginia (Van Hassel, 2007).B

- 44

Bill Goodfellow Same comment as for Question 11.

Jim Pletl The freshwater ammonia criteria must appropriately address the pH of the juvenile freshwater mussel environment to be reliable. Although the criteria must be protective they must also be representative of aquatic life sensitivities and implemented in a way that does not overstate those sensitivities. Being protective is straight

- forward using conservative assumptions but criteria development and implementation already have multiple layers of conservatism. These l ayers include the return frequency (once in 3 years) of the criteria, the assumption that exposure is continuous instream for a time period equivalent to the duration of the tests, an acute criterion averaging period of one hour, the use of the 90 th percen tile of temperature and pH to apply the ammonia criteria, the reasonable potential process of EPA’s Technical Support Document for Water Quality

- based Toxics Control which relies on a very high percentile (95 th or 99 th ) of the distribution of the highest c oncentration measured for an effluent to issue permit limits, the 303(d) listing process which is dependent on the vast minority of data exceeding the criteria to conclude that a water body is not in compliance with standards, etc. The conservatism built into criteria development and implementation demands that pH instream be accurately assessed and applied to the criteria to reliably determine compliance with water quality standards.
EPA has not adopted data quality objectives to limit the amount of unc ertainty that can exist when applying water quality criteria instream, therefore a consensus standard to determine if water based data can be used to estimate sediment based responses is not available and scientific defensibility cannot be assessed. Howev er, there is an approach that will likely provide consensus agreement in scientific defensibility: to directly address the pH of the instream sediment within the context of a toxicity test rather than trying to compensate for pH differences between water t ests and the instream sediment. This approach will directly address concerns that juvenile test results are a function of the presence/absence of sediment and avoid the uncertainty of mechanisms attempting to extrapolate response from water only exposures . It is recognized that this recommendation would not allow the use of current data to derive ammonia water quality criteria inclusive of freshwater mussel sensitivities. It would ensure that the unique ecology of these mussels’ life stages is accurately captured (within the constraints of conventional testing methods) and that when the resulting criteria are implemented management actions will be reliable.B

- 45

13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?

Alex Barron Results of toxicity tests without sediment provide good information regarding the toxicity of ammonia via exposure to ammonia concentrations in water. Acceptable control mortality in a test remains a significant indicator that the test conditions and the general overall health of the tested organisms are reasonable and the increasing mortality observed in the test chambers with higher concentrations of the tested substances is a direct result of the toxicity of the toxic substance. If the mortality in the control chambers remains low then, this is good evidence that the tested organisms are not experiencing unacceptable stress during the test. In several water

- only 28

- day tests, 100% to 90 % survival was observed in controls. This provides evidence that

w ater

- only tests with juvenile mussels can provide acceptable conditions for assessing the toxicity of a contaminant.

Water

- only exposure tests provide relatively comparable and reproducible exposure conditions that are useful for assessing toxicity sens itivity and to distinguish effects of pH temperature or hardness etc. Toxicity testing in sediment will not change what we know about the sensitivity of the species to ammonia via a water

- only exposure as used and summarized in this criteria document. As shown in Tables 1, 2 and 4, the variability of toxicity values within a species and within genera is all within a factor of 10. Water

- only toxicity tests methods have shown good intra and inter laboratory variability for toxicity tests with ammonia, as we ll as copper and chlorine. This lends confidence in these data for use in criteria development. Toxicity data from water

only exposures are most relevant to the purposes that water quality criteria are most often employed; assessments of natural waters b y comparison to the criteria and for use in developing regulatory limits for permitted discharges of the pollutant.
Concerns that tests conducted in sediment might produce different toxicity results are speculation. Some speculation is that water

- only exposure stresses the test organisms and may make the test organisms more sensitive to the toxicant. However the good survival in the control chambers indicates that the tested mussels are not overly stressed during the test. Also, test conditions are de signed to provide optimum dissolved oxygen and temperature conditions. Natural conditions often are less than optimum with lowered oxygen levels and other conditions. The sediment may be a more natural environment for the juvenile mussels, making them mo re hearty and resistant to the toxic substance, or the sediment may act to absorb the toxic substance and make it less bioavailable and less toxic. On the other hand, if the sediment may absorb the toxic substance and over time increase the concentration in the interstitial pore water in relation to the overlying water. Most of these issues are site specific in nature and beyond the scope of national recommended criteria.
Natural sediments are highly variable and even a standardized reconstituted “clea n” sediment to be used in laboratory tests may prove variable in some unknown way perhaps in response to some unrecognized factor, however this is simply speculation. Use of sediment in toxicity tests will likely introduce added variability without provid ing additional value for judging sensitivity and this will introduce added complexity to interpreting the tests’ results for criteria development.
Several studies (Whiteman et. al., 1996, Hickey and Martin, 1999) have reported that ammonia in interstiti al pore water can often be higher than concentrations in the water. Overall, these concerns about increased ammonia concentrations in interstitial pore water are not so much related to toxicity sensitivity or issues that directly influence the derivati on of criteria. These concerns and observations are more related to anB

- 46 exposure assessment of potential risk to the sediment dwelling organisms and the related physical factors that influence the exposure conditions.
I believe that toxicity data from wate r

- only tests that were conducted under acceptable conditions (with acceptable control mortality) provide the most useful data to judge the quantitative toxic effects of the toxic substance. These water

- only data can then be used to assess potential for t oxicity in the water column. Currently, there is insufficient information available to indicate that the toxicity of ammonia should be considered fundamentally different in interstitial pore water as compared to the toxicity observed in water

- only tests u nder the same pH and temperature conditions. An ammonia toxicity test with water

- only exposures provides reliable test results that can be applied to exposures if the juvenile mussels are exposed to concentrations of ammonia in water. Water exposure te sts should be used in the derivation of water quality criteria and this information should apply equally well to water column or interstitial pore water. If there is a special need to assess interstitial pore water at a site, the criteria can be applied to an assessment of the interstitial pore water. Samples of interstitial water can be collected and analyzed for ammonia concentrations and pH can be measu red in the field. If monitoring of interstitial pore water is conducted, care should be taken concerning the depth at which the samples are taken. Juvenile mussels are likely to be within a few centimeters of the surface and juveniles and adult mussels a re capable of some control over maintaining their position in the sediment and should not be assumed to be regularly found buried in deeper sediments where low dissolved oxygen, reduced food availability and other stress factors may overshadow risk due to ammonia toxicity. Water samples for ammonia analysis and pH field measurements should be taken at the surface interface or at a depth where mussels are likely to be found.

Steven Canton If the test is designed to address water column concerns, then the exposure test using

juvenile mussels should be conducted without sediment in the test chamber. The use of sediment in water quality toxicity tests introduces too many confounding factors in tests designed to evaluate the needs to be protective of aquatic life in the water column. For many species of mussels, the juveniles primarily feed on particles in the sediment and filter the pore water, rather than directly siphon from the overlying water column. Depending upon the composition of the sediment (i.e., organic matter content) the sediment layer can have profound influences on the pH and ammonia concentration within the pore water of the sediment. In addition, the metabolic byproducts of juvenile organisms that live in sediments also influence the level of ammonia observed within pore waters of sediment. For these reasons alone it is difficult to separate the effects solely due to conditions within the water column versus the effects of the sediment layer.
Furthermore, in static renewal tests that inco rporate the use of sediment, there is the potential for decreased ammonia concentrations, as well as pH, in the pore water of sediment when compared to the water column conditions (Allan and Maguire 1992); whereas in natural conditions this relationship is often reversed, such that pH levels and ammonia concentrations are greater in the sediments than the overlying water column. The sediment based toxicity studies of Whiteman et al. (1996) tried to address these gradient issues between the sediment and wa ter column, by introducing nitrogen via sediment and maintaining overlying water concentrations in a non

- toxic range. However, the mobility of the test organisms allowed them to seek out the zones of lower ammonia concentration at the interface rather tha n within the sediment, which also confounded the test results.B

- 47 This concludes my responses to the Charge Questions. Given the time constraints, I feel somewhat limited in what I could provide. I hope this review was helpful

– and thank you for the oppor tunity.

Jerome Diamond Tests using juvenile mussels should be conducted using sediment. Several researchers reported better survival and growth of juvenile mussels with the addition of sediment (e.g., Gatenby et al. 1996, Yeager et al. 1994) and I would note that many of the

mu ssel culturing facilities that produce juveniles for transplants, use sediment in their culturing systems. The ASTM standard for glochidia and juvenile mussel testing (E 24550

- 06) indicates this in several places including sections 10.4.2.4, 10.5.2.1, 10. 5.2.5, 10.6.3.3, and 10.6.3.13. Laboratory studies by Yeager et al. (1994) observed that young juveniles burrow immediately to < 1cm deep in the sediment, and that they were not exposed to overlying water. Others also have reported rapid burrowing of

new juveniles in sediment. This information suggests to me that some sediment in test containers may help ensure that juvenile mussels are unstressed and respond appropriately to contaminants. This might be a good check to see whether ammonia concentrations shown to be toxic in water

- only exposures are also toxic when the tests are conducted with some sediment (clean, sterile, etc.) as well.

George Dixon My comments here are limited to laboratory tests where issues of pH variability

between the sediment and the water column could be eliminated (to avoid direct measurement as discussed above) by experimental manipulation. The main reason for including sediment would presumably be to reduce extraneous stress on the experimental organisms, stress which could po ssibly interact with the toxicant to produce an anomalous result. I have no opinion as to whether the presence or absence of sediment would stress juvenile mussels in an experimental setting. This will have to be determined experimentally.

Jerry Farris I f the need is greater at this point with the reassessment to consider data from tests that offer repeatability and precision of results, than to ensure inclusion of test conditions reflecting the life history and habitat selection for this life stage of th e mussel, then the test should exclude the sediment considerations. There is a growing body of evidence from tests without sediment that suggests comparable juvenile shell growth can be measured in control treatments in static, static

- renewal, and flow

- th rough conditions. This is somewhat contradictory to earlier studies that recognized the importance of sediment in distinguishing effects during longer term exposures with freshwater mussels and fingernail clams. For this reason, the information still see ms lacking in determining how much of a confounding effect is introduced by the effects of sediment characteristics (e.g., particle size, microbial mobilization and organic carbon content). More specific information on the survival and physiological condit ion of benthic organisms when tested in a water

- only medium seems necessary for testing beyond 96 hours. Burton et al. (2002) describes a range of toxicity assays using benthic invertebrates in sediment

- free systems such as interstitial water, elutriate p hase, or spiked waters, with each possibly differentiating toxicant uptake, pathway, and hazard. It would seem prudent to be consistent with current conventional approaches to sediment evaluations for their consideration of appropriate phases to be tested (extractable, elutriate, interstitial, whole sediment, and in situ ) relating to appropriate conditions for the species or substrate of interest. The complexB

- 48 interactions between mussel, water, and sediment that influence their responses has been cited to reinforce those considerations (Thorsen et al., 2007).

Bill Goodfellow I believe this question is very similar to the Hyalella azteca question. If adequate test

results that are representative to the toxicity of ammonia can be performed in water only, that is how they should be performed. If sediment is necessary for the test to be performed adequately, then they should use sediment. However, it should be cautioned that when the variable of sediment is added to the toxicity testing methodology, the com

plexity of the test increases considerably as to the interpretation. In my opinion the agency should caution the use of sediment in the test my lead to positive and negative bias of the test results. The agency should try to stay with water column only whe never possible.

Jim Pletl It is clear from Newton and Bartsch (2007) that the potential for juvenile freshwater mussels to be stressed in the absence of sediment exists. Wang et al. (2007) found that sediment did not appear to impact shell growth and may have slightly reduced survival, but only one test using sediment was conducted and this test only consisted of two replicates per treatment. As stated previously toxicity tests must represent the stress experienced by a single stressor to be used in deve loping water quality criteria. Therefore for this particular use, until more information is available, juvenile tests of any duration must include sediment in the test vessels in order to be used in deriving water quality criteria.B

- 49

Additional Comments

Steven Canton References:

Allan, G. L. and G. B. Maguire. 1992. Effect of pH and salinity on survival, growth and osmoregulation in Penaeus monodon Fabricius. Aquaculture 107:33

- 47.

Anderson , K.B., R.E. Sparks, and A.A. Paparo. 1978. Rapid Assessment of Water Quality, Using the Fingernail Clam, Musculium transversum. WRC Research Report No. 133. University of Illinois, Water Resources Center, Urbana, IL.
Arid West Water Quality Research Proje ct (AWWQRP). 2006. Evaluation of EPA Recalculation Procedure in Arid West Effluent

- Dependent Waters:Final Report. Prepared for the Arid West Water Quality Research Project by URS Corporation, Chadwick Ecological Consultants, Inc., and Parametrix, Inc., P ima County Wastewater Management Department, Tucson, AZ.
Arthur, J.W., C.W. West, K.N. Allen, and S.F. Hedtke. 1987. Seasonal toxicity of ammonia to five fish and nine invertebrate species. Bull. Environ. Contam. Toxicol. 38:324

- 331.
Augspurger, T., A.E. K eller, M.C. Black, W.C. Cope, and F.J. Dwyer. 2003. Water quality guidance for protection of freshwater mussels (Unionidae) from ammonia exposure. Environmental Toxicology and Chemistry 22:2569

- 2575.
Bartsch, M.R. et al. 2003. Effects of pore water ammonia on in situ survival and growth of juvenile mussels (Lampsilis cardium) in the St. CroixRiverway. Environ Toxicol Chem 22:2561

- 2568
Calamari, D., R. Marchetti, and G. Vailati. 1977. Effects of prolonged treatments with ammonia on stages of development of S almo gairdneri. (English Translation) Nuovi Annali d’Igiene e Microbiologia. 28:333

- 345.
Colt, J.E., and G. Tchobanoglous. 1978. Chronic exposure of channel catfish, Ictalurus punctatus, to ammonia: effects on growth and survival. Aquaculture 15:353

- 372.

D ankers N, K. Koelemaij. 1989. Variations in the mussel population of the Dutch Wadden Sea in relation to monitoring of other ecological parameters. Helgolan d Marine Research 43: 529

- 535
Khatami, S. H., D. Pascoe, M. A. Learner. 1998. The acute toxicity of phenol and unionized ammonia, separately and tog ether, to the ephemeropteran Baetis rhodani (Pictet). Environmental Pollution 99:379

- 387.
Mount, D.I. 1982. Ammonia toxicity tests with Ceriodaphnia acanthine and Simocephalus vetulus. Memorandum to Dr. R.C. Russo, ESEPA, August 6, 1982.
Reinbold, K.A., an d S.M. Pescitelli. 1982. Acute Toxicity of Ammonia to the White Sucker. Final Report to EPA. Center for Aquatic Ecology, Illinois Natural History Survey, Champaign, Illinois.
Rubin, A.J., and M.A. Elmaraghy. 1977. Studies on the toxicity of ammonia, nitrat e and their mixtures to guppy fry. Water Research 11:927

- 935.B

- 50 Russo, R.C., D.J. Randall, and R.V. Thurston. 1988. Ammonia toxicity and metabolism in fishes. In R.C. Ryans, ed. Protection of River Basins, Lakes, and Estuaries . pp. 159

- 173. American Fisherie s Society, Bethesda, Maryland.
Sangli, A. B. and V. V. Kanabur. 2001. Toxicity of ammonia to a freshwater fish, Gambusia affinis and its effect on oxygen consumption. Geobios 28:56

- 58.
Sparks, R.E. and M.J. Sandusky. 1981. Identification of Factors Respon sible for Decreased Production of Fish Food Organisms in the Illinois and Mississippi Rivers. Final Report for Project No. 3

- 291

- R, Illinois Natural History Survey, River Research Laboratory, Havana, Illinois.
Stephan, C.E., D.I. Mount, D.J. Hansen, J.H. Gentile, G.A. Chapman, and W.A. Brungs. 1985. Guidelines for Deriving Numerical National Water Quality Criteria for the Protection ofAquatic Organisms and Their Uses . PB

- 85

- 227049. U.S. Environmental Protection Agency, Office of Research and Developmen t, Duluth, Minnesota.
Thurston, R.V., R.J. Luedtke, and R.C. Russo. 1984. Toxicity of Ammonia to Freshwater Insects of Three Families. Technical Report No. 84

- 2, Fisheries Bioassay Laboratory, Montana State University, Bozeman, MT. 26 pp.
Thurston, R.V., R .C. Russo, and G.A. Vinogradov. 1981. Ammonia Toxicity to Fishes. Effect of pH on the Toxicity of the Un

- ionized Ammonia Species. Environ. Sci. Technol. 15:837

- 840.
Tomasso, J.R., C.A. Goudie, B.A. Simco, and K.B. Davis. 1980. Effects of environmental pH and calcium on ammonia toxicity in channel catfish. Transactions of the American Fisheries Society 109:229

- 234.
U.S. Environmental Protection Agency (USEPA). 1999 Update of Ambient Wate r Quality Criterion for Ammonia. Office of Water, Washington, DC. EPA

- 822

- R

99

- 014.
U.S. Environmental Protection Agency (USEPA). 2001. 2001 Update of Ambient Water Quality Criteria for Cadmium . EPA

- 822

- R

- 01

- 001. Office of Water, Washington, DC.
Yeager , M.M., D.S. Cherry and R.J. Neves. 1994. Feeding and burrowing behavior of juvenile rainbow mussels, Villosa iris (Bivalvia: Unionidae). J.N. Am. Benthol. Soc. 13(2):217

- 222.
Wallen, I.E., W.C. Greer and R. Lasater. 1957. Toxicity to Gambusia affinis of c ertain pure chemicals in turbid waters. Sewage Ind. Wastes. 29: 695

- 711.
Whiteman, F.W., G.T. Ankely, M.D. Kahl, D.M. Rau, and M.D. Balcer. 1996. Evaluation of interstitial porewater as a route of exposure for ammonia in sediment tests with benthic macroin vertebrates. Environmental Toxicology and Chemistry. 15(5):794

- 801.

Jerome Diamond Other Comments Regarding Appendex A Under “USEPA’s Aquatic Life Criteria Coordinating Committee” comments, point #2 makes it appear that EPA is willing to alter a national criterion due to a commerciallyB

- 51 valuable species (e.g., trout) but not an ecologically critical species? Point #3: I a gree there is no evidence that glochidia in conglutinates are exposed to water

- borne pollutants but there’s no evidence that they are not exposed either. Also, uptake of pollutants by planktonic invertebrates lacking gills (e.g., Daphnia ) occurs via absor ption through the carapace, not just through food. Point #8: “For glochidia that attach to hosts,”

– all glochidia are meant to attach to hosts. What I think you mean here is “In terms of the duration of free

- living glochidia, there is a major differenc e....” Other comments regarding acute data in the Reassessment Document Daphnia GMAV is Incorrect I believe the document reports an incorrect GMAV for Daphnia (p. 9) as well as test data in Table 1 (p. 49

- 50). It appears that the unionized ammonia values re ported by Russo et al. (1985) and reported as such in EPA’s 1999 document, were incorrectly transcribed as total ammonia values (see p. 109, Appendix 4 of 1999 document as well as p. 38, Table 4 of the 1999 document). Even with the temperature correction for invertebrates in the Reassessment Document, Daphnia couldn’t possibly go from Rank 19 in acute sensitivity to 8 th in the draft document. This error has other ramifications as well: assuming the Daphnia value is incorrect in the draft, when mussels we re absent, the four most sensitive genera are Corbicula
, Potamopyrous , Pleurocera , and Prosopium (mountain whitefish). If Corbicula is removed along with the unionids as EPA proposed (and which I disagree with), then Deltistes (Lost River sucker) becomes the 4 th most sensitive genera. Either way, invertebrates no long occupy the four most sensitive genera. Although I did not do a comprehensive check, it appears that the other acute values in the draft document are consistent with the 1999 document.
Tempe rature Equation for Invertebrate and Acute Data should be re

- examined The temperature

- ammonia sensitivity relationship applied by EPA to invertebrate acute data in the draft document is based on the slope provided in the 1999 document, based on Arthur et a l. (1987), but not used for the acute criterion in that document. As the 1999 document makes clear, the temperature relationships from Arthur et al. (1987) were based on seasonal tests of experimental stream water, including condition, life stage, etc. of test organisms collected during each season. As Arthur et al. (1987) notes, several water quality characteristics other than temperature varied with season and, even more importantly perhaps, organism condition was unlikely to be similar

across seasons a nd at the least was unknown. EPA provided what I believe was a poor rationale for using Arthur’s et al. data for chronic invertebrate data. The fact that Arthur did not observe a clear temperature relationship with fish (which are also cold blooded) woul d weaken the case that temperature was what was driving the apparent ammonia sensitivity in invertebrates in his study (and which Arthur himself acknowledges in his paper). The temperature

- toxicity relationship derived by EPA for ammonia in the 1999 docume nt had a fairly steep slope; e.g., a difference between 20 and 25  C resulted in 2

- 3 fold decrease in the LC50. There are a few invertebrate species in the draft document for which it appears possible to reanalyze a temperature toxicity relationship, and to determine if one actually exists.
I have not had time to do a formal analysis of the invertebrate acute data, however, my initial reaction is that temperature may be somewhat weakly related to acute ammonia toxicity in invertebrates. For example, for t he snail species PotamopyrgusB

- 52 antipodarum , Hickey and Vickers (1994) reported an LC50 of 8.71 mg N/L at pH = 8.2 and 15  C and an LC50 of 4.08 mg N/L at pH = 8.2 and 25  C (a factor of 2 over a 10  C difference). The LC50 they reported at the same pH and 20  C was nearly identical to the LC50 at 25  C (4.73 mg N/L). LC50’s for the fatmucket mussel at pH = 8.1 and 20  C ranged from 5.2 mg N/L to 11 mg N/L (Wang et al. 2008) a factor of about 2, whi ch could be within the range specified by a 5  C difference in EPA’s temperature equation. Even larger differences in LC50’s are commonly reported for other invertebrates based on tests at the same pH and temperature (e.g., Villosa iris LC50 = 3.0

– 8.9 m g N/L at pH = 8.3 and 20  C). These data suggest that a temperature adjustment factor may be premature for acute invertebrate data. Acute data for Ceriodaphnia dubia may perhaps provide the best information for invertebrates thus far. At pH = 8.2 and te mperature of 7  C, the LC50 = 16.65 mg N/L (Nimmo et al. 1989). At pH = 8.16 and 22  C, the LC50 = 30.08 mg N/L (Black 2001), however, at pH = 8.0 and 25  C, the LC50 = 14.52 mg N/L (Scheller 1997), similar to the LC50 reported at 7  C. EPA should reanal yze current acute data, similar to the way they did for the 1999 document, to determine the relationship between temperature and acute ammonia toxicity to invertebrates.

LITERATURE CITED
Arthur, J.W., C.W.West, K.N. Allen and S.F. Hedtke. 1987. Seasonal to xicity of ammonia to five fish and nine invertebrates species. Bull. Environ. Contam. Toxicol. 38(2): 324

- 331.
Black, M. 2001.Water quality standards for North Carolina's endangered mussels. Department of Environmental Health Science, Athens, GA.

Burton, G.A. 1992. Sediment collection and processing factors affecting realism. In: G.A. Burton, Ed., Sediment Toxicity Testing. Lewis, Boca Raton, FL, pp. 37

- 64.
Catherine M. Gatenby, Richard J. Neves and Bruce C. Parker 1996. Influence of Sediment and A lgal Food on Cultured Juvenile Freshwater Mussels. Journal of the North American Benthological Society, Vol. 15: 597

- 609
Dodd, BJ, MC Barnhart, CL Rogers

- Lowery, TB Fobian, RV Dimock, Jr, 2006. Persistence of host response against glochidia larvae in Micropterus salmoides. Fish & Shellfish Immunology 21:473

- 484
Hickey, C.W. and M.L. Vickers. 1994. Toxicity of ammonia to nine native New Zealand freshwater invertebrate species. Arch. Environ. Contam. Toxicol. 26(3): 292

- 298.
Kernaghan, N. J., Gross, T. S., Bishop, C.D., Wang, N., Roberts, A., and Ingersoll, C. G.. 2005. “Laboratory Toxicity Testing with Freshwater Unionid Mussels,” Freshwater Bivalve Ecotoxicology, van Hassel, J. H., and Farris, J. L., eds., SETAC Press, Pensacola, FL.
M. M. Yeager, D. S. Cherry and R. J. Neves. 1994. Feeding and Burrowing Behaviors of Juvenile Rainbow Mussels, Villosa iris (Bivalvia:Unionidae) Journal of the North American Benthological Society, Vol. 13: 217

- 222
Neves, R. J., and Widlak, J. C. 1988. Occurrence of Gloc hidia in Stream Drift onB

- 53 Fishes of the Upper North Fork Holston River, Virginia. Am Midland Naturalist, 119, 1988, pp. 111

- 120.
Nimmo, D.W.R., D. Link, L.P. Parrish, G.J. Rodriguez, W. Wuerthele and P.H. Davies. 1989. Comparison of on

- site and laboratory toxicity tests: Derivation of site

- specific criteria for un

- ionized ammonia in a Colorado transitional stream. Environ. Toxicol. Chem. 8(12): 1177

- 1189.
Rogers

- Lowery, CL and RV Dimock, Jr. 2006. Encapsulation of attached ectoparasitic larvae of freshwa ter mussels by epithelial tissue on fins of naive and resistant host fish. Biol. Bull. 210:51

- 63
Salmon, A. and R.H. Green. 1983. Environmental determinants of unionid clam distribution in the Middle Thames River, Ontario. Canadian Journal of Zoology 61: 832

- 838.
Sardia, N. and G. Burton. 1995. Ammonia variation in sediments: Spatial, temporal and method

- related effects. Environ. Toxicol. Chem. 14(9):1499

- 1506. Scheller, J.L. 1997. The effect of dieoffs of Asian clams ( Corbicula fluminea ) on nativ e freshwater mussels (unionidae). Virginia Polytechnic Institute and State University, Blacksburg, VA.
Schwal, A. and M. Pusch 2007. Horizontal and vertical movements of unionid mussels in a lowland river Journal of the North American Benthological Socie ty 26(2):261

– 272.
Stemmer, B.L., G.A. Burton, Jr. and G. Sasson

- Brickson. 1990. Effect of sediment spatial variance and collection method on cladoceran toxicity and indigenous microbial activity determinations. Environ. Toxicol. Chem. 9:1035

- 1044.

Str ayer, D. L., Downing, J. A., Haag,W. R., King, T. L., Layzer, J. B., Newton, T. J., and Nichols, S. J. 2004. Changing Perspectives on Pearly Mussels, North America’s Most Imperiled Animals. BioScience, 54:2004, pp. 429

- 439.

U.S. Environmental Protection Agency. 1994. Interim Guidance on Determination and Use of Water Effect Ratios for Metals, Appendix B: The Recalculation Procedure. EPA

- 823

- B

- 94

- 001. Office of Water, Washington, DC.
USEPA. 2000. Ambient Aquatic Life Water Q uality Criteria for Dissolved Oxygen (Saltwater): Cape Cod to Cape Hatteras. EPA

- 822

- R

- 00

- 012 Office of Water, Washington, D.C. November 2000
USEPA. 2002. Clinch and Powell Valley Watershed Ecological Risk Assessment. 2002. EPA

- 600

- R

- 01

- 050. NCEA, O RD, Washington, D.C.
Wang, N., C.G. Ingersoll , D.K. Hardesty, C.D. Ivey, J.L. Kunz, T.W. May, F.J. Dwyer, A.D. Roberts, T. Augspurger, C.M. Kane, R.J. Neves and M.C. Barnhart. 2007. Contaminant sensitivity of freshwater mussels: Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mussels (Unionidae). Environ. Toxicol. Chem. 26(10):2036

- 2047.
Wang, N., J.T. Augspurger, M.C. Barnhart, J.R. Bidwell, WG. Cope, F.J. Dwyer, S. Geis, I.E. Greer, C.G. Ingersoll, C.M., Kane, T .W. May, R.J. Neves, T.J.B

- 54 Newton, A.D. Roberts and D.W. Whites. 2007. Contaminant sensitivity of freshwater mussels: Intra

- and inter

- laboratory variability in acute toxicity tests with glochidia and juveniles of freshwater mussels (Unionidae). Environ. To xicol. Chem. 26(10):2029

- 2035.
Watters, G. A., 2005. A Brief Look at Freshwater Mussel (Unionacea) Biology. Freshwater Bivalve Ecotoxicology, van Hassel, J. H., and Farris, J. L., eds., SETAC Press, Pensacola, FL.
Watters, G. T., S. H. O’dee, And S. Chordas. 2001. Patterns of vertical migration in freshwater mussels (Bivalvia: Unionidae). Journal of Freshwater Ecology 16:541

– 549.
Way, C.M., A.C. Miller and B.S. Payne. 1990. The influence of physical factors on the distribution and abundance of fresh water mussels (Bivalvia:Unionidae) in the lower Tennessee River. The Nautilus 103(3): 96

- 98.

George Dixon I have reviewed the document listed above as requested in the Charge to Reviewers

dated 24 July 2009. In addition I have gone through the background document “1999 Update of Ambient Water Quality Criteria for Ammonia”. Let me state at the outset that I fin d the Draft Reassessment to be a well conceived, well developed and carefully written document. The acute and chronic ammonia criteria for freshwaters with mussels present, and separate criteria for waters with mussels absent, are well founded and scientif

ically defensible. I have presented my review comments as responses to the thirteen questions posed in the Charge to Reviewers Those questions are repeated here for clarity.

Jerry Farris References:
American Society for Testing and Materials. 2006. Stan dard guide for conducting laboratory toxicity tests with freshwater mussels. E2455

- 06. In Annual Book of ASTM Standards, Vol 11.06. Philadelphia, PA.
Augspurger, T. 2007. Advances and opportunities in assessing contaminant sensitivity of freshwater mussel (Unionidae) early life stages. Environ Toxicol Chem 26:2025

- 2028.
Bartsch MR, Newton TJ, Allran JW, O’Donnell JA, Richardson WB. 2003. Effects of po re

- water ammonia on in situ survival and growth of juvenile mussels ( Lampsilis cardium ) in the St. Croix riverway, Wisconsin, USA. Environ Toxicol Chem 22:2561

– 2568.
Burton, GA, PM Chapman, and EP Smith. 2002. Weight of evidence approaches for assessing ecosystem impairment. Human and Ecological Risk Assessment: An international Journal 8:1657

- 1673.
Cherry, DS, JL Scheller, N.L. Cooper and J.R. Bidwell. 2005. The potential for Asian clam ( Corbicula fluminea ) dieoffs to impact native freshwater mussels (Unionidae) I: Water column ammonia levels and ammonia toxicity. Journal of the North American Benthological Society, 24:369

- 380.B

- 55 Cooper, NL. JR Bidwell and DS Cherry. 2005. The potential for Asian clam ( Corbicula fluminea ) die

- offs to impact native freshwater mussels (Unionidae) II: Pore

- water ammonia. Journal of the North American Benthological Society, 24:381

- 394.
Mummert, AK, Neves RJ, Newcomb TJ, Cherry DS. 2003. Sensitivity of juvenile freshwater mussels ( Lampsilis f asciola
, Villosa iris ) to total and un

- ionized ammonia. Environ Toxicol Chem 22:2545

– 2553.
Newton TJ, Allran JW, O’Donnell JA, Bartsch MR, Richardson WB. 2003. Effects of ammonia on juvenile unionid mussels ( Lampsilis cardium ) in laboratory sediment toxic ity tests. Environ Toxicol Chem 22:2554

– 2560.
Salazar, MH and SM Salazar. 2007. Linking bioaccumulation and biological effects to chemicals in water and sediment: A conceptual framework for freshwater bivalve ecotoxicology. In Farris JL, Van Hassel JH , eds, Freshwater Bivalve Ecotoxicology. SETAC, Pensacola, FL, USA, pp 215

- 250.

Scheller JL. 1997. The effect of die

- offs of Asian clams ( Corbicula fluminea ) on native freshwater mussels (Unionidae). Masters thesis. Virginia Polytechnic Institute and Sta te University, Blacksburg, VA, USA.
Stephan CE, Mount DI, Hansen DJ, Gentile JH, Chapman GA, Brungs WA. 1985. Guidelines for deriving numerical national water quality criteria for the protection of aquatic organisms and their uses. EPA 822/A85/100. U.S. E nvironmental Protection Agency, Office of Research and Development, Washington, DC.
Thorsen, WA, WG Cope, and D Shea. 2007. Toxicokinetics of environmental contaminants in freshwater bivalves. In Farris JL, Van Hassel JH, eds, Freshwater Bivalve Ecotox icology. SETAC, Pensacola, FL, USA, pp 169

- 207.
Van Hassel, JH. 2007. Case study: Discrimination of factors affecting Unionid mussel distribution in the Clinch River, Virginia, USA. In Farris JL, Van Hassel J, eds, Freshwater Bivalve Ecotoxicology. S ETAC, Pensacola, FL, USA, pp 311

- 331.
Wang N, Ingersoll CG, Hardesty DK, Ivey CD, Kunz JL, Dwyer FJ, Roberts AD, Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007. Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mu ssels (Unionidae). Environ Toxicol 26:2048

- 2056.
Wang N, Ingersoll CG, Greer IE, Hardesty DK, Ivey CD, Kunz JL, Dwyer FJ, Roberts AD, Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007. Chronic toxicity of copper and ammonia to juvenile freshwater mussels (Unionidae). Environ Toxicol Chem 26:2048

- 205.

Bill Goodfellow Re: Review of EPA’s Draft Reassessment of the 1999 Ambient Water Quality Criteria for Ammonia

- Freshwater.

Dear Ms. Waite:
I have found the criteria document to be well organized and very well thought out. I also feel that the scientific justification laid out in the document to be excellent.B

- 56 I have several questions or comments presented below as well as imbedded in the Charge Questions section (my comments are presented in Bold).

I do question why only standards consistent with ASTM Standards for testing (page 14) are the only relevant test procedures that were used in the document. I believe that the EPA’s Whole Effluent Testing (WET) toxicity testing guidelines for testing are also re levant. Did this exclude any additional data? From my review of the references, I do not believe so.
I also question when the Nile tilapia was place in the other chronic toxicity data (and I agree this was appropriate), why is the Asiatic clam included in the data used for development of the criteria (and in fact it is the third most sensitive species for the acute criterion). The Asiatic clam is also a nonindigenous species (or exotic). In my opinion I don’t believe that the Asiatic clam should not be use d in the development of the criteria regardless that this exotic is found throughout United States. I believe elimination of this species from the dataset will still generate a protective acute criterion.

The Table A in text of the GMAV for the Mountain Whitefish does not match Table 1. The Text table reports 12.11 and Table 1 reports 12.09. I am expecting that the Table 1 is correct and is what was used for the FAV. Please verify.
I thank you for the opportunity to allow me to be a peer reviewer for thi s important freshwater water quality criteria document. If you have any questions or require additional information, please let me know.
Sincerely,

William L. Goodfellow, Jr. Vice President and Senior Scientist
Clarification question from EPA for the following comment made by Dr. Bill Goodfellow:
"I also question when the Nile tilapia was place in the other chronic toxicity data (and I agree this was appropriate), why is the Asiatic clam included in the data used for development of the criteria (and i n fact it is the third most sensitive species for the acute criterion). The Asiatic clam is also a nonindigenous species (or exotic ). In my opinion I don't believe that the Asiatic clam should not be used in the development of the criteria regardless that this exotic is found throughout United States. I believe elimination of this species from the dataset will still generate a protective acute criterion

. "
EPA Question: “...it sounds like he's questioning why we included the clam data but then the text I highlighted in red sounds like he's saying that we should include it

- maybe there's a typo and the double negative sentence is wrong? If you could get clarification from him for me on this I would appreciate it.

Clarification from Dr. Bill Goodfellow: I n my opinion the Asiatic clam (an exotic) should not be included in the database usedB

- 57 for development of the criteria. Rather it should be placed in the Table with other data. If the Agency decides to include the Asiatic Clam in the criteria development, I believe considerable justification will need to be provided in the written justification of the criteria document.
Hopefully this makes it clearer. Let me know if you have any other questions or require additional information.

Jim Pletl LITERATURE CITED

1. R.B Bringolf, W.G. Cope, S. Mosher, M.C. Barnhart, D. Shea. 2007a. Acute and Chronic Toxicity of Glyphosate Compounds to Glochidia and Juveniles of Lampsilis siliquoidea (Unionidae). Env. Tox. Chem.26(10), p.2094

2. R.B Bringolf, W.G. Cope, M.C. Bar nhart, S. Mosher, M.C. Barnhart, P. Lazaro, D. Shea. 2007b. Acute and Chronic Toxicity of Pesticide Formulations (Atrazine, Choropyrifos and Permethrin) to Glochidia and Juveniles of Lampsilis siliquoidea (Unionidae). Env. Tox. Chem.26(10), p.2101

3. T.J N ewton and M.R. Bartsch. 2007. Lethal and Sublethal Effects of Ammonia to Juvenile Lampsilis Mussels (Unionidae) in Sediment and Water Only Exposures. Env. Tox. Chem. (26(10), p.2057

4. N. Wang, C.G. Ingersoll, I.E. Greer, D.K. Hardesty, C.D. Ivey, J.L. Kunz, W.G. Brumbaugh, F.J. Dwyer, A.D. Roberts, T. Augspurger, C.M. Kane, R.J. Neves, M.C. Barnhart. 2007. Chronic Toxicity of Copper and Ammonia to Juvenile Freshwater Mussels (Unionidae). Env. Tox. Chem. 26(10), p. 2048C

- 1 

## SECTION C
Individual Peer Reviewer CommentsC

- 3 PEER REVIEW COMMENTS FROM



### Alex Barron Virginia Dep artment of Environmental Quality Office of Water Quality Criteria and Standards

629 East Main Street Richmond, VA 23218 804

- 698

- 4119 Email: Alex.Barron@deq.virginia.govAlex Barron

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- 5 EPA’s Draft Reassessment of the 1999 Ambient Water Quality

Criteria for Ammonia

– Freshwater

July 14, 2009
Review Comments by Alex Barron Virginia Department of Environmental Quality

August 18, 2009

CHARGE QUESTIONS
Acute criteria in fresh waters: Question: 1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other rele vant data that were not used?

Review Comment : Yes, all the toxicity tests that influence the derivation of the new criteria for ammonia appear to be scientifically defensible. All relevant data that I am aware of has been considered in this reassessment
document

.

Question: 2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?

Review Comment: Table B compares Final Acute Value (FAV) and Criterion Maximum Concentration (CMC) values calculated with the entire dataset including data for bivalve mollusks and also with the dataset with the data for bivalve mollusks removed. The Asiatic clam ( Corbicula fluminea ) is in a different Family ( Cobiculidae ) an d Order ( Veneroida ), and is not a Unionid mussel. Corbicula has shown very similar sensitivity to ammonia compared to Unionid mussels however. Table B really represents a non

- bivalve mollusk dataset, which would be appropriate where no bivalve mollusks o f any kind are present. The
FAV and CMC calculated without the data for bivalve mollusks is less than a factor of two different than the FAV using the entire dataset, which is 62.75 % higher. The 1999 ammonia criteria also provided separate recommended acute criteria based on observed sensitivity of salmonid species . The 1999 criteria
document also presented separate acute criteria that are approximately 50 % differe nt between the salmonids present or absent acute criteria . T h e recommendations in the 20 09 reassessment document providing separate criteria , calculated with and without a group of sensitive species is in keeping with precedents set in the earlier ammonia criteria for calculating separate criteria when one group of species appears to be espe cially sensitive to ammonia. This can be considered appropriate for ammonia criteriaAlex Barron

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- 6

because of the relatively large database and the fact that ammonia toxicity is usually caused by mg/L concentrations as compared to ug/L concentrations as seen with man y other toxic pollutants.

The implementation of criteria based on the dataset without freshwater mussels will require some way of determining whether or not a water body is not likely to contain freshwater mussels, before such
criteria can be consider ed appropriate. The presence or absence of freshwater mussels in a water body may be difficult to ascertain without a survey that specifically targets this issue. Most monitoring techniques for collecting benthic macroinvertibrates such as the widely use d EPA Rapid Bioassessment Protocol (RBP) are not designed or intended to regularly collect mussels that are often tightly imbedded in the sediment. Thus a typical RBP survey that did not collect freshwater mussels cannot be accepted as proof of absence of mussels in that waterbody . I would recommend a default of assuming the presence of
freshwater mussels unless several surveys that specifically targeted the identification of mussels have been conducted on that water body. A brief review of biological su rvey data in Virginia showed that the presence of freshwater mussels was detected in 83 % of water bodies that had been surveyed with methods capable of finding mussels. However, in some waterbodies the presence of mussels was
confirmed only after several surveys . Because the distribution of freshwater mussels is often dependent on fish for dispersal, it might be suspected that mussels may not be present in some headwater or intermittent steams where it can be demonstrated that fish are not present due to physical barriers preventing fish migration.

Question: 3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe there is an alternative approach to the use of this data that would be more scientifically sound?

Review Comment: The position statement in Appendix B of the reassessmen t document acknowledges several

c oncerns about the use of toxicity data for glochidia and recommends that these data should not be used for criteria development at the present time. This position seems reasonable given the uncertainties regarding glochidia testing as outlined in Appendi x B. Glochidia, with such short

- lived life stages and with variable lengths of natural viability in the water column
, ranging from a few hours to several days, it is difficult to know how to use some of these data for developing criteria. For some specie s where the natural viability of glochidia, or where the actual exposure to the water column is short, the result of 48 hour tests may not provide a realistic exposure scenario , while a shorter exposure may be more important.

.Alex Barron

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- 7 There are some additiona l short

- term (6 and 24

- hour) exposure data available that may provide
some additional information on this issue. Table 2 of the Reassessment Document provides acute values
based on 48

- hour exposures for tests conducted with glochidia in several publication s. For some of these
same tests, a dditional information on 6 hour and 24

- hour exposures are also contained in Wang et al 2007b, in their table 3. These 6 and 24

- hour data indicate that glochidia are less sensitive to ammonia during 6 and 24

- hour exposur es than during a 48 hour exposure. For the species where glochidia may only be viable or exposed to the water column for a few hours, the results of 6 hour and 24 hours tests may provide useful information and these data could be normalized to the proper s tandard conditions and included in Table 2, to provide additional information for comparison

. As reported in Wang et al 2007b; the data suggest that at 6 to 24 hour test durations, the sensitivity of glochidia for most species of
freshwater mussels is les s than or equal to the sensitivity of the juveniles of the same species at 96 hours. This provides some support that the revised ammonia acute criteria (based on 96 hour dat a for juveniles) may also provide protection to glochidia at exposure durations of 24 hours or less. The fact the acute criterion is applied as a one

- hour average should also provide some additional level of protection.

Question: 4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scientifically defensible principle for structuring the population exposure duration problem and designing further research to quantify such a duration?

Review Comment: I believe that the approach outlined in appendix B is defensible. Given the significant uncertainties
concerni ng the appropriate exposure durations for glochidia as discussed in the appendix B, attempting to use toxicity data currently available for glochidia to derive defensible criteria at the present time appears
to be untenable. This would be especially impor tant if the final criteria were significantly influenced by glochidia data with the recognized uncertainties about practical ecological importance. Water quality criteria should be based on the best information available in order to gain acceptance and av oid continued uncertainty. Additional information as discussed in appendix B would be needed to provide sufficient confidence in utilizing glochidia data to d e rive water quality criteria.Alex Barron

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- 8

Hyalella azteca position statement and proposed rationale (s ee Appendix B):
The EPA workgroup developed a position statement and proposed supporting rationale describing the concerns over using Hyalella azteca toxicity test data in criteria development due to the uncertain health of the test organisms in different test water compo sition. The rationale defines the specific concerns and uncertainties supporting the recommended exclusion of the Hyalella data from use in criteria derivation, at this time; the position statement is based on the workgroup’s review of a number of toxicit y tests on Hyalella, referenced in the rationale.
Question: 5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?

Review Comment: Yes. The EPA Guidelines for Diving Numerical National Water Quality Criteria for the Protection of Aquatic Organisms and Their Uses, in section IV. 5. H specifies that in order to be useful for calculating a Final Acute Value, the must be relativ ely good agreement of the data within a species. Generally “if the acute values differ by more than a factor of 10, some or all of the values probably should not be used in the calculation” of a Species Mean Acute Value (SMAV). This applies to the avai lable
data set for Hyalella azteca, where the normalized acute values listed in Table 2 range from 1.58 mg/L to 83.9 mg/L, differing by as much as a factor of 53. This wide range of acute values for H. azteca required
more careful review before determinin g what if any of the data for this species is acceptable for criteria development. EPA has conducted an additional review of these data and has determined that there are significant uncertainties with data for this species , enough to preclude their use i n deriving criteria. The position statement provides a summary of the information known concerning possible factors that could cause the acute toxicity values for H. azteca to be affected by some water quality characteristics not previously recognized or considered and not fully understood at this time . Based on the current information available, it appears reasonable that until these factors are better understood, the data for H

. azteca should not be used in the calculation of a SMAV or a FAV.Alex Barron

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- 9 Chronic Freshwater Criterion:
Question: 6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such use? Are you aware of other relevant data that were not used?

Review Comment: Yes. The toxicity studies referenced all appear to be good quality studies and provide useful information on the chronic toxicity of ammonia to aquatic organisms. The use of EC 20 v alues calculated by regression analysis is excellent and provides a consistent estimation of low

- level effects under chronic
conditions.
All toxicity data relevant to ammonia toxicity that I am aware of are included in the references and we r e considered i n the reassessment document.

Question: 7. Is the freshwater chronic criterion scientifically defensible with mussels present and mussels absent?

Review Comment: Yes. Similar distinctions for the presence or absence of certain sensitive organisms sho wing
different sensitivities to ammonia have been used before. The 1999 ammonia criteria recommended different chronic criteria based on the presence or absence of early life stages of fish in the water body
, setting a precedent of allowing different crit eria for different situations. These different criteria were based on a similar magnitude of difference in sensitivity between the early life stages and the older life stages of fish (in the 1999 reassessment criteria) as is seen between the sensitivities of freshwater mussels and other species (in the 2009 reassessment) . This policy of providing these separate criteria based on the presence absence of freshwater mussels will provide some flexibility in implementing the criteria in some
instances and is r easonable.

Given the wide spread nature of ammonia in natural environments and the difficulties and expense imposed on municipalities in treating for ammonia in sewage treatment facilities
, a significant lowering of the ammonia criteria is likely to hav e important consequences for municipalities . Providing these two criteria may help alleviate concerns that any additional restrictions on ammonia discharges are targeted to providing the needed level of protection for the waterbody, based on whether or no t the more sensitive mussels are present. Providing two EPA recommended criteria that already take these issues
into account could help reduce the potential for requests for consideration of developing site

- specific criteria based on the recalculation pr ocedure, which can require significant resources to pursue.Alex Barron

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- 10

Table C shows the four most sensitive genera and the calculation of the CCC for the two different criteria (with and without Unionid mussel data). The data under “excluding freshwater mussel data (family Uniondale)” includes the GMCV for the fingernail clam, Musculium transversum as the third most sensitive genera and this GMCV is used in the calculation of the CCC for “non

- Unionid containing
waters” . While it is true that M

. transversum spec ies is not in the family Unionidae
, it is a bivalve
mollusk.

In the recommendations for developing separate acute criteria, the calculation of the FAV and CMC as shown in Table B, the Asiatic clam was removed from the dataset to produce a criteria based on
a “non

- bivalve mollusk” dataset (as commented on under question # 2 above). There may be water bodies with physical habitats (such as some headwater streams) that are unsuitable for bivalve mollusks of any kind, not just Unionids. EPA should consider whether or not to base their recommendations regarding separate criteria based on removing data for Unionid genera only, or for all bivalve mollusks. The same approach should be used for both the acute and chronic criteria.

(Note: there is a small typo in Table C; the far right hand column should be labeled GMCV, rather than GMAV)

Use of 28

- day Juvenile Test Data (see Appendix C):
Water quality criteria for the protection of aquatic life are derived using toxicity endpoints that relate to population level impacts. In general, these endpoints relate to survival, growth and/or reproduction. The 28

- day test with juvenile mussels, while similar in duration to a standard chronic test, is not technically an early

- life stage test according to the 1985 Guidelines for Aquatic Life Criteria, as much of the early development will have already occurred.
Question: 8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defensible as "chronic" test data for criteria development?

Review Comment : I do not believe that growth measured during a 28

- day test can be considered a true measure of a
chronic effect as intended by the requirements in the 1985 Guidelines for Aquatic Life Criteria, such that they can be related to significant population impact s. Given the life span of the organism and the time needed to reach maturity, a 20% difference in growth observed over 28 days may be reversible and may not be relevant on a population level and should not be used to derive criteria.

Question:Alex Barron

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- 11 9. Should toxicity studies on the growth rates of mussel shells during 28

- day tests be considered quantitatively when developing water quality criteria?

Review Comment: No. I do not believe that differences in growth rates measured over a 28

- day period should b e considered to provide acceptable quantitative chronic endpoint data for freshwater mussels that should be
used to calculate chronic criteria for ammonia. Chronic criteria for ammonia must be based on clearly recognized adverse effects in order to be acceptable. Reductions in survival are irreversible and are clearly adverse effects, while slight reductions in growth may not be significant to the populatio n , or the organisms may be able to compensate over time and reverse the difference in growth. These type tests do not meet the requirements of the EPA Guidelines for use as a quantitative value in driving a chronic criterion. Using such data to derive chronic criteria, especially if it significantly influences the final
criteria would likely increase uncertainty about the final criteria.

Question: 10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?

Review Comment: Yes. Although t oo many questions surround the use of growth effects during a 28 day test in relation to long lived species such as freshwater mussels to consider using differences in growth as data that directly influence the calculation of the final chronic criteria, thes e data still deserve consideration and inclusion in the EPA criteria document. These data do provide useful information to help judge the potential for “other” effects that have a potential for concern. Including such data under the category of
“other dat a” can provide additional information that can lend confidence that the criteria based on survival can provide an adequate amount of protection. The magnitude of difference in growth may make a difference; a 20 to 25 % reduction in growth may not be of mu ch ecological significance, especially if there is a thought that the effect may be short term and reversible, while a > 75% reduction in growth may be considered more of a concern. The magnitude of difference between the effects on survival and the
effec t on growth can also provide useful information. Altogether, I see value in presenting these growth
data under the classification of “other data” in the criteria document

.

Question

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?Alex Barron

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- 12

Review Comment : I would agree that this statement could a lso be true. We simply do not know whether or not an effect on growth observed over a 28

- day exposure can be considered reversible or indicative of a permanent effect. As discussed above, I believe the magnitude in difference in the growth and the magnit ude of difference between the survival end point verses the growth endpoint could influence the level of concern regarding such data.

Question: 11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Juvenile and adult muss els, as sediment

- dwelling organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have suggesti ons on how states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?

Review Comment : The toxicity data currently available for freshwater mussels are for glochidia and juveniles. The

exposu re to unencysted glochidia will be primarily via water exposure, and once encysted on a host fish they will be in a less exposed condition. Juvenile mussels will also be initially exposed to ammonia in the
water column following dropping off (excystment) from the host fish. A s the juvenile mussels are deposited onto the substrate they may remain on the surface or may become situated within a few centimeters of the surface. They will need to maintain a position on or near the surface in order to e nsu r e

acc ess to well

- oxygenated water as well as filterable food. In all cases, the exposure to ammonia will be over the gills via water; either surface water or interstitial water and the water quality criteria can be adjusted to account for any differences in pH between the water column, the surface

- sediment interface or pore water at a specific depth in the sediment.

For normal use in establishing permit limits, or assessment of waterbodies; the criteria should be applied
to the water column. If there is a special concern at a site, the option of monitoring the pH of interstitial water can be investigated, and the pH characteristic of the interstitial water can be used to assess the criteria. Care should be taken to determine the pH in the interstitial wate r at depths where mussels are likely to be found at the site.Alex Barron

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- 13 Question: 12. In general, should the criteria include a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically defensible way to account for these differences when deriving protective water quality criteria?

Review Comment: The potential differences in pH between water and sediment is not so much directly related to the criteria development, as it is an issue related to how and wh ere the criteria may be applied. The water quality criteria should provide recommendations for protective criteria based on ammonia concentrations in water and adjusted for pH and temperature. On a site

- specific basis, if in sediment there are significan t differences between pH in the interstitial pore water in sediment and the overlying surface water
, this ca n be assessed by site

- specific measurement of pH levels in the sediment (at depths where juvenile mussels are likely to be found ) and then adjustin g the criteria to those pH conditions . Another concern is whether
or not the pH at the appropriate depth is relatively constant. These issues will likely vary considerably depending on the characteristics of the sediment , and perhaps seasonally . The cri teria can be adjusted to the pH in the interstitial water to assess the potential for adverse effects at the site

.

Question: 13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?

Review Comment: Results of toxicity tests without sediment provide good information regarding the toxicity of ammonia via exposure to ammonia concentrations in water. A cceptable control mortality in a test remains a significant indicator that the test conditions and the general overall health of the tested organisms are reasonable and the increasing mortality observed in the test chamber s with higher concentrations of the tested substances is a direct result of the toxicity of the toxic substance. If the mortality in the control chambers remains low then
, this is good evidence that the tested organisms are not experiencing unacceptable stress during the test. In several water

- only 28

- day tests, 100% to 90 % survival was observed in controls. This provides evidence that w ater

- only tests with juvenile mussels can provide acceptable conditions for assessing the toxicity of a contaminant.

Water

- only exposure tests provide relatively comparable and reproducible exposure conditions that are useful for assessing toxicity sens itivity and to distinguish effects of pH temperature or hardness etc. Toxicity testing in sediment will not change what we know about the sensitivity of the species to
ammonia via a water

- only exposure as used and summarized in this criteria document. As shown in Tables 1, 2 and 4, the variability of toxicity values within a species and within genera is all within a factorAlex Barron

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of 10. Water

- only toxicity tests methods have shown good intra and inter laboratory variability for toxicity tests with ammonia, as we ll as copper and chlorine. This lends confidence in these data for use in criteria development. T oxicity data from w ater

- only exposures are most relevant to the purposes that water quality criteria are most often employed; assessments of natural waters b y comparison to the criteria and for use in developing regulatory limits for permitted discharges of the pollutant.

Concerns that tests conducted in sediment might produce different toxicity results are speculation. Some speculation is that water

- only exposure stresses the test organisms and may make the test organisms more sensitive to the toxicant. However the good survival in the control chambers indicates that the tested mussels are not overly stressed during the test. Also, test conditions are de signed to provide optimum dissolved oxygen and temperature conditions. Natural conditions often are less than optimum with lowered oxygen levels and other conditions. The sediment may be a more natural environment for the juvenile mussels, making them mo re hearty and resistant to the toxic substance, or the sediment may act to absorb the toxic substance and make it less bioavailable and less toxic. On the other hand, if the sediment may absorb the toxic substance and over time increase the concentration in the interstitial pore water in relation to the overlying water. Most of these issues are site specific in nature and beyond the scope of national recommended criteria.

Natural sediments are highly variable and even a standardized reconstituted “clea n” sediment to be used in laboratory tests may prove variable in some unknown way perhaps in response to some unrecognized factor, however this is simply speculation. Use of sediment in toxicity tests will likely introduce added variability without provid ing additional value for judging sensitivity and this will introduce added complexity to interpreting the tests’ results for criteria development.

Several studies (Whiteman et. al., 1996, Hickey and Martin, 1999) have reported that ammonia in interstitial pore water can often be higher than concentrations in the water. Overall, these concerns
about increased ammonia concentrations in interstitial pore water are not so much related to toxicity sensitivity or issues that directly influence the derivation of criteria. These concerns and observations are more related to an exposure assessment of potential risk to the sediment dwelling organisms and t he related physical factors that influence the exposure conditions.

I believe that toxicity data from water

- only tests that were conducted under acceptable conditions (with acceptable control mortality) provide the most useful data to judge the quantita tive toxic effects of
the toxic substance. These water

- only data can then be used to assess potential for toxicity in the water column. Currently, there is insufficient information available to indicate that the toxicity of ammoniaAlex Barron

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- 15 should be considered fundamentally different in interstitial pore water as compared to the toxicity
observed in water

- only tests under the same pH and temperature conditions. An ammonia toxicity test
with water

- only exposures provides reliable test results that can be applied to exposures if the juvenile mussels are exposed to concentrations of ammonia in water. Water exposure tests should be used in the derivation of water quality criteria and this information should apply equally well to water column or interstitial pore water.

If there is a special need to assess interstitial pore water at a site, the criteria can be applied to an assessment of the interstitial pore water. Samples of interstitial water can be collected and analyzed for ammonia concentrations and pH can be measured in the field. If monitoring of interstitial pore water is conducted, care should be taken concerning the depth at which the samples are taken. Juvenile mussels are likely to be within a few centimeters of the surface and juveniles and adult mussels are capable of some control over maintaining their position in the sediment and should not be assumed to be regularly found buried in deeper sediments where low dissolved oxygen, reduced food availability and other stress factors may overshadow ris k due to ammonia toxicity. Water samples for ammonia analysis and pH field measurements should be taken at the surface interface or at a depth where mussels are likely to be found.C

- 17 PEER REV IEW COMMENTS FROM



### Steven P. Canton . MS Senior Aquatic Ecologist GEI Consultants 5575 S Sycamore Street

– Suite 101 Littleton, CO 80120 303

- 794

- 5530 Email: scanton@geiconsultants.comSteven Canton

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- 19 CHARGE QUESTIONS
Acute criteria in fresh waters:

1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?
Yes, in general the toxicity tests used to derive the water column criteria , as cited in the draft update, are scientifically defensible . Plus, it appears they provide sufficient data to evaluate inter and intra

- species comparisons using the guidelines established for deriving water quality criteria.

Additiona lly, a few years ago, we conducted a review of a number of ambient water quality criteria (AWQC) as part of the Arid West Water Quality Research Project (AWWQRP 2006). One aspect of that project included a review and update to the 1999 EPA Ammonia criteri a document, similar to this draft. Checking those updates side

- by

- side, it appears there are a few more studies that could be included in this

re

- evaluation:

Augspurger, T., A.E. Keller, M.C. Black, W.C. Cope, and F.J. Dwyer. 2003. Water quality guidance for protection of freshwater mussels (Unionidae) from ammonia exposure. Environmental Toxicology and Chemistry 22:2569

- 2575.
Khatami, S. H., D. Pascoe, M. A. Learner. 1998. The acute toxicity of phenol and unionized ammonia, separately and together, to the ephemeropteran Baetis rhodani (Pictet). Environmental Pollution 99:379

- 387.
Rubin, A.J., and M.A. Elmaraghy. 1977. Studie s on the toxicity of ammonia, nitrate and their mixtures to guppy fry. Water Research 11:927

- 935.
Russo, R.C., D.J. Randall, and R.V. Thurston. 1988. Ammonia toxicity and metabolism in fishes. In R.C. Ryans, ed. Protection of River Basins, Lakes, and Estu aries . pp. 159

- 173. American Fisheries Society, Bethesda, Maryland.
Sangli, A. B. and V. V. Kanabur. 2001. Toxicity of ammonia to a freshwater fish, Gambusia affinis and its effect on oxygen consumption. Geobios 28:56

- 58.
Tomasso, J.R., C.A. Goudie, B.A. Simco, and K.B. Davis. 1980. Effects of environmental pH and calcium on ammonia toxicity in channel catfish. Transactions of the American Fisheries Society 109:229

- 234.

I should also note that in that prior review of the 1999 Ammonia document (AWWQRP 200 6), we raised concerns regarding the appropriateness of some studies/ data used in the 1999 EPA document ( see Table 1
below ). These studies may need an additional evaluation to determine if they provide toxicity data meeting EPA data quality guidelines (St ephan et al. 1985).Stev en Canton

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- 20 Table 1 Deletions of Inappropriate Data Presented in the EPA 1999 AWQC Ammonia Document (from AWWQRP 2006)

Species References Comments Acute Simocephalus vetulus Mount 1982 Insufficient data to validate results Oncorhynchus mykiss Calamari et al. 1977 Unable to validate citation or data

- other data available by same author with this species Oncorhynchus mykiss Thurston et al. 1981 Unable to validate citation or data Oncorhynchus mykiss Reinbold & Pescitelli 1982 Unable to validate citation or data Pimephales promelas Thurston et al. 1981 Unable to validate citation or data Pimephales promelas Reinbold & Pescitelli 1982 Unable to validate citation or data Gambusia affinis Wallen et al . 1957 Insufficient data to validate results Lepomis macrochirus Reinbold & Pescitelli 1982 Unable to validate citation or data Sander vitreum Reinbold & Pescitelli 1982 Unable to validate citation or data Chronic Ictalurus punctatus Colt & Tchobanoglous 1978 Insufficient data to validate results Source: ( AWWQRP 2006)

2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?
Interestingly, we made a similar recommendation for this particular situation (i.e., the concept of with and
without mussels

- based criteria for ammonia) in that prior report ( AWWQRP 2006, Chapter 4). I have attached a copy of the relevant chapter from that analysis, which may be useful, as least in refer ence to how this has been looked at by other parties.

In addition to a with and without mussel approach, the document could also include other “implementation” issues, such as evaluating additional site

- specific information needed to provide in

depth tec hnical recommendations. For example:

 Is there a definitive cause and effect relationship between ammonia sources and the diversity and abundance of mussels downstream of the source?

 How have the manageable sources (i.e., Agriculture, WWTP) of nitrogen ch anged over the last decade within the study waters in comparison to measured changes in mussel communities ?

 Is there evidence of anthropogenic nitrogen loading that is equal to or greater than the natural occurrence of decomposition due to heterotrophic bacteria?

 What is the within

- site spatial and temporal variability in NH 3 of the pore water and is this variability linked to season, storm events, temperature, or flow conditions.Steven Canton

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- 21 In addition it would be advisable to include a statement to recognize the importance of developing “site

specific standards”, since mussels now play an important role and are the most sensitive species in the
list. Site

- specific standards reflect the reality of which species are needed to be protected in a particular waterbody, particularly with mussels that may be more abundant in some parts of the country, where as in some other regions their presence can be very limited. In this case, some guidance on when and how to do mussel surveys to determine when to apply a “with mussels” or “without mussels” criterion would be a
useful addition to the criteria document.

3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe there is an alternative approach to the use of this data that would be more scientific ally sound?
Given the rationale provided
, in combination with EPA criteria development guidance, general ecotoxicological principles, and my own views on general invertebrate biology, I agree it is scientifically defensible to exclude glochidia data in am monia criteria development for the following reasons.

 First, the document notes that despite having four acute toxicity studies that consider five different mussel species ( with the glochidia stage)
, with the majority of test having a duration of 2 days, there are still several questions to be answered when testing these particular life stages :
o H ow long do they stay attached to the host?

o H ow long do they stay in conglomerates and d oes this jelly (for those species) protect them from exposure to contaminan ts?

o I f they sink to sediment how long do they stay there in the conglomerate before transforming to the juvenile mussel ?

o I agree with the authors that these valid questions and also note that the questions will probably have different answers for differ ent species of mussels, which makes the data of even more uncertain general ecological relevant and, therefore, use of glochidia data even more problematic and difficult to apply generally to this group for criteria development purposes

.  Second, concernin g the mechanisms of ammonia toxicity in this particular stage of development there is also a long list of unanswered questions: would glochidia be able to take up ammonia if available in the host?, what is the mechanism of glodichia exposure to ammonia whe n they are released in conglomerates?

- Again

– great uncertainty with unknown ecotoxicological significance.

 Third, based on the results from those four studies it seems that if the glochidia acute data were included in the calculations, they would drama tically reduce the acute criteria and probably be over conservative for other fresh water species without actually knowing if the information has any ecological relevance.

Given these uncertainties

– which are well articulated in the draft document, th ere appear to be t wo scientifically defensible approaches : 1) Do not use the glochidia acute toxicity data 2) Do a final acute
criteria calculation “ with and without glochidia ” . The first approach would be the easiest to avoid
confusion and possible errors in development of safe levels for ammonia. The second approach wouldStev en Canton

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give flexibility

– although it would be necessary to include a strong caveat to a “with glochidia” value, given the questions noted in the criteria document and my thoughts above.

4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scientifically defensible principle for structuring the population exposure duration problem a nd designing further research to quantify such duration?
The proposed approach will give researchers and scientific community time to come up with some answers and provide relevant information concerning the appropriate time of duration for glochidia

expo sure testing

– if such testing is determined to be necessary for this short

- lived, parasitic life stage. However, if this approach is proposed, it will also create a limbo period, in which this issue will have to
be addressed while decisions are made for application of a criteria that some may fight as “non

protective” since the criteria document has “acknowledged” that a sensitive life stage was not considered!

I’ll be the first to admit i t is a difficult to task to structure a criterion that includes po pulation exposure approach to criteria development when dealing with a wide variety of fresh water mussel species that have different life spans ( few months to several years ) , not to mention a glochidia life

- stage that can be released individually or in co nglomerates, and that can get attached to fish, rocks, sediment or plants.

Such variability could mean that for the particular issue of including glochidia data or not , we perhaps
should be t reating these mussels individually, by genus or species, rather than thinking of them simply as the “mussel” group. Regardless, i t will take a considerable amount of time and effort to develop some way to make “uniform” relevant exposure toxicity testing time for the glochidia life stage that will be relevant for all if not the majority of fresh water mussels.

Hyalella azteca position statement and proposed rationale (see Appendix B):

5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?
I support the position statement based on the workgroup’s review of a num ber of toxicity tests on Hyalella, referenced in the rationale. I can’t help but wonder if this recommendation holds for Hyalella test data for other EPA criteria documents, as well

– for example, Hyalella is the most sensitive species in
EPA’s chronic ca dmium database (EPA 2001). Is this position statement generic to all EPA criteria databases, or simply this ammonia update?

I should note that our prior review of the study also recommended removal of Hyalella from the ammonia database, primarily becau se of the poor control performance (AWWQRP, 2006), which it now appearsSteven Canton

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- 23 may be due to the ionic balance issue noted by the position statement. So, I support non

- use of Hyalella toxicity data for this ammonia update.

Most of the recommendations provided h ave scientific basis. It appears that ongoing studies will provide quality data that will widen the knowledge for water quality requirements for H. azteca husbandry. However, the statement concerning the complications due to possible H. azteca genetic or t axonomic diversity is not relevant because the way this information is used in criteria development, which only considers appropriate data at genus level (i.e., GMAVs) not new species or subspecies. In addition, the issue of a wide range of surface waters inhabited by H. azteca could be easily addressed setting a site

specific standard, which will consider only the species present at a particular site. Thus, inclusion of this information may add confusion rather than help explain a decision on non

- use for criteria development.

Chronic Freshwater Criterion:

6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such use? Are you aware of other relevant data that were not used?
While , the toxicity tests used to derive the criteria are generally scientifically defensible (see discussion below) ; I am uncomfortable with the calculation because there is insufficient data to evaluate intra

- species comparisons using the guidelines establish ed for deriving water quality criteria. More specifically, the
2009 data set still does not fully meet the EPA guidelines for developing water quality criteria. A salmonid representative has been added to the data set (which was missing in the 1999 docum ent) , but a representative from the Class Insecta is still missing. It appears that the option for using acute

- to

- chronic ratios for determining chronic criteria would be more relevant than simply using a “hypothetical GMAV
for insects”.

One of the studi es noted in Question 1 may provide additional relevant chronic data.
It is also important to point out that there are only two studies (Anderson et al. 1978, Sparks and Sandusky 1981) available for Musculium genus. Interestingly, those two studies provide substantially different estimates of what level of ammonia is protective to fingernail clams

– creating uncertainty in the true chronic value. A closer analysis of these two papers reveals that while the la ter Sparks and Sandusky study was designed to confirm Anderson et al’s findings, the researchers faced some serious methodology deficiencies, as the authors recognize: “Although it would have been desirable to perfect the culture methods first and then emp loy them in the toxicity tests, methods were developed as testing proceed because of limited time” (Sparks and Sandusky 1981). As part of their study design, Sparks and Sandusky used clinoptilolite to remove ammonia, but note this compound could also have remove potassium and chlorinated hydrocarbons

– which raises the question that toxic effects observed in MusculiumStev en Canton

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transversum in this particular study may be also attributed to other confounding factors in the test water and not exclusively to ammonia exp osure. In addition, authors conclude that also biological processes (i.e., naturally occurring bacteria that converts ammonia into a relatively non

- toxic nitrate) may be involved in ammonia removal rather than physico

- chemical processes. Thus, it would be advisable to re

analyze the relevance of these studies for this update. As it stands, I cannot fully support calculating a GMAV for this fingernail clam using these two studies.

Another issue that is relevant to mention is the continued inclusion of a te mperature relationship for chronic ammonia criteria originally used by EPA (1999), which was derived from a single study. It appears the EPA used the Arthur et al. (1987) study, which evaluated acute ammonia toxicity to 14 species (9 invertebrate and 5 fis h species), to incorporate temperature dependence into the chronic equations. There are three problems with incorporating temperature in the chronic relationship, but not acute. First, there is lack of chronic ammonia toxicity studies for fish or invertebr ate species to demonstrate the relationship between temperature and chronic ammonia toxicity. Second, the rationale for deriving an invertebrate chronic temperature slope from acute data (i.e., fish and invertebrates) is unclear. Finally, the assumption th at chronically exposed invertebrates will have similar temperature dependence compared to acutely exposed individuals is based on the Thurston et al. (1984) study and those authors
indicated that a 96

- h period is insufficient to determine an acute toxic co ncentration of ammonia for insects. Consequently, it appears there are no appropriate data to support incorporation of a temperature component to the chronic ammonia standards, just as EPA concluded with the acute equations. The position statement notes th at the temperature

- dependent toxicity model now only applies to invertebrates.

I have to admit I was hoping the actual equations, inflection points, and pH and temperature relationships
would be thoroughly re

- evaluated as part of this update , given the a bundance of new data included not in
the 1999 document . I would strongly recommend re

- evaluation of the chronic temperature component of the equations rather than just accept the 1999 update evaluation. Although, I have to admit, the draft document doesn’t actually provide any equations to test

– just recalculations of acute and chronic endpoints. This is another disappointment.

7. Is the freshwater chronic criterion scientifically defensible with mus sels present and mussels absent?
The freshwater chronic criterion based on a mussels present/absent approach maybe scientifically
defensible

– but will potentially result in very different approaches, even in regions of the United States
where mussels a re known to be absent or rare. In fact, the primary question will become “what level of data is required” to determine whether the habitat is or is not suitable to support mussels and/or whetherSteven Canton

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- 25 mussels have ever occurred within the site historically. Gu idance on such data needs questions would be
useful to include in the criteria document to support a with/without mussel criterion.

Additionally, even when the “mussel present” option is applied, a whole other series of issues arise,
because there is of

ten a poor correlation between the water column NH 3 concentration and the pore water
NH 3 concentration where most juvenile mussels reside. Site

- specific sediment conditions can greatly influence the availability of NH 3 , and in the case of the upper Missis sippi River, as much as 6

- 30 times greater than the surface waters (Bartsch et al. 2003).

In such cases, criteria developed without considering the complete affects of sediment on the
accumulation of NH 3 , or other confounding affects on mussel communiti es (i.e., the fact that mussel beds
can self

- produce nutrients
, such as ammonia) (Dankers & Koelemaij 1989), may greatly underestimate, or even overestimate, a criterion that is protective of mussels. In addition, this also brings up the question of
wheth er the use of sediment is appropriate in chronic toxicity tests designed to be protective of aquatic life in the water column, and what endpoint should be evaluated using such methodologies.

Addressing the “Use of 28

- day Juvenile Test Data” (see Appendi x C):

8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defensible as "chronic" test data for criteria development?
Water quality criteria for the protection of aquatic life are derived using toxicity endpoints that relate to population level impacts. In general, these endpoints relate to survival, growth and/or reproduction. I would agree that the 28

- day test with juvenile mussels, while similar i n duration to a standard chronic test for many other groups of organisms, is not technically an early

- life stage test according to the 1985 Guidelines for Aquatic Life Criteria, and as such, should perhaps not be included in the chronic database.

If this is carried forward, I would support the approach of the 28

- day exposure tests being considered as
“other data’

– if the test are acceptable in all other respects. Reasons for not including in chronic criteria calculations could follow and build on the arg uments presented in the position paper:

 First, it is not representative. If the duration of the life stage is 2

- 6 years for juvenile life stage of fresh mussels, 10% of this amount of time would be 73

- 219 days. Thus, a longer exposure (i.e., 90

- day) wo uld be more representative for a “chronic” study for juvenile fresh water mussels than 28

- day tests.

 Second, extending the exposure time would allow the tested animals to adapt better to laboratory conditions, thus reducing the stress and decrease the ris k of having test condition

- biased results.

 Third, although it will be more challenging to keep all the water quality parameters under control in a longer exposure it could also provide other avenues for future research on juvenile musselsStev en Canton

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(i.e., if captiv ity conditions and pollutant exposure would enhance growth over longer periods of time, if they may need additional food source, among others)
Once possible conflicting issue is that other species used in EPA criteria development have “allowable”
chronic test conditions that also represent a small portion of their total life span

– e.g., 60

- day post hatch ELS for salmonids, which can live for years. The difference, conceptually, is that the 28

- day test for mussels does not include the earliest life stage prior to juvenile testing commences

– i.e., it does not start with egg, then hatch, then juvenile exposure more typical of an ELS for fish. If we can conceptually
overcome this “weakness” for a 28

- day juvenile mussel test, then I suppose growth

- effects da ta in a 28

- day juvenile mussel test could be at least evaluated in the context of other reported data

– and if comparable to effects data from longer

- duration testing, then perhaps it could be used in some form for
criteria calculations.

9. Should toxicit

y studies on the growth rates of mussel shells during 28

- day tests be considered quantitatively when developing water quality criteria?

As described in the previous question, the studies on growth rates of mussels shells during 28

- day tests should only be used as information to better evaluate appropriate longer

- term chronic studies (i.e., 90

- day studies). The results from longer chronic tests could certainly be used quantitatively for the developing the water quality criteria. Unless 28

- day test data prov ide similar endpoints to long

- term testing, it would be necessary to keep the results separate and potentially have two conflicting sets of data (i.e., 28

- day and
90

- day data)

– which will only make the calculations of the new criteria more complicated, an d can lead to confusion when trying to implement. And I have to say that ammonia criteria development is confusing enough as is.

10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?
My understanding of data included as “other data” in the criteria do cument is that these data are provided just as a reference to make the interested parties know that these data exist. They can certainly be presented that way and not considered in the calculations of the chronic criteria for the reasons outlined in the p revious two questions.Steven Canton

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- 27  Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?
As noted in the position paper, it is not possible with the available data to conclude that impaired growth
after 28

- day exposure will necessarily lead to continued reductions in growth over time. The position paper rightly points out the possibility of organisms to “recover” from potentially short term stress and show no further decline. In fact, these organisms may exhibit normal growth during a more relevant long

term test. However, given the lack of informa tion available to compare short

- term and long

- term toxicity studies in fresh water mussels, it would be advisable to note, as appropriate, that any of the three outcomes: continued decline, stabilize, or recovery is possible. In addition, the propensity fo r any of these three outcomes will most likely depend upon the particular mussel species and the stage of development used in the toxicity tests.

11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Juvenile and adult mussels, as sediment

- dwelling organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have sugg estions on how states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?
Given that juvenile mussels of most species reside completely in the sediment, filter pore water, and feed
on the sed iment, I would suggest one approach might be determining a mean burial depth for the juveniles of the selected mussel species within the 2009 data set. In laboratory tests, juvenile Villosa iris are reported to bury <1cm, yet were not exposed to the overl ying water (Yeager et al. 1994). Perhaps it would be useful to use this burial depth and evaluate the average pH conditions within a burial zone, per se. In addition, it is likely any measures of pH in the mussel micro

- environment (i.e., taking measures of water quality over a mussel bed) may also show elevated ammonia levels simply from the metabolic activity of those organisms, themselves. I can’t help but wonder how to determine attainment of “safe”
ammonia levels in future field monitoring , given the potential for organism

- generated ammonia values.

12. In general, should the criteria include a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically defensible way to account for these diffe rences when deriving protective water quality criteria?
Because adult mussels siphon directly from the water column, there is no need for a pH differential calculation. However, to be protective of the juvenile life stage which typically filters pore wat er, there may be a need for differential pH measurements. B ut how does one account for all of the biotic and abiotic factors that affect pH in the sediment? Because site

- specific sediment conditions greatly influenceStev en Canton

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the pH levels, adjusting the pH should only be an option for site

- specific calculations (or considered as “other data”) rather than within the national water quality criteria.

13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?

If the test is designed to address water column concerns, then the exposure test using juvenile mussels should be conducted without sediment in the test chamber. The use of sediment in water quality toxicity tests introduces too many confounding factors in t ests designed to evaluate the needs to be protective of aquatic life in the water column. For many species of mussels, the juveniles primarily feed on particles in the sediment and filter the pore water, rather than directly siphon from the overlying wate r column. Depending upon the composition of the sediment (i.e., organic matter content) the sediment layer can have profound influences on the pH and ammonia concentration within the pore water of the sediment. In addition, the metabolic byproducts of ju venile organisms that live in sediments also influence the level of ammonia observed within pore waters of sediment. For these reasons alone it is difficult to separate the effects solely due to conditions within the water column versus the effects of the sediment layer.

Furthermore, in static renewal tests that incorporate the use of sediment, there is the potential for decreased ammonia concentrations, as well as pH, in the pore water of sediment when compared to the water column conditions (Allan and M aguire 1992); whereas in natural conditions this relationship is often reversed, such that pH levels and ammonia concentrations are greater in the sediments than the overlying water column. The sediment based toxicity studies of Whiteman et al. (1996) tr ied to address these gradient issues between the sediment and water column, by introducing nitrogen via sediment and maintaining overlying water concentrations in a non

- toxic range. However, the mobility of the test organisms allowed them to seek out the zones of lower ammonia concentration at the interface rather than within the sediment, which also confounded the test results.

This concludes my responses to the Charge Questions. Given the time constraints, I feel somewhat limited in what I could provid e. I hope this review was helpful

– and thank you for the opportunity.

References:
Allan, G. L. and G. B. Maguire. 1992. Effect of pH and salinity on survival, growth and osmoregulation in Penaeus monodon Fabricius. Aquaculture 107:33

- 47.
Anderson, K.B., R.E. Sparks, and A.A. Paparo. 1978. Rapid Assessment of Water Quality, Using the Fingernail Clam, Musculium transversum. WRC Research Report No. 133. University of Illinois, Water Resources Center, Urbana, IL.Steven Canton

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- Dependent Waters:Final Report. Prepared for the Arid West Water Quality Research Project by URS Corporation, Chadwick Ecological Consultants, Inc., and Parametrix, Inc., Pim a County Wastewater Management Department, Tucson, AZ.
Arthur, J.W., C.W. West, K.N. Allen, and S.F. Hedtke. 1987. Seasonal toxicity of ammonia to five fish and nine invertebrate species. Bull. Environ. Contam. Toxicol. 38:324

- 331.
Augspurger, T., A.E. Kel ler, M.C. Black, W.C. Cope, and F.J. Dwyer. 2003. Water quality guidance for protection of freshwater mussels (Unionidae) from ammonia exposure. Environmental Toxicology and Chemistry 22:2569

- 2575.
Bartsch, M.R. et al. 2003. Effects of pore water ammonia o n in situ survival and growth of juvenile mussels (Lampsilis cardium) in the St. CroixRiverway. Environ Toxicol Chem 22:2561

- 2568
Calamari, D., R. Marchetti, and G. Vailati. 1977. Effects of prolonged treatments with ammonia on stages of development of Sal mo gairdneri. (English Translation) Nuovi Annali d’Igiene e Microbiologia. 28:333

- 345.
Colt, J.E., and G. Tchobanoglous. 1978. Chronic exposure of channel catfish, Ictalurus punctatus, to ammonia: effects on growth and survival. Aquaculture 15:353

- 372.

Dan kers N, K. Koelemaij. 1989. Variations in the mussel population of the Dutch Wadden Sea in relation to monitoring of other ecological parameters. Helgoland Marine Research 43: 529

- 535
Khatami, S. H., D. Pascoe, M. A. Learner. 1998. The acute toxicity of phenol and unionized ammonia, separately and toget her, to the ephemeropteran Baetis rhodani (Pictet). Environmental Pollution 99:379

- 387.
Mount, D.I. 1982. Ammonia toxicity tests with Ceriodaphnia acanthine and Simocephalus vetulus. Memorandum to Dr. R.C. Russo, ESEPA, August 6, 1982.
Reinbold, K.A., and S.M. Pescitelli. 1982. Acute Toxicity of Ammonia to the White Sucker. Final Report to EPA. Center for Aquatic Ecology, Illinois Natural History Survey, Champaign, Illinois.
Rubin, A.J., and M.A. Elmaraghy. 1977. Studies on the toxicity of ammonia, nitrate and their mixtures to guppy fry. Water Research 11:927

- 935.
Russo, R.C., D.J. Randall, and R.V. Thurston. 1988. Ammonia toxicity and metabolism in fishes. In R.C. Ryans, ed. Protection of River Basins, Lakes, and Estuaries . pp. 159

- 173. American Fisheries Society, Bethesda, Maryland.
Sangli, A. B. and V. V. Kanabur. 2001. Toxicity of ammonia to a freshwater fish, Gambusia affinis and its effect on oxygen consumption. Geobios 28:56

- 58.
Sparks, R.E. and M.J. Sandusky. 1981. Identification of Factors Responsi ble for Decreased Production of Fish Food Organisms in the Illinois and Mississippi Rivers. Final Report for Project No. 3

- 291

- R, Illinois Natural History Survey, River Research Laboratory, Havana, Illinois.
Stephan, C.E., D.I. Mount, D.J. Hansen, J.H. Ge ntile, G.A. Chapman, and W.A. Brungs. 1985. Guidelines for Deriving Numerical National Water Quality Criteria for the Protection ofAquaticStev en Canton

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Organisms and Their Uses . PB

- 85

- 227049. U.S. Environmental Protection Agency, Office of Research and Development, Duluth, Minnesota.
Thurston, R.V., R.J. Luedtke, and R.C. Russo. 1984. Toxicity of Ammonia to Freshwater Insects of Three Families. Technical Report No. 84

- 2, Fisheries Bioassay Laboratory, Montana State University, Bozeman, MT. 26 pp.
Thurston, R.V., R.C . Russo, and G.A. Vinogradov. 1981. Ammonia Toxicity to Fishes. Effect of pH on the Toxicity of the Un

- ionized Ammonia Species. Environ. Sci. Technol. 15:837

- 840.
Tomasso, J.R., C.A. Goudie, B.A. Simco, and K.B. Davis. 1980. Effects of environmental pH and calcium on ammonia toxicity in channel catfish. Transactions of the American Fisheries Society 109:229

- 234.
U.S. Environmental Protection Agency (USEPA). 1999 Update of Ambient Water Quality Criterion for Ammonia. Office of Water, Washington, DC. EPA

- 822

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- 99

- 014.
U.S. Environmental Protection Agency (USEPA). 2001. 2001 Update of Ambient Water Quality Criteria for Cadmium . EPA

- 822

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- 01

- 001. Office of Water, Washington, DC.
Yeager, M.M., D.S. Cherry and R.J. Neves. 1994. Feeding and burrowing behavior o f juvenile rainbow mussels, Villosa iris (Bivalvia: Unionidae). J.N. Am. Benthol. Soc. 13(2):217

- 222.
Wallen, I.E., W.C. Greer and R. Lasater. 1957. Toxicity to Gambusia affinis of certain pure chemicals in turbid waters. Sewage Ind. Wastes. 29: 695

- 711.

W hiteman, F.W., G.T. Ankely, M.D. Kahl, D.M. Rau, and M.D. Balcer. 1996. Evaluation of interstitial porewater as a route of exposure for ammonia in sediment tests with benthic macroinvertebrates. Environmental Toxicology and Chemistry. 15(5):794

- 801.C

- 31 PEER REVIEW COMMENTS FROM

Jerome M. Diamond , Ph.D. Principle Aquatic Ecologist and Director of Ecotoxicology

Tetra Tech 400 Red Brook Boulevard

- Suite 200 Owning Mills, MD 21117 410

- 356

- 8993 Email: jerry.diamond@tetratech.comJerome Diamond

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- 33 Peer Review Ammonia
Acute criteria in fresh waters:

1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?
Response: In general, the toxicity tests used to derive ammonia criteria in the draft Reassessment Document (RD) are scientifically defensible given EPA’s criteria development guidelines (Stephan et al. 1985). How ever, I have reservations regarding the use of snail data derived from field

- collected organisms as explained below under Chronic Freshwater Criterion . I am not aware of other relevant freshwater toxicity test data that were not either used or considered in the RD.

2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?
Response: Attempting to differentiate freshwater ammonia criteria on the basis of mussels being present or absent is in my opinion a very tenuous and probably unsupportable idea scientifically. First, the acute
data indicate that Corbicula (Asiatic Clam) is nearly as acutely sensitive as the most sensitive unionids.
Corbicula is not a unionid and has a totally different life cycle than unionids. Furthermore, they are now widespread in the U.S. so it may prove fairly inefficient to separate criteria on the basis of presence of bivalves in general.

As an implementation issue, disting uishing between surface waters with mussels and those without is not nearly as straightforward as it is for salmonids or “cold water” species presence/absence because the latter are dependent on a fairly known, measurable water characteristic (water temper ature regime). Also, while most states have cold water as well as warm water aquatic life uses in their water quality standards, I’m not aware of many (perhaps any) that have specifically a mussels use. The only example of which I’m aware is Virginia DEQ , which distinguishes different application of the chlorine standard based on whether federally listed mussels are known to be present in a given stream segment. They apply a “halogen ban” (no chlorine or other halogenated disinfection chemicals are allowe d to be used for wastewater treatment) in those segments for those permittees that do not have intermittent dischargers and have discharges < 20,000 gallons per day. However, in this case the water quality standard was not changed but rather a risk mana gement approach was taken to reduce the major point sources of chlorine or other halogenated disinfection chemicals to the stream.

Mussels can and do occur (or have occurred) in a wide variety of habitat types and water quality regimes and so their presenc e is not often known without specifically sampling for them. Such sampling may need to be fairly intensive to “prove” the absence of mussels. In addition, mussels have been extirpatedJerome Diamond

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from a number of streams in the U.S. due to historic land use and habi tat changes, toxic spills, and poorly treated wastewater and nonpoint runoff (e.g., see EPA 2002). If today, a state finds that mussels are not present but historically they were, what then should be the ammonia criteria? I believe such
differentiations in criteria suggested in the RD be relegated to EPA’s recalculation of site

- specific criteria (USEPA 1994). That procedure was designed to identify whether certain types of species are present or could occur in a given site or waterbody. This would be a more efficient and scientifically defensible way to handle the apparent sensitivity of freshwater mussels to ammonia.

3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe there is an alternative approach to the use of this data that would be more scientifically sound?
Response: I agree with the RD that glochidia data should not be included in criteria derivation at this
time but not en tirely for the reasons given by EPA. First, a female mussel often produces hundreds or even thousands of glochidia in a single season. This helps counteract the fact that a high percentage of the glochidia will not survive because they have not encysted on a host fish within a reasonable amount of time. As many researchers have documented, glochidia are incapable of surviving and maturing on their

own

— they require nourishment from a host organism typically within days of being produced. It could be argu es that basing criteria on a 50% reduction in glochidia survival would be similar to basing criteria on a 50% reduction in egg survival of other species that broadcast an abundance of eggs (e.g., marine barnacles) in which only a small fraction of the larv ae are likely to survive and become established under natural, unstressed conditions. I am not aware that EPA has ever based acute criteria on egg survival (even fertilized egg survival) and I believe this might contradict EPA’s criteria development guide lines for establishing acute criteria.

A second reason I don’t think glochidia should be included at this time is that the majority of mussel species at risk today (i.e., threatened, endangered, or species of concern) appear to have relatively narrow speci ficity in terms of a vertebrate host and have evolved structures (e.g., lures) or behaviors that enable very rapid encysting in the host (e.g., see Rogers

- Lowery and Dimock 2006). Of the mussel species actually used in glochidia testing that are discussed in the RD, most of them have evolved adaptations to enable rapid infestation of a host (often < 6 hours), resulting in a very short free

- living glochidia stage. Thus, for the majority of mussel species at risk, exposure periods > 24h are probably inappro priate, regardless of whether control survival is > 90%.

A third reason is that while the ASTM glochidia test procedure appears to be reasonably robust in terms
of intra and inter

- laboratory testing, the procedure is still a “Guide”, not a “Method”, which indicates thatJerome Diamond

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- 35 there are still many aspects requiring expert judgment, trial and error, and research, especially in terms of culturing and maintaining glochidia under laboratory conditions. Also the inter

- lab study reported by Wang et al. (2007) used the same water source and glochidia stocks for all labs. This would tend to underestimate the true interlab variability, where each lab uses their own source of glochidia (and techniques for obtaining glochidia) as well as water source. In addition, at the present time there are still relatively few laboratories that have performed this test (e.g., compare with daphnia or amphipod tests). Much of the glochidia data under consideration were produced by a few laboratories. This may be acceptable for tests th at use species closely resembling those for which a standard EPA or ASTM method has been developed and used extensively; e.g., an acute test with a different species of minnow or Daphnia but the identical test design and organism life stage as used for the standard fathead minnow or Daphnia acute test. However, such is not the case with the ASTM glochidia test, which uses a fairly different test design and the endpoint is dependent on sensitivity to a sudden stress (i.e., rapid valve closure to NaCl exposu re). While the ASTM procedure appears to address these concerns for the most part, there is still some uncertainty in my mind regarding defensibility of the test endpoint. As noted in the ASTM method, exposures beyond 24h may not achieve 90% control surv ival, indicating the precarious nature of keeping this lifestage alive without a host. Given this, is the apparent increase in ammonia sensitivity between 6 and 48h a true difference in sensitivity or is it an artifact of the method (i.e., glochidia are u nder increased stress). I also question the ecological importance of having valve closure within one minute or less to the introduction of a NaCl solution. I’m not altogether sure this endpoint is similar to other acute endpoints relied upon by EPA in th eir water quality criteria. In addition, the ASTM Guide points out that live or dead mussels could be open or closed at a given time and some may respond more slowly to the NaCl shock than others (Kernaghan et al. 2005). If there was a more direct way to determine mortality of glochidia to ammonia exposure, for example, that would be preferable in terms of using such data for criteria development.

I wonder if better use of glochidia survival data couldn’t be made by considering the level of juvenile

recru itment necessary to maintain a viable mussel population, similar to the EPA fish larval recruitment model for dissolved oxygen in coastal waters in the Virginian Province (USEPA 2000). Perhaps a similar type of framework could be used based on the mussel species (or, if feasible and defensible, genus or even family), when glochidia are typically produced in a given region, and a function specifying ammonia toxicity to glochidia given ambient temperatures and pH at that time. The modeling framework could b e made general so that a user could input necessary temperature and pH data, recruitment timing (e.g., season, month) and certain assumptions regarding host abundance. This would not be unlike the larval recruitment model EPA has developed for dissolved o xygen with the exception that the abundance ofJerome Diamond

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another species (i.e., a host) is not needed or relevant for the dissolved oxygen model. However, if such a model was considered (albeit simplistic but using conservative assumptions based on expert malacolog ist judgment), it could put glochidia toxicity testing results into an ecological context in a more useful way.

4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scientifically defensible principle for structuring the population exposure duration problem and designing further research to quantify such a duration?
Response: I am not sure I concur entirely with the proposed approach for further research discussed in Appendix A of the RD. I do agree that it would be useful to have better information regarding the natural life expectancy of free

- living glochidia in nature for several representative species covering the range of different reproduc tion strategies observed. Such information would help indicate whether a 6, 24, or 48h exposure is warranted. I do not, however, understand EPA’s proposal specifying the free

- living duration based on 95% of the glochidia that attach to a host. This appe ars to be a fairly restrictive proposition and it is not clear to me how this would be determined. As Rogers

- Lowery and Dimock (2006) observed, the encapsulation process in fish varies with the species and organism history of exposure to mussel
infestatio ns. Many researchers have demonstrated poor encapsulation rates of glochidia of certain mussel species (including several listed as threatened or endangered) with several common fish species (e.g., Dodd et al. 2006). In other words, the number of glochid ia that encyst on a host has as much to do with the host as it does glochidia viability in and of itself. Somehow, there would need to be the presence of a known host with the glochidia to determine an answer to EPA’s proposal. However, viable host speci es are not known for many mussel species. Therefore, this evaluation would require testing with a few mussel species for which host species are known with certainty. Other comments regarding Appendix A Under “USEPA’s Aquatic Life Criteria Coordinating Committee” comments, point #2 makes it appear that EPA is willing to alter a national criterion due to a commercially valuable species (e.g., trout) but not

an ecologically critical species? Point #3: I a gree there is no evidence that glochidia in conglutinates are exposed to water

- borne pollutants but there’s no evidence that they are not exposed either. Also, uptake of pollutants by planktonic invertebrates lacking gills (e.g., Daphnia ) occurs via absor ption through the carapace, not just through food. Point #8: “For glochidia that attach to hosts,”

– all glochidia are meant to attach to hosts. What I think you mean here is “In terms of the duration of free

- living glochidia, there is a major differenc e....”Jerome Diamond

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- 37 Other comments regarding acute data in the Reassessment Document Daphnia GMAV is Incorrect I believe the document reports an incorrect GMAV for Daphnia (p. 9) as well as test data in Table 1 (p.
49

- 50). It appears that the unionized ammonia values reported by Russo et al. (1985) and reported as such in EPA’s 1999 document, were incorrectly transcribed as total ammonia values (see p. 109, Appendix 4 of 1999 document as well as p. 38, Table 4 of the 1999 document). Even with the tempe rature correction for invertebrates in the Reassessment Document, Daphnia couldn’t possibly go from Rank 19 in acute sensitivity to 8 th in the draft document. This error has other ramifications as well: assuming the Daphnia value is incorrect in the draf t, when mussels were absent, the four most sensitive

genera are Corbicula
, Potamopyrous , Pleurocera , and Prosopium (mountain whitefish). If Corbicula is removed along with the unionids as EPA proposed (and which I disagree with), then Deltistes (Lost Rive r sucker) becomes the 4 th most sensitive genera. Either way, invertebrates no long occupy the four most sensitive genera. Although I did not do a comprehensive check, it appears that the other acute values in
the draft document are consistent with the 19 99 document.

Temperature Equation for Invertebrate and Acute Data should be re

- examined The temperature

- ammonia sensitivity relationship applied by EPA to invertebrate acute data in the draft document is based on the slope provided in the 1999 document, ba sed on Arthur et al. (1987), but not used for the acute criterion in that document. As the 1999 document makes clear, the temperature relationships from Arthur et al. (1987) were based on seasonal tests of experimental stream water, including condition, l ife stage, etc. of test organisms collected during each season. As Arthur et al. (1987) notes, several water quality characteristics other than temperature varied with season and, even more importantly perhaps, organism condition was unlikely to be simila r across seasons and at the least was unknown. EPA provided what I believe was a poor rationale for using Arthur’s et al. data for chronic
invertebrate data. The fact that Arthur did not observe a clear temperature relationship with fish (which
are also cold blooded) would weaken the case that temperature was what was driving the apparent ammonia sensitivity in invertebrates in his study (and which Arthur himself acknowledges in his paper).

The temperature

- toxicity relationship derived by EPA for ammonia in the 1999 document had a fairly steep slope; e.g., a difference between 20 and 25  C resulted in 2

- 3 fold decrease in the LC50. There are a few invertebrate species in the draft document for which it appears possible to reanalyze a temperature toxicity relationship, and to determine if one actually exists.

I have not had time to do a formal analysis of the invertebrate acute data, however, my initial reaction is that temperature may be somewhat weakly related to acute ammonia toxicity in invertebrates. ForJerome Diamond

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example, for the snail species Potamopyrgus antipodarum , Hickey and Vickers (1994) reported an LC50 of 8.71 mg N/L at pH = 8.2 and 15  C and an LC50 of 4.08 mg N/L at pH = 8.2 and 25  C (a factor of 2
over a 10  C difference). The LC50 they reported a t the same pH and 20  C was nearly identical to the
LC50 at 25  C (4.73 mg N/L). LC50’s for the fatmucket mussel at pH = 8.1 and 20  C ranged from 5.2 mg N/L to 11 mg N/L (Wang et al. 2008) a factor of about 2, which could be within the range specified by a 5  C difference in EPA’s temperature equation. Even larger differences in LC50’s are commonly reported for other invertebrates based on tests at the same pH and temperature (e.g., Villosa iris LC50 =
3.0

– 8.9 mg N/L at pH = 8.3 and 20  C). These data suggest that a temperature adjustment factor may be premature for acute invertebrate data. Acute data for Ceriodaphnia dubia may perhaps provide the best information for invertebrates thus far. At pH = 8.2 and temperature of 7  C, the LC50 = 16.65 mg N/L (Nimmo et al. 1989). At pH = 8.16 and 22  C, the LC50 = 30.08 mg N/L (Black 2001), however, at pH =
8.0 and 25  C, the LC50 = 14.52 mg N/L (Scheller 1997), similar to the LC50 reported at 7  C. EPA should reanalyze current acu te data, similar to the way they did for the 1999 document, to determine the relationship between temperature and acute ammonia toxicity to invertebrates.

Hyalella azteca position statement and proposed rationale (see Appendix B):
The EPA workgroup develo ped a position statement and proposed supporting rationale describing the concerns over using Hyalella azteca toxicity test data in criteria development due to the uncertain health of the test organisms in different test water composition. The rationale d efines the specific concerns and uncertainties supporting the recommended exclusion of the Hyalella data from use in criteria derivation, at this time; the position statement is based on the workgroup’s review of a number of toxicity tests on Hyalella, ref

erenced in the rationale.

5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?
Response: I have reviewed the Hyalella data in a previous outside peer review for EPA and agreed with EPA’s concerns regarding Hyalella test data for ammonia. I agree with the position statement in Appendix B that Hyalella toxicity data should not be used in water quality criteria development . There is a clear need to resolve water quality requirements and other methodological issues for this species in

water

- only tests.Jerome Diamond

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- 39 Chronic Freshwater Criterion:

6. Are the toxicity tests and other studies used to derive the crite rion scientifically defensible for such use? Are you aware of other relevant data that were not used? Response: Most of the chronic toxicity tests relied on by EPA for chronic freshwater criterion development are consistent with EPA’s Guidelines and other water quality criteria developed. I am not convinced that the 28 day juvenile test data for the snails included in Table C should be used. Both of
these tests used mixed

- aged organisms, and in the case of the O zark spring snail, organisms were also
field

- collected adding uncertainty in terms of their condition and acclimation to laboratory test conditions. In general, EPA has not relied on such tests for chronic criteria development, and I believe for good

reas on. I am not aware of any other relevant data that EPA did not already consider in this document.

7. Is the freshwater chronic criterion scientifically defensible with mussels present and mussels absent?
Response: I don’t think it is. See answer to question 2. The chronic value used for L. fasciola in Table 4 (0.39 mg N/L), and which must have been used in the GMCV calculation in Table C, doesn’t appear to make sense given an IC25 at the same pH and temperature reported of 0.38 mg N/L on p. 20 and t he IC25 based on the actual data (pH = 8.2 and 20  C) of 0.39 mg N/L. The IC or EC20 should be lower than the IC or EC25. The IC pin value of 0.23 mg N/L appears more in line with the data for this species. EPA should at least defend why 0.39 mg N/L is a more appropriate value than 0.23 mg N/L for this species. In addition, wouldn’t it be more consistent to use the ICp value for both Lampsilis species because the L. siliquoidea chronic value could only be calculated using the ICpin method?

Use of 28

- day Juvenile Test Data (see Appendix C):
Water quali ty criteria for the protection of aquatic life are derived using toxicity endpoints that relate to population level impacts. In general, these endpoints relate to survival, growth and/or reproduction. The 28

- day test with juvenile mussels, while similar in duration to a standard chronic test, is not technically an early

- life stage test according to the 1985 Guidelines for Aquatic Life Criteria as much of the early development will have already occurred.

8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defensible as "chronic" test data for criteria development?
Response: The question posed here by EPA implies that a 28

- d test may not be sufficiently long for
freshwater mussels that live 2

- 6 years (actually, many unionid species live far longer than that

— 20 years is not uncommon). If this is the case, I fail to see how EPA justifies use of 28

- d tests f or fish such as
salmonids that live 3

- 5 years or sturgeon that live > 30 years. I realize there’s McKim’s 1977 paper butJerome Diamond

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that was based on outdated test methods and has questionable relevance to data collected in more recent

years.

The discussion concerni ng the usability of 28

- d mollusk survival and growth tests in chronic criteria development (pages 15

- 16 of the draft document) appears to have contradicting statements and questionable rationale in my view. The discussion begins with the statement that 28

- d tests do not qualify
as life

- cycle, partial life cycle, or early life stage tests as defined in EPA’s Guidelines. According to those guidelines, 28

- d tests can only be used for fish because it has not been demonstrated that a 28

- d exposure
is a reasona ble predictor of invertebrate sublethal chronic effects (defined as at least 90d for vertebrates and presumably for invertebrates such as mussels which live for several years). However, the draft document goes on to say that 28

- d survival information coul d be used to inform the chronic criterion because obviously, lethality is not reversible. Yet in footnote #2 on page 15 of the document, as well as in Appendix C, uncertainties regarding the 28

- d mussel test method are discussed which include optimal qual ity and quantity of food, exposure apparatus, etc. needed to sustain juvenile mussels in good health and maintain adequate growth throughout the test. Based on my limited experience testing bivalves, I believe it may not be as challenging as one might exp ect to keep control juvenile mussels alive for 28

- d given adequate oxygen and clean water but it may be quite another thing to document that organism condition (or alternatively, susceptibility to stress) is truly adequate. Therefore, the fact that juveni le mussel survival is lower with exposure to certain levels of ammonia may be due to having an already stressed population due to inadequate food or other factors.

A similar finding is now being made by EPA after reviewing chronic Hyalella chronic test dat a (reported to be one of the most sensitive species in the 1999 ammonia criteria document). In that case, control survival was apparently satisfactory but test organisms were in fact ultrasensitive to the chemical due to suboptimal test conditions.

A seco nd point I would raise regarding the chronic mussel tests is that they were only conducted once (based on published information) for a given species in one laboratory. While this in itself is not a reason to exclude test data, it is clear that there is co nsiderable variability in acute endpoints among juvenile mussel tests using the same species and conditions (even within the same lab as mentioned previously), which should apply to chronic survival with ammonia as well. Given that certain mollusks appear to be far more sensitive to ammonia than any species tested thus far, there should be some replication of these results to be sure we’re not going to find out that EPA needs to do yet another reassessment because of issues similar to the ones they are dea ling with now for Hyalella

.Jerome Diamond

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- 41 It is important to note that while interlaboratory testing was conducted for acute juvenile tests (Wang et al. 2007), similar interlab testing has not been conducted (or at least published) to my knowledge for 28

- d
mussel chroni c tests. In addition, one should view the acute interlaboratory results cautiously because, as mentioned previously, a single culture and water source was used by all laboratories in that study. As Wang et al. (2007) point out: “However, the present stu dy used the same batch of test organisms and the same dilution water, which were different from the referenced inter

- laboratory studies and might have contributed to the lower variability in test results. The present study was designed to determine the
inh erent variability in the test. Higher variability would be likely if the inter

- laboratory tests were conducted with different sources of dilution water and organisms (e.g., from different populations or watersheds). Therefore, additional study is needed to further characterize potential variability associated with the newly developed ASTM standard methods for conducting acute toxicity tests with early life stages of freshwater mussels.” Therefore, the CVs reported by Wang et al. (2007) are likely to be
und erestimates of true test method variability. This is borne out by the acute data generated for the same species by different labs as noted previously in my comments.

9. Should toxicity studies on the growth rates of mussel shells during 28

- day tests be co nsidered quantitatively when developing water quality criteria?

Response: I don’t believe there is sufficient information or adequate test method development and standardization at this time to be able to use growth rates of mussel shells in 28

- d tests f or criteria development. Growth rates, based on shell measurements, are not sufficiently large over a 28

- d period to have much confidence in the results. Based on the studies reported in this draft, as well as others of which I’m aware, shell length is t oo coarse a measurement to be used as an indicator of mussel growth over such a short time period. Furthermore, it is generally not known what the expected growth rate should be for a given species under these test conditions. As I indicated earlier, wit hout having such information, I don’t believe that survival data from such tests can be used with confidence as well.

10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?
Response: I agree w ith EPA’s position that mussel growth data should be categorized as “other data” until it is demonstrated that there is a repeatable, defensible methodology for generating adequate growth data and that the growth endpoint calculated is ecologically meaning ful. For this reason, I don’t think itJerome Diamond

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matters whether impaired growth observed in a 28

- d test is indicative of effects at longer exposures or not. However, I would recommend that EPA not consider 28

- d mussel growth as a valid endpoint based
on methodolo gical and perhaps biological uncertainties, as I mentioned under (9) above, rather than because reduced growth rate is theoretically a reversible effect. I see no reason why reduced growth would not be an even bigger effect with increased exposure duratio n to a toxicant.

11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Juvenile and adult mussels, as sediment

- dwelling organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have suggestions on how states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?
Response: I am not so sure that applying ammonia criteria to juvenile mussels at least, and possibly adults as well, should consider sediment pH at all for several reasons. First, larger juvenile and adult mussels are reported to be exposed to waterborne contaminants via siphoning the overlying water and not as much via sediment ingestion or pore water exposure during their growth season (Strayer et al. 2005). This is particularly true during times when water temperature exceeds 50  F (Watters et al. 2001; Schwal and Pusch 2007). In fact, many mussel sampling protocols specify a minimum water temperature of 50

- 60  F because mussels are near or on the surface under those conditions and more readily seen (e.g., Pennsylvania DEP and Army Corps sam pling protocol in the Allegheny River, PA). During this time (typically spring, summer, and fall in most waterbodies of the temperate U.S.), mussels are actively feeding via siphoning the water column and sediment pH is probably not a factor affecting wat er column ammonia toxicity. Rather it is the overlying water pH that is probably more important.

Second, many researchers report that sediment pH and ammonia can vary substantially from one location to another within even small areas and may be dependent on very complex redox gradients that are difficult to measure much less predict (Burton 1992; Stemmer et al. 1990; Sarda and Burton 1995). It is no easy matter to identify an appropriate sediment pH that should be used to apply an ambient water
ammonia cr iterion.

Third, many of the mussel species tested thus far, or that are species of concern, inhabit well

- aerated sediments that often have either fairly coarse particle size (gravels

– cobbles) or sand, both of which have interstitial water closely resembl ing the water quality conditions of the overlying water (Salmon and Green 1983; Neves and Widlak 1988; Way et al. 1990). In these cases one would not expect a large difference between sediment and overlying water pH.Jerome Diamond

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- 43 Fourth, it seems premature to consider complex pH adjustments based on sediments when it hasn’t been demonstrated that juvenile or adult mussels are as sensitive to sediment ammonia as they appear to be to water column ammonia in lab tests. If indeed sediment pH needs to be considered, then i t seems likely that mussels are not being exposed to surface water (including surface water ammonia) but rather sediment and interstitial ammonia. If this is the case, there is no need to adjust water column ammonia criteria based on sediment pH.

Finally, basing a water column criterion on sediment water characteristics represents a major departure for EPA in terms of criteria development. While I would encourage the Agency to consider more holistic criteria that take into account multiple media (e.g., se diment and water), I don’t believe there is sufficient information at this time to warrant altering a water quality criterion, which was derived using water exposure test methods exclusively, due to sediment characteristics.

12. In general, should the cri teria include a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically defensible way to account for these differences when deriving protective water quality criteria?
Response: I don’t think criteria should consider sediment pH or the difference between sediment and water pH in deriving water quality criteria. Rather, EPA should establish sediment ammonia criteria, using toxicity test data for mussels and other benthic species ( e.g., Hyalella
, Hexagenia , etc.) exposed to
sediments with ammonia.

13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?
Response: Tests using juvenile mussels should be conducted using sediment. Se veral researchers reported better survival and growth of juvenile mussels with the addition of sediment (e.g., Gatenby et al. 1996, Yeager et al. 1994) and I would note that many of the mussel culturing facilities that produce juveniles for transplants, us e sediment in their culturing systems. The ASTM standard for glochidia and juvenile mussel testing (E 24550

- 06) indicates this in several places including sections 10.4.2.4, 10.5.2.1, 10.5.2.5, 10.6.3.3, and 10.6.3.13. Laboratory studies by Yeager et al. (1994) observed that young juveniles burrow immediately to < 1cm deep in the sediment, and that they were not exposed to overlying water. Others also have reported rapid burrowing of new juveniles in sediment. This information suggests to me that some sediment in test containers may help ensure that juvenile mussels are unstressed and respond appropriately to contaminants. This might be a good check to see whether
ammonia concentrations shown to be toxic in water

- only exposures are also toxic when the tests are conducted with some sediment (clean, sterile, etc.) as well.Jerome Diamond

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LITERATURE CITED Arthur, J.W., C.W.West, K.N. Allen and S.F. Hedtke. 1987. Seasonal toxicity of ammonia to five fish and nine invertebrates species. Bull. Environ. Contam. Toxicol. 38(2): 324

- 331.
Black, M. 2001.Water quality standards for North Carolina's endangered mussels. Department of Environmental Health Science, Athens, GA.
Burton, G.A. 1992. Sediment collection and processing factors affecting realism. In: G.A. Burton, Ed., Sediment Toxicity Testing. Lewis, Boca Raton, FL, pp. 37

- 64.
Catherine M. Gatenby, Richard J. Neves and Bruce C. Parker 1996. Influence of Sediment and Algal Food on Cultured Juvenile Freshwater Mussels. Journal of the North American Benthologica l Society, Vol. 15: 597

- 609
Dodd, BJ, MC Barnhart, CL Rogers

- Lowery, TB Fobian, RV Dimock, Jr, 2006. Persistence of host response against glochidia larvae in Micropterus salmoides. Fish & Shellfish Immunology 21:473

- 484
Hickey, C.W. and M.L. Vickers. 19 94. Toxicity of ammonia to nine native New Zealand freshwater invertebrate species. Arch. Environ. Contam. Toxicol. 26(3): 292

- 298.
Kernaghan, N. J., Gross, T. S., Bishop, C.D., Wang, N., Roberts, A., and Ingersoll, C. G.. 2005. “Laboratory Toxicity Testi ng with Freshwater Unionid Mussels,” Freshwater Bivalve Ecotoxicology, van Hassel, J. H., and Farris, J. L., eds., SETAC Press, Pensacola, FL.
M. M. Yeager, D. S. Cherry and R. J. Neves. 1994. Feeding and Burrowing Behaviors of Juvenile Rainbow Mussels, V illosa iris (Bivalvia:Unionidae) Journal of the North American Benthological Society, Vol. 13: 217

- 222
Neves, R. J., and Widlak, J. C. 1988. Occurrence of Glochidia in Stream Drift on Fishes of the Upper North Fork Holston River, Virginia. Am Midland Nat uralist, 119, 1988, pp. 111

- 120.
Nimmo, D.W.R., D. Link, L.P. Parrish, G.J. Rodriguez, W. Wuerthele and P.H. Davies. 1989. Comparison of on

- site and laboratory toxicity tests: Derivation of site

- specific criteria for un

ionized ammonia in a Colorado trans itional stream. Environ. Toxicol. Chem. 8(12): 1177

- 1189.
Rogers

- Lowery, CL and RV Dimock, Jr. 2006. Encapsulation of attached ectoparasitic larvae of freshwater mussels by epithelial tissue on fins of naive and resistant host fish. Biol. Bull. 210:51

- 63
Salmon, A. and R.H. Green. 1983. Environmental determinants of unionid clam distribution in the Middle Thames River, Ontario. Canadian Journal of Zoology 61: 832

- 838.
Sardia, N. and G. Burton. 1995. Ammonia variation in sediments: Spatial, temporal and method

- related effects. Environ. Toxicol. Chem. 14(9):1499

- 1506. Scheller, J.L. 1997. The effect of dieoffs of Asian clams ( Corbicula fluminea ) on native freshwater mussels (unionidae). Virgi nia Polytechnic Institute and State University, Blacksburg, VA.
Schwal, A. and M. Pusch 2007. Horizontal and vertical movements of unionid mussels in a lowland river Journal of the North American Benthological Society 26(2):261

– 272.Jerome Diamond

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- 45 Stemmer, B.L., G.A. Burton, Jr. and G. Sasson

- Brickson. 1990. Effect of sediment spatial variance and collection method on cladoceran toxicity and indigenous microbial activity determinations. Environ. Toxicol. Chem. 9:1035

- 1044.
Strayer, D. L., Downing, J. A., Haag,W. R. , King, T. L., Layzer, J. B., Newton, T. J., and Nichols, S. J. 2004. Changing Perspectives on Pearly Mussels, North America’s Most Imperiled Animals. BioScience, 54:2004, pp. 429

- 439.
U.S. Environmental Protection Agency. 1994. Interim Guidance on Dete rmination and Use of Water Effect Ratios for Metals, Appendix B: The Recalculation Procedure. EPA

- 823

- B

- 94

- 001. Office of Water, Washington, DC.
USEPA. 2000. Ambient Aquatic Life Water Quality Criteria for Dissolved Oxygen (Saltwater): Cape Cod to Cap e Hatteras. EPA

- 822

- R

- 00

- 012 Office of Water, Washington, D.C. November 2000
USEPA. 2002. Clinch and Powell Valley Watershed Ecological Risk Assessment. 2002. EPA

- 600

- R

- 01

- 050. NCEA, ORD, Washington, D.C.
Wang, N., C.G. Ingersoll , D.K. Hardesty, C. D. Ivey, J.L. Kunz, T.W. May, F.J. Dwyer, A.D. Roberts, T. Augspurger, C.M. Kane, R.J. Neves and M.C. Barnhart. 2007. Contaminant sensitivity of freshwater mussels: Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mu ssels (Unionidae). Environ. Toxicol. Chem. 26(10):2036

- 2047.
Wang, N., J.T. Augspurger, M.C. Barnhart, J.R. Bidwell, WG. Cope, F.J. Dwyer, S. Geis, I.E. Greer, C.G. Ingersoll, C.M., Kane, T.W. May, R.J. Neves, T.J. Newton, A.D. Roberts and D.W. Whites. 20 07. Contaminant sensitivity of freshwater mussels: Intra

- and inter

- laboratory variability in acute toxicity tests with glochidia and juveniles of freshwater mussels (Unionidae). Environ. Toxicol. Chem. 26(10):2029

- 2035.
Watters, G. A., 2005. A Brief Look at Freshwater Mussel (Unionacea) Biology. Freshwater Bivalve Ecotoxicology, van Hassel, J. H., and Farris, J. L., eds., SETAC Press, Pensacola, FL.
Watters, G. T., S. H. O’dee, And S. Chordas. 2001. Patterns of vertical migration in freshwater mussels (Bivalvia: Unionidae). Journal of Freshwater Ecology 16:541

– 549.
Way, C.M., A.C. Miller and B.S. Payne. 1990. The influence of physical factors on the distribution and abundance of freshwater mussels (Bivalvia:Unionidae) in the lower Tennessee River. The Nautilus 103(3): 96

- 98.C

- 47 PEER REVIEW COMMENTS FROM

George Dixon, Ph.D. Professor, Department of Biology

and Vice President of University Research University of Waterloo 200 University Avenue West Waterloo, ON N2L 3G1 Canada 519

- 888

- 4567 Email: gdixon@uwaterloo.caGeorge Dixon

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- 49 11 August 2009

Peer Review of “Draft Reassessment of the 1999 Ambient Water Quality Criteria for Ammonia

– Freshwater” by D. George Dixon. EPA Contract No. EP

- C

- 07

- 059. Work Assignment No. 1

- 04.
I have reviewed the document listed above as requested in the Charge to Reviewers dated 24 July 2009. In addition I have gone through the background document “1999 Update of Ambient Water Quality
Criteria for Ammonia”. Let me state at the outset that I find the Draft Reassessment to be a well conceived, well developed and carefully written docu ment. The acute and chronic ammonia criteria for freshwaters with mussels present, and separate criteria for waters with mussels absent, are well founded and scientifically defensible. I have presented my review comments as responses to the thirteen questi ons posed in the Charge to Reviewers Those questions are repeated here for clarity.

Acute criteria in fresh waters:

1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?
I am familiar with the majority of the literature cited and, in my opinion, the tests used to develop the acute criteria are scientifically defensible. I am, however, relying on the fact that the Draft Reassessment states that a process to identify data meeting the minimum requirements for inclusion was followed. I
have not obta ined all of the cited literature and reviewed each paper for suitability of inclusion of the findings in the data sets; this type of detailed audit is beyond the scope of this review. I follow the ammonia literature closely, and I am unaware of any additio nal data that could be of use

.

2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?
The document is silent on the appropriate methods for determ ining whether mussels are present or absent in a given receiving environment, and therefore which criteria should be applied. I expect that a guidance document will have to be developed to assist those applying the revised criteria. My expertise is in aqua tic toxicology, while the question of presence or absence is based more in field

- based aquatic ecology, particularly mussel ecology. As such I do not have detailed recommendations for the determination. I would, however, expect that some consideration w ould be given to a method that was able to deal with environments where mussels were once present, but have now been expatriated.George Dixon

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3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test du ration time for this life stage? Do you believe there is an alternative approach to the use of this data that would be more scientifically sound?

Exclusion of the glochidia data , as detailed in Appendix A, is fully appropriate and well explained. Until tests are completed to determine (on a species by species basis) the duration of the period of longevity (and by inference viability) for glochidia , LC50s obtained with those life stages will be suspect. I am unaware of any other approach which would be more scientifically valid

4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is th e approach a scientifically defensible principle for structuring the population exposure duration problem and designing further research to quantify such a duration?
I am not a mussel ecologist, but the approach taken, and its implications for further res earch appear valid. Having said that, it is likely that modifications to the approach will have to be made as such research proceeds. It is also apparent that there is sufficient species variability in the life habits of glochidia that a single approach wi ll not fit all species.

Hyalella azteca position statement and proposed rationale (see Appendix B):
The EPA workgroup developed a position statement and proposed supporting rationale describing the concerns over using Hyalella azteca toxicity test data in criteria development due to the uncertain health of the test organisms in different test water composition. The rationale defines the specific concerns and uncertainties supporting the recommended exclusion of the Hyalella data from use in criteria der ivation, at this time; the position statement is based on the workgroup’s review of a number of toxicity tests on Hyalella, referenced in the rationale.

5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?

The exclusion of Hyalella azteca data, as summarized on page 13, is fully defensible. I would suggest that while all of the points are relevant, the pivotal consideration is the impact of chloride and bromide ion on the viability of the species and the potential interaction of the concentration of these ions with ammonia
toxici ty. The Hyalella data will be suspect until that information is obtained.

Chronic Freshwater Criterion:
6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such u se? Are you aware of other relevant data that were not used?
Please see the response to question 1 above. That statement is also applicable to the chronic data base.George Dixon

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- 51 7. Is the freshwater chronic criterion scientifically defensible with mussels present and mussels absent?
The chronic criterion is defensible, provided that suitable guidelines are developed to differentiate environments where mussels are present from those where they are absent. Please see my response to question 2 above.

Use of 28

- day Juvenile Test Data (see Appendix C):
Water quality criteria for the protection of aquatic life are derived using toxicity endpoints that relate to population level impacts. In general, these endpoints relate to survival, growth and/or reproduction. The 28

- day test with juvenile mussels, whi le similar in duration to a standard chronic test, is not technically an early

- life stage test according to the 1985 Guidelines for Aquatic Life Criteria as much of the early development will have already occurred.

8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defensible as "chronic" test data for criteria development?
This is well covered in Appendix C, and the arguments are convincing. I agree that the 28

- d test does not qualify as a chronic test, since it is neither a life

- cycle test nor a partial life

- cycle test. I a lso agree that a concentration causing a greater than 20% reduction in survival could be used as a n upper limit for the Species Mean Chronic Value, assuming the test met the requirement for good lab practice.

9. Should toxicity studies on the growth rates of mussel shells during 28

- day tests be considered quantitatively when developing water quality criteria?
The approach taken in the document (Appendix C), that of not using data for growth of organisms in non

life

- cycle tests with durations of less than 90 days in the derivation of a Species Mean Chronic Value (SMCV), is in my opinion perfectly appropriate. The justification giv en in paragraph 5 of Appendix C centers on two points: lack of direct comparisons of growth between shorter tests and life

- cycle tests and uncertainties around the relationship between short

- term growth reductions and growth effects over a full life cycle. Both are valid reasons to exclude the data from calculations of SMCV.

10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?
With respect to the first point above, inclusion of the data as “other data” is perfectly appropriate. This is

co nsistent with the practice for short

- term growth results with other species, and I see no reason whyGeorge Dixon

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mussel data should be treated differently. With respect to the second question, this is a statistically valid point, but it does not reduce our level of un certainty around the actual meaning of the data. The issue will only be resolved when someone undertakes 90

- day tests.

11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Juvenile and adult mussels, as sediment

- dwell ing organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have suggestions on how states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?
While mussels live in sediment, the pH of importance from an ammonia criteria perspective is the pH of the respiratory environment, which in th is case is the pH at the sediment

- water interface. The sediment pH is of importance only to the extent that it influences the pH of the water at the interface. Remember these are water quality criteria, not sediment quality criteria. I have no suggestion a s to the appropriate method for determining the pH at the interface. The methodology, more in the realm of environmental chemistry, falls outside of my area of expertise.

12. In general, should the criteria include a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically defensible way to account for these differences when deriving protective water quality criteria?
My opinion here is partially covered in the response to question 11. The pH of the sediment is only of consequence to the degree that it influences the pH in the water column at the sediment

- water interface. I am not aware of any methodologies, other than direct measurement, that would allow estimation of the interface pH if the only data available were the sediment pH and the water

- column pH.

13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test
chamber?

My comments here are limited to laboratory tests where issues of pH variability between the sediment and the water column could be eliminated (to avoid direct measurement as discussed above) by experimental manipulation. The main reason for including sedim ent would presumably be to reduce extraneous stress on the experimental organisms, stress which could possibly interact with the toxicant to produce an anomalous result. I have no opinion as to whether the presence or absence of sediment would stress juven ile mussels in an experimental setting. This will have to be determined experimentally.C

- 53 PEER REVIEW COMMENTS FROM

Jerry L. Farris , Ph.D. Professor of Environmental Biology Arkansas State University 3613 Alabama Road Jonesboro, AR 72401 870

- 680

- 8151 Email: jlfarris@astate.eduJerry Farris

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- 55 EPA’s Draft Reassessment of the 1999 Ambient Water Quality

Criteria for Ammonia

– Freshwater
Acute criteria in fresh waters:

1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?
With greater recent attention dedicated to standardization of test techniques suitably addressing the
fea sibility of repeatability and precision of results from juvenile mussel responses (Augspurger, 2007), data available at the time of this reassessment meet a higher standard of acceptability for guidance modified from the US EPA (Stephan et al., 1985) and f ollow consensus test protocols and quality assurance recommendations sufficient to support water quality criteria development. The majority of tests used to derive the criteria seem scientifically defensible for such use. Upon review
of those included a nd others not specifically included in the calculation, but cited in Table 1 of the draft addendum, there are tests that present concern for acclimation procedures of test organisms and culturing (either reported or unreported) and lack of reference testin g that can insure level of expertise or consistency associated with such field collection, organism transfer, and culturing that can significantly influence test responses. Specifically, sufficient acclimation information for water quality and temperature was not included in Mummert et al., 2003 and Scheller, 1997.

I am not aware of other data from standardized toxicity tests with ammonia that could be considered
relevant to this reassessment.

2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?
With mussels presenting such an extreme sensitivity and complex life history in comparison to other groups, not only will their presence establish con sideration for site variance demonstrations related to specific media, habitat or host fish interactions, but their spatial positions and ecological requirements against the backdrop of a stream’s ability to meet water quality standards

- will require some unique approaches to:

 Short

- term authorizations to discharge

 Compliance with permits that account for ammonia levels, but in streams or those segments that fail to meet water quality standards

 Designated mixing zones and how “shock effect” associated with ammonia will be considered

 The applicability of biocriteria and use of in

- stream community assessments related to the concept of mussels present and absent that would incorporate differences between degree of colonization and distribution rates for fish, other benthic organisms of conventional use in stream surveys, and freshwater mussels. These considerationsJerry Farris

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- 56 coupled with only a recent attempt to describe how certain stream characteristics may serve to structure mussel communities, will need to be care fully approached when and if such recommendation is made.
Since many of the ‘mussel present’ instances may involve listed species, the ability to qualify broad testing and culturing of suitable surrogate species for the time and seasonal limits related to any testing related to permit or enforcement actions, will likely present an additional set of laboratory qualifying or certification considerations related to accessing suitably sited mussel species for testing.
Laboratories listed in reference to th e range of studies cited with this reassessment and that have implemented the new test standards (ASTM, 2006) have certainly advanced the methodologies needed for risk assessments and management decisions. However, this relatively limited number of

resear ch and agency laboratories will likely be challenged to fulfill the rigorous requirements of consistency with organism culturing and quality assurance that may be expected of more conventional test demands. While consideration of these outcomes may reach beyond the scope of the question for mussel presence, increased demand as a technical consideration would be expected to impact population availability.

Relative use of reference sites in determining relationships between areas of reduced freshwater

mus sel diversity and abundance and known significant ammonia sources will also likely become a
technical consideration. Definitive cause

- and

- effect relationships have not been documented in such instances (Bartsch et al., 2003) and such considerations would require more detailed attention between the field exposure and effects data.

3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe th ere is an alternative approach to the use of this data that would be more scientifically sound?
Information from tests with glochidia can be supportive of the consideration for site variance information, but should not be used to provide stand alone estim ates in the absence of support ive acute juvenile estimates. Multiple lines of evidence comprised of testing with more than one mussel life stage that include comparisons with glochidia responses, would seem more scientifically sound, and may remove much o f the seasonal dependence upon culturing and provision of test organisms with less invasive techniques in instances mentioned in the above mentioned technical considerations.Jerry Farris

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- 57 4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scientifically defensible principle for structuring the population exposure duration problem a nd designing further research to quantify such a duration?
The support of life history information and exposure information can support this contention, but the pertinence of this endpoint would continue to be challenged for its scientific defensibility a s relates to what we currently understand of exposure durations related to this life stage and variety of adaptations.

Hyalella azteca position statement and proposed rationale (see Appendix B):
The EPA workgroup developed a position statement and proposed supporting rationale describing the concerns over using Hyalella azteca toxicity test data in criteria development due to the uncertain health of the test organisms in different test water compo sition. The rationale defines the specific concerns and uncertainties supporting the recommended exclusion of the Hyalella data from use in criteria derivation, at this time; the position statement is based on the workgroup’s review of a number of toxicit y tests on Hyalella, referenced in the rationale.

5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?
Those points are very critical given the consideration for osmoregulation by this organism and the points cited in the workgroup’s review. This seems well illustrated in the background material
furnished for this review.

Chronic Freshwater Criterio n:

6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such use? Are you aware of other relevant data that were not used?

The tests utilized for the criterion are scientifically defensible for such use and I’m not aware of other
relevant data that were not used.

7. Is the freshwater chronic criterion scientifically defensible with mussels present and mussels absent?

T he criterion does not seem defensible for locations where mussels are present as would relate to considerations under the Endangered Species Act, but would likely be defensible under considerations with the Clean Water Act. I pose this statement as an ex ample of the difficulty inJerry Farris

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relying on scientific defensibility in the face of a very broad range of considerations in risk and decision management that may rely on these estimates.

Use of 28

- day Juvenile Test Data (see Appendix C):
Water quality criter ia for the protection of aquatic life are derived using toxicity endpoints that relate to population level impacts. In general, these endpoints relate to survival, growth and/or reproduction. The 28

- day test with juvenile mussels, while similar in durati on to a standard chronic test, is not technically an early

- life stage test according to the 1985 Guidelines for Aquatic Life Criteria as much of the early development will have already occurred.

8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defensible as "chronic" test data for criteria development?
Research that validates effective endpoints related to the range of pollutant effects in freshwate r mussels have shown that 28

- 30

- day exposure tests in both field and laboratory settings can be included to predict long term impacts related to biomarker responses, bioaccumulation, and even comparisons to conventional “chronic” test data. The 28

- day t est exposure period will continue to be temporal as relates to the life cycle, but can be relational to long

- term considerations and offer critical information when combined with multiple lines of evidence. Inclusion of more than one life stage

response a lso advances mussel toxicity assessments, rather than providing semantic arguments over longevity for a life stage and whether the exposure period qualifies as “chronic”. Data from the 28

- day test can be defensible when supported by “other” data. Other data should also be scientifically
defensible.

9. Should toxicity studies on the growth rates of mussel shells during 28

- day tests be considered quantitatively when developing water quality criteria?
Growth rates of mussels during the 28

- day tests should not be considered quantitatively until more information is available on the validity of this test. Variability is still attributable to diet and holding conditions and has been cited broadly.

10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?
Growth should not be considered as defensible for the above mentioned reasons relating to lack of reliable test conditions, but can be used as supportive information that might influence
the criteria. S tudies that include recovery and or stability in growth or even degrowth withJerry Farris

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- 59 freshwater bivalves, have produced more reliable estimates of pollutant effects and could
therefore be used to influence the criteria when included with broader scale studies.

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potentia l outcome only)?
As stated, the above consideration seems very confusing and should be clarified to relate to the defensibility as considered with “other data”.

11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Ju venile and adult mussels, as sediment

- dwelling organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have suggestions on how states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?
Even if an adequate amount of information was available for any benthic invertebrates for chronic

to xicity related more specifically to the generic relationship of ammonia toxicity to pH, larger issues challenge this consideration with the reassessment for changes related to pH shifts, which may increase when phase waters are isolated for testing. These relationships have only recently been examined for juvenile freshwater mussels, without sufficient detail furnished as to the pH adjustment used with diluter systems in those tests or their effect upon alkalinity changes measured during the assays (Wang e t al., 2007). Although these tests seem suitably performed to establish the influence of pH on the acute toxicity of ammonia and even the pertinence of normalizing ammonia toxicity data for mussels to a common pH end point (total ammonia nitrogen at pH 8. 0), information is still lacking for the sediment relations specific to mussel responses. As relates to this consideration, the bulk of the studies that have examined sediment and phase water relationships to date rely heavily on marine field monitoring, and not standardized laboratory testing or field monitoring with freshwaters (Salazar and Salazar, 2007). Any scientifically defensible way to account for these differences would greatly benefit from studies with comparable methods examining habitat pref erences, feeding, and mobility of a sensitive mussel life stage (juvenile) among sediment compartments, since the synthesis of ammonia toxicity data confirmed its status as a sensitive species. Data from these studies would also be needed to generate pert inent modeling of the ammonia fluxes from sediment and the concentration profiles in the phases that would have consequence to mussel impact.Jerry Farris

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12. In general, should the criteria include a consideration for the potential pH difference between sedimen t and the water? If so, what is the most scientifically defensible way to account for these differences when deriving protective water quality criteria?
In addition to the aforementioned comments regarding the need for additional studies involving mussel responses before including a consideration for this difference, site

- specific variance procedures inclusive of sediment characteristics for a “mussels pre sent “ site might be especially applicable in this instance. Should these site criteria account for such differences in testing of site waters with and without sediment, they would be expected to be applicable for the species (or suitable surrogate) and s ediment of interest. Use of a site

- specific criterion for copper that incorporated such broad test considerations has been utilized to insure protection of mussel species for almost two decades within the Clinch River, Virginia (Van Hassel, 2007).

1 3. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?
If the need is greater at this point with the reassessment to consider data from tests that offer repeatability and precision of results, than to ens ure inclusion of test conditions reflecting the life history and habitat selection for this life stage of the mussel, then the test should exclude the sediment considerations. There is a growing body of evidence from tests without sediment that suggests

c omparable juvenile shell growth can be measured in control treatments in static, static

- renewal, and
flow

- through conditions. This is somewhat contradictory to earlier studies that recognized the importance of sediment in distinguishing effects during lon ger term exposures with freshwater mussels and fingernail clams. For this reason, the information still seems lacking in determining how much of a confounding effect is introduced by the effects of sediment characteristics (e.g., particle size, microbial mobilization and organic carbon content). More specific information on the survival and physiological condition of benthic organisms when tested in a water

- only medium seems necessary for testing beyond 96 hours. Burton et al. (2002) describes a range of toxicity assays using
benthic invertebrates in sediment

- free systems such as interstitial water, elutriate phase, or spiked waters, with each possibly differentiating toxicant uptake, pathway, and hazard. It would seem prudent to be consistent with curren t conventional approaches to sediment evaluations for their consideration of appropriate phases to be tested (extractable, elutriate, interstitial, whole sediment,
and in situ ) relating to appropriate conditions for the species or substrate of interest. The complex interactions between mussel, water, and sediment that influence their responses has been cited to reinforce those considerations (Thorsen et al., 2007).Jerry Farris

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- 61 Referenc es: American Society for Testing and Materials. 2006. Standard guide for conducting laboratory toxicity tests with freshwater mussels. E2455

- 06. In Annual Book of ASTM Standards, Vol 11.06. Philadelphia, PA.

Augspurger, T. 2007. Advances and opportuniti es in assessing contaminant sensitivity of freshwater mussel (Unionidae) early life stages. Environ Toxicol Chem 26:2025

- 2028.
Bartsch MR, Newton TJ, Allran JW, O’Donnell JA, Richardson WB. 2003. Effects of pore

- water ammonia on in situ survival and growt h of juvenile mussels ( Lampsilis cardium ) in the St. Croix riverway, Wisconsin, USA. Environ Toxicol Chem 22:2561

– 2568.
Burton, GA, PM Chapman, and EP Smith. 2002. Weight of evidence approaches for assessing ecosystem impairment. Human and Ecological R isk Assessment: An international Journal 8:1657

- 1673.
Cherry, DS, JL Scheller, N.L. Cooper and J.R. Bidwell. 2005. The potential for Asian clam ( Corbicula fluminea ) dieoffs to impact native freshwater mussels (Unionidae) I: Water column ammonia levels an d ammonia toxicity. Journal of the North American Benthological Society, 24:369

- 380.
Cooper, NL. JR Bidwell and DS Cherry. 2005. The potential for Asian clam ( Corbicula fluminea ) die

- offs to impact native freshwater mussels (Unionidae) II: Pore

- water ammon ia. Journal of the North American Benthological Society, 24:381

- 394.
Mummert, AK, Neves RJ, Newcomb TJ, Cherry DS. 2003. Sensitivity of juvenile freshwater mussels ( Lampsilis fasciola
, Villosa iris ) to total and un

- ionized ammonia. Environ Toxicol Chem 22 :2545

– 2553.
Newton TJ, Allran JW, O’Donnell JA, Bartsch MR, Richardson WB. 2003. Effects of ammonia on juvenile unionid mussels ( Lampsilis cardium ) in laboratory sediment toxicity tests. Environ Toxicol Chem 22:2554

– 2560.
Salazar, MH and SM Salazar. 200 7. Linking bioaccumulation and biological effects to chemicals in water and sediment: A conceptual framework for freshwater bivalve ecotoxicology. In Farris JL, Van Hassel JH, eds, Freshwater Bivalve Ecotoxicology. SETAC, Pensacola, FL, USA, pp 215

- 250

.
Scheller JL. 1997. The effect of die

- offs of Asian clams ( Corbicula fluminea ) on native freshwater mussels (Unionidae). Masters thesis. Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
Stephan CE, Mount DI, Hansen DJ, Gentile JH, Chapman GA, Brungs WA. 1985. Guidelines for deriving numerical national water quality criteria for the protection of aquatic organisms and their uses. EPA 822/A85/100. U.S. Environmental Protection Agency, Offic e of Research and Development, Washington, DC.
Thorsen, WA, WG Cope, and D Shea. 2007. Toxicokinetics of environmental contaminants in freshwater bivalves. In Farris JL, Van Hassel JH, eds, Freshwater Bivalve Ecotoxicology. SETAC, Pensacola, FL, USA, pp 169

- 207.Jerry Farris

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Van Hassel, JH. 2007. Case study: Discrimination of factors affecting Unionid mussel distribution in the Clinch River, Virginia, USA. In Farris JL, Van Hassel J, eds, Freshwater Bivalve Ecotoxicology. SETAC, Pensacola, FL, USA, pp 311

- 331.
Wang N, Ingersoll CG, Hardesty DK, Ivey CD, Kunz JL, Dwyer FJ, Roberts AD, Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007. Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mussels (Unionidae). Environ Toxicol 26 :2048

- 2056.
Wang N, Ingersoll CG, Greer IE, Hardesty DK, Ivey CD, Kunz JL, Dwyer FJ, Roberts AD, Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007. Chronic toxicity of copper and ammonia to juvenile freshwater mussels (Unionidae). Environ Toxicol Chem 26 :2048

- 205.C

- 63 PEER REVIEW COMMENTS FROM

William L. Goodfellow Jr., MS EA Engineering, Science, and Technology, Inc.

15 Loveton Circle Sparks, MD 21152 410

- 329

- 5121 Email: bgoodfellow@eaest.comWilliam Goodfellow

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- 65 26 August 2009

Ms. Laurie Waite ERG 110 Hartwell Avenue Lexington, MA 02421

- 3136
Re: Review of EPA’s Draft Reassessment of the 1999 Ambient Water Quality Criteria for Ammonia

Freshwater.

Dear Ms. Waite:
I have completed my review of the above reference EPA water quality criteria document for ammonia. I have found the criteria document to be well organized and very well thought out. I also feel that the scientific justification laid out in the document t o be excellent.

I have several questions or comments presented below as well as imbedded in the Charge Questions section (my comments are presented in Bold).

I do question why only standards consistent with ASTM Standards for testing (page 14) are the on ly relevant test procedures that were used in the document. I believe that the EPA’s Whole Effluent Testing (WET) toxicity testing guidelines for testing are also relevant. Did this exclude any additional data? From my review of the references, I do not be lieve so.

I also question when the Nile tilapia was place in the other chronic toxicity data (and I agree this was appropriate), why is the Asiatic clam included in the data used for development of the criteria (and in fact
it is the third most sensitive species for the acute criterion). The Asiatic clam is also a nonindigenous species (or exotic). In my opinion I don’t believe that the Asiatic clam should not be used in the development of the criteria regardless that this exotic is found throughout United States. I believe elimination of this species from the dataset will still generate a protective acute criterion.

The Table A in text of the GMAV for the Mountain Whitefish does not match Table 1. The Text table reports 12.11 and Table 1 reports 12.09. I am expecting that the Table 1 is correct and is what was used for the FAV. Please verify.William Goodfellow

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As requested, please find below my addresses as they related to the Charge Questions as presented in ERG’s Charge to Peer Reviewers:

PEER REVIEW CHARGE QUESTIONS
Acute criteria in fresh waters:

1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?
From my review, I believe that the acute toxicity testing data is r elevant. My only concern is that this document was careful to use data that was from published references which I believe is a strength of this document as compared to earlier water quality criteria documents. Since the only unpublished data used
that demo nstrates a somewhat low toxicity response to ammonia was from Keller 1999 for the Pondshell Mussel, maybe putting a synopsis as an attachment would be warranted. This would help make the document transparent as the other Attachments help do.

The Table A in text of the GMAV for the Mountain Whitefish does not match Table 1. The Text table reports 12.11 and Table 1 reports 12.09. I am expecting that the Table 1 is correct and is what was used for the FAV. Please verify.

2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended acute criteria?
I believe that EPA has adequately defended their position for when mussels were used or not use d for the generation of the two phased acute criterion.

I do feel that EPA should include guidance that the lower mussel criterion should only be used when mussels are currently present in the water system and not historic or wished to be in the system. I can see States using the lower mussel numbers without a strong technical reason to use them because someone wished them to be in the system, etc. When States choose to go this way arbitrarily, it will weaken the acceptability of the criterion for every one.William Goodfellow

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- 67 3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe there is an alternative approach to the use of this data that would be more s cientifically sound?
Not only is it scientifically defensible, I believe that it is the right way to go. Too much is unknown with regards to the biology of glochidia and the associated toxicology or appropriate methods to evaluate the toxicity of various chemicals for this life stage.

4. Regarding the proposed approach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scientifically defensible principle for structuring the population exposure duration problem and designing further research to quantify such duration?
See comment for Question 3, the same comment is relevant for Question 4.

Hyalella azteca position statement and prop osed rationale (see Appendix B):
The EPA workgroup developed a position statement and proposed supporting rationale describing the concerns over using Hyalella azteca toxicity test data in criteria development due to the uncertain health of the test organisms in different test water compo sition. The rationale defines the specific concerns and uncertainties supporting the recommended exclusion of the Hyalella data from use in criteria derivation, at this time; the position statement is based on the workgroup’s review of a number of toxicit y tests on Hyalella, referenced in the rationale.

5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?

I believe that the position statement and supporting rationale is appropriate and justified.

Chronic Freshwater Criterion:

6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such use? Are you aware of other relevant data that were not used?

I believe that they are scientifically defensible for the use in the development of the chronic
criterion. I am not aware of other relevant data.William Goodfellow

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7. Is the freshwater chronic criterion scientifically def ensible with mussels present and mussels absent?
Similar to the acute criterion, I believe it is scientifically defensible to develop the criterion with mussels present and mussels absent. I do believe that the EPA should have a cautionary note as in
th e acute criterion as to when and how to determine if mussels are present or absent.

Use of 28

- day Juvenile Test Data (see Appendix C):
Water quality criteria for the protection of aquatic life are derived using toxicity endpoints that relate to population level impacts. In general, these endpoints relate to survival, growth and/or reproduction. The 28

- day test with juvenile mussels, whi le similar in duration to a standard chronic test, is not technically an early

- life stage test according to the 1985 Guidelines for Aquatic Life Criteria as much of the early development will have already occurred.

8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scientifically defensible as "chronic" test data for criteria development?
I do not believe that 28 day mussel studi es should be considered as chronic test data for the national database as described in the position statement and supporting documentation. I believe this is very well thought out and applaud the developers.

9. Should toxicity studies on the growth rates of mussel shells during 28

- day tests be considered quantitatively when developing water quality criteria?
The problem with using shell growth rates, it doesn’t necessarily link to a smaller organism being any less viable or having less reproductive benefi ts. Since mussels are not a commercially or recreational important organism, I believe that the use of growth rates are not valid as compared to a fish species or commercially important bivalve.

10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?

I b elieve that it should remain in the other data section so the relevant data can be used in site specific evaluations.

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to declin e in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?
I don’t believe that data supports this and such a statement should not be made.William Goodfellow

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- 69 11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Juvenile and adult mussels, as sediment

- dwelling organisms, inhabit a medium that may have vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposu re conditions. For applying a criterion protecting mussels, do you have suggestions on how states and EPA might determine the pH applicable to the sediment micro

- environment to which mussels are typically exposed?
It is much easier to do this for water column organisms. I believe that the agency will open themselves up for tougher battle if they attempt to make this issue even more complex than it already is. I believe that noting that this is the case and stating t hat pH dependence vs. the criteria as it relates to mussels was not developed at this time given the uncertainty about what the appropriate pH assignment should be.

I also argue whether pH in sediment is a relevant question with regards to criteria sinc e the management tool for water quality is going to be primarily the NPDES permit. How will anyone be able to control sediment pH to manage it? I am not sure what this does for development of a
defensible criterion.

12. In general, should the criteria in clude a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically defensible way to account for these differences when deriving protective water quality criteria?
Same comment as for Question 11.

13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?
I believe this question is very similar to the Hyalella azteca question. If adequate test results that are representative to the toxicity of ammonia can be performed in water only, that is how they should be performed. If sediment is necessary for the test to be performed adequately, then they should use sediment. However, it should be cautioned that when the variable of sediment is
added to t he toxicity testing methodology, the complexity of the test increases considerably as to the interpretation. In my opinion the agency should caution the use of sediment in the test my lead to positive and negative bias of the test results. The agency shoul d try to stay with water column only whenever possible.William Goodfellow

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I thank you for the opportunity to allow me to be a peer reviewer for this important freshwater water quality criteria document. If you have any questions or require additional information, please l et me know.

Sincerely,

William L. Goodfellow, Jr. Vice President and Senior Scientist

Clarification question from EPA for the following comment made by Dr. Bill Goodfellow:
"I also question when the Nile tilapia was place in the other chronic toxicity data (and I agree this was appropriate), why is the Asiatic clam included in the data used for development of the criteria (and in fact it is the third most sensitive sp ecies for the acute criterion). The Asiatic clam is also a nonindigenous speci es (or exotic ). In my opinion I don't believe that the Asiatic clam should not be used in the development of the criteria regardless that this exotic is found throughout United States. I believe elimination of this species from the dataset will still generate a protective acute criterion

. "
EPA Question: “...it sounds like he's questioning why we inc luded the clam data but then the text I highlighted in red sounds like he's saying that we should include it

- maybe there's a typo and the double negative s entence is wrong? If you could get clarification from him for me on this I would appreciate it.

Clarification from Dr. Bill Goodfellow: In my opinion the Asiatic clam (an exotic) should not be included in the database used for development of the criteria . Rather it should be placed in the Table with other data. If the Agency decides to include the Asiatic Clam in the criteria development, I believe considerable justification will need to be provided in the written justification of the criteria document.
Hopefully this makes it clearer. Let me know if you have any other questions or require additional information.C

- 71 PEER REVIEW COMMENTS FROM

James J. Pletl , Ph.D. Chief, Technical Services Division Hampton Roads Sanitation District 1436 Air Rail Avenue

– 2 nd Floor Virginia Beach, VA 23455 757

- 460

- 4246 Email: jpletl@hrsd.comJames Pletl

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- 73 EPA’s Draft Reassessment of the 1999 Ambient Water Quality Criteria for Ammo nia

– Freshwater (July 14, 2009)

CHARGE TO PEER REVIEW QUESTIONS
Acute criteria in fresh waters:

1. Are the toxicity tests used to derive the criteria scientifically defensible for such use? Are you aware of other relevant data that were not used?
The question requires that the term “scientifically defensible” be defined within the context of the use or the consequences of the use. The use is to support development of water quality criteria that will be promulgated by states as legally enforceable water quality standards. Failure to meet water quality standards instream and/or at the edge of respective mixing zones will result in TMDLs, implementation plans, or NPDES permit limits for dischargers to the respective stream. Failure to meet water qual ity standards also infers that designated uses are not being supported. In the case of ammonia criteria, the designated use is aquatic life. The Clean Water Act also states that violations of such permit limits can result in significant monetary fines an d/or jail time for responsible individuals. Therefore the use, in this case, is quite demanding of the data.

The level of scientific defensibility set by EPA for this use is limited to that defined by EPA’s 1985 “Guidelines For Deriving Numerical Natio nal Water Quality Criteria For The Protection of Aquatic Organisms And Their Uses”, hereafter referred to as the Guidelines, and the published, peer reviewed toxicity test methods used in each case. The tests in question appear to meet these standards; ho wever the data quality objectives set by these documents may not be sufficient for the data use. The toxicity test methods and the Guidelines do not require what are considered to be critical elements of scientifically defensible toxicity tests. Acute te sts typically only have one quality objective: minimum survival in controls. Other than control survival there are no other quality objectives that must be met for the results of these tests to be considered reliable. For example, 1) the methods and Guid elines do not require a
concentration

- response curve for data to be deemed acceptable for any use. There can be little certainty in a toxicity test result where response does not change predictably with the concentration exposure. If
demonstration of thi s curve is not required of the test or EPA, data of questionable quality may be used to derive water quality criteria. 2) Analysis of acute toxicity test data typically also does not address the
intra

- treatment variability that commonly occurs in these te sts; providing results with biased estimates on uncertainty. 3) The Guidelines also do not require replication of any test result for the most sensitive species. This would allow a single test result to significantly bias the calculation of water quality criteria.James Pletl

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ASTM E2455

- 06 for testing freshwater mussels recommends that “different batches of the same species and the same life stage should be collected and tested over time in order to obtain a measure of the variability associated with testing the par ticular species”. This goal was not accomplished for the pink mucket, the third most acutely sensitive genus of the database.

Aside from universal issues for acute tests 4) there is also concern that the acute juvenile mussel tests may be biased toward s findings of more sensitivity and toxicity. The tests proposed to be used for this reassessment are conducted without sediment in the test vessels. Newton and Bartsch (2007) found that without sediment juvenile organisms did not grow in 96 hour toxicity tests. This clearly shows that the organisms are stressed by the lack of sediment in the test vessels. This also strongly suggests that the survival endpoints from acute tests may be biased low (falsely indicating greater toxicity) due to the
added stre ss of failing to provide sediment in the test vessels. All of these issues characterize a significant level of uncertainty which is indirectly proportional to scientific defensibility. EPA should address these uncertainties before using new data to updat e this criteria document.

I am not aware of other relevant data that were not used for this reassessment.

2. What are the technical considerations that EPA should evaluate when mussels are present and mussels are absent with respect to the recommended a cute criteria?
Generally, water quality criteria are designed to protect populations. The first step in determining whether mussels are present or absent is to define a standard for the conditions describing that a population exists at a site. This is n ot a straight

- forward exercise and can be quite difficult as seen in
various site

- specific criteria projects around the country. There must be guidance as to what parameters and measures of those parameters are to be used to define presence/absence of a p opulation. Quantitative evidence of reproduction would be required at a minimum, as well as the presence of various developmental stages. The normal abiotic conditions documented for a species should also be compared to that of a particular surface water to determine whether a conclusion of presence or absence is supported. Certainly the availability of host species could influence the determination of absence/presence.

Once absence/presence is established EPA should only use mussel data to develop wa ter quality criteria for waters where mussels are deemed present. EPA should ensure that conditions of the site where absence has been concluded have not been anthropogenically changed to a state where mussels cannot reproduce. Historic records of water quality and biological surveys would help support this determination. It is clear that EPA and the states will share the burden of the presence/absenceJames Pletl

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- 75 determination and that there will be differences of opinion as to whether, when absence seems apparent, conditions changed due to human activity that could have been reasonably precluded or the species simply never successfully established a population at the site due to species

- specific factors.

EPA should also determine whether simply the presence of m ussels is important or the presence of specific species is important. Given that the sensitivity of mussel species in acute tests spans a six

- fold concentration range the presence of only certain species could have a similar effect on the resulting water quality criteria; which then could have significant impacts on water quality management decisions. Further, different species have different life cycle durations. If individual species were used to develop criteria rather than the presence/absence of a t axonomic group the differences in life cycles may also impact criteria implementation.

3. Is it scientifically defensible to exclude the glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Do you believe there is an alternative approach to the use of this data that would be more scientifically sound?
The alternative question would be: Is it scientifically defensible to include glochidia data at this time due to the uncertainty of appropriate test duration time for this life stage? Again, EPA has not defined standards in the Guidelines for this determination other than requiring acute tests be at least 48 hours in duration and test organisms not be fed (except for mysids). These requirements were not developed to address the duration of the lifestage unique to glochidia. Toxicity data for glochidia appear to show that duration of tests with this lifestage significantly affect the test results when tests are 24 hours in duration or longer. Theref ore it seems that the glochidia are stressed in some fashion when tests meet the 48 hour requirement of the Guidelines. The test design for water quality criteria development is intended to measure stress due to the treatment (ammonia) independent of all other stressors. Tests of 48 hour duration and more fail to meet this goal. Since tests of duration less than 48 hours do not meet the Guidelines’ requirements but tests of duration equal to or greater than 48 hours provide biased results it
seems that a new standard for test duration unique to these mussels must be first developed. However the literature indicates a great deal of diversity among freshwater mussels in terms of their glochidia ecology.
The extent of this issue demands either more detail in this criteria document or more opportunities for
site

- specific ammonia criteria implemented by the States for only the mussel species present. The States will also require guidance on how to develop site

- specific ammonia criteria based on the issues un ique to glochidia.

To support the scientific defensibility of EPA’s decision it would be appropriate to first develop guidance for the appropriate test duration to be used for developing water quality criteria relative to theJames Pletl

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duration of the life stage. Currently acute test duration commonly is within an order of magnitude of life stage duration. To accomplish this goal for glochidia a test duration of 12 hours or less may be necessary. Such a guideline would help defend the scientific defensibility o f using glochidia data for developing water quality criteria.

One should conclude that EPA’s decision to not use glochidia data is logical and correct without more information. There is a great deal of uncertainty associated with test results using gloc hidia. The question that must be answered is whether the level of uncertainty realized by using glochidia data is so great that it compromises the integrity of the resulting water quality criteria. This, in fact, seems the case; however
it should be stre ssed that scientific defensibility should not be based on judgment but minimum standards of quality. Without these standards of quality the magnitude of uncertainty is unknown and probability of
erroneous actions is elevated.

4. Regarding the proposed ap proach to glochidia data in the 2009 draft position statement as it relates to ecological relevance and practicality

- Is the approach a scientifically defensible principle for structuring the population exposure duration problem and designing further rese arch to quantify such a duration?
As discussed earlier the uncertainties of using juvenile mussel data for deriving water quality criteria are significant, but EPA has identified additional concerns associated with tests of glochidia. The sensitivity of glochidia as a function of exposure duration, within the constraints of the Guidelines, precludes the use of glochidia data to derive water quality criteria. This is primarily due to the relatively short duration of instream exposure that can occur with this lifestage and the sensitivity of glochidia to that duration of exposure. The issue of exposure duration instream versus that of lab toxicity tests is critical to the use of all toxicity data in deriving water quality criteria and is not unique to fre shwater mussels and their glochidia lifestage. Decapod crustacean larvae are sensitive to duration of test exposure without food for durations required by the Guidelines. The literature refers to the “Point of No Return” or the PNR (Klaus Anger, various papers), where larvae will not survive beyond a certain time period without food even if food is provided to the larvae. Toxicity tests of duration approaching the PNR are likely stressing the test organisms in addition to the toxicant in question. The s ame can be said of glochidia tests with durations of 48 hours and more and likely durations even greater than 24 hours.

Development of criteria must consider all test factors that might bias test results in comparison to responses that are expected instre am, and exposure constitutes one of the most important factors. Exposure is a function of frequency, magnitude and duration. The default frequency assumption for water quality criteria toxicity tests is that exposure is continuous. Magnitude of exposure is usually dependant on concentration, which may also be a function of other factors like pH, alkalinity, DOC, etc.James Pletl

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- 77 The magnitude of exposure for glochidia will also be affected by the species

- specific conditions unique to glochidia of different species. For example, some glochidia are released into the water column singly; some are released in groups encased in a gelatinous shell where exposure would be mitigated. Duration of test exposure should be related to the duration of the lifestage, unless it c an be demonstrated that a different test duration is representative of that lifestage. This is a fundamental of the Guidelines although it is not clearly delineated. In the case of glochidia 48 hour or longer test durations have been shown to
result in r esponses not expected instream. This is a defensible reason to reconsider use of the acute glochidia data to derive water quality criteria and defines well the research needs for this taxonomic group and lifestage. Shorter duration tests will be necessar y to provide reliable test results for deriving water quality criteria.

Hyalella azteca position statement and proposed rationale (see Appendix B):

5. Are the position statement and supporting rationale regarding use of toxicity data for Hyalella azteca in criteria development reasonable and scientifically sound recommendations?
The position statement is written to address the use of H. azteca data for the ammonia update; however the issues presented would likely be important for many other wat er quality criteria using data for this species. Therefore the position statement is not reasonable and scientifically sound if H. azteca data are considered within the context of all water quality criteria calculations. There is a concern that
decisions such as this do not have associated rules as normally defined in the Guidelines. EPA is being reasonable and responsible in considering factors that may bias the development of water quality criteria in this case, but it is unclear whether EPA has a proc ess to identify such factors as new information is collected over time. Perhaps there was insufficient data to identify the H. azteca issues when data for this species was considered for use, but EPA needs to develop and implement a system to periodically review data for issues such as this and publish the system as part of the Guidelines.

Based on this comment the position statement may be reasonable but the scientific basis of the recommendation is questionable without a well defined system of review. Such a system should include review of data relative to standards of quantity and quality required to make such an assessment. EPA needs to determine how much information is necessary to make this type of recommendation as well as the qualitative nature of the data (measured vs nominal concentrations, static vs flow through tests, etc.) Without a system it will become increasingly difficult to be consistent in making these types of decisions
as more variances arise.James Pletl

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Chronic Freshwater Criterion:

6. Are the toxicity tests and other studies used to derive the criterion scientifically defensible for such use? Are you aware of other relevant data that were not used?

Using the standards established by the Guidelines and the re spective toxicity test methods the toxicity tests used to derive the chronic criterion are scientifically defensible. However, this does not mean that the data generated from these tests and the resulting test endpoints are scientifically defensible
for t he use. As stated before, there are standards that should be met to establish that the test results are of sufficient quality to be used in deriving water quality criteria. These standards include an appropriate concentration response curve and limits on variability within and between test treatments, replication of tests and use of reference toxicant tests to gauge organism sensitivity, as well as defensible control response across tests. One of the shortcomings of the chronic freshwater mussel method i s that it does not include minimum organism size (weight, length) requirements that must be met at the end of the test in controls. It is unknown whether the growth expressed in the controls of these tests is acceptable in a
natural environment where stre ss does not exist. The tests only can establish weights attainable in controls in the lab environment when organisms in the controls meet the minimum survival requirement. The organisms may be stressed in all test vessels, resulting in a bias of the test endpoint. The combination of treatment stressors (ammonia and vessels without sediment, for example) may produce a synergistic effect resulting in a lower ammonia IC20 for growth and survival than that resulting from a test which has only one stressor (a mmonia). In this case, the control response cannot account for the synergism, even though the control is intended to account for all factors independent of the tested

treatment.

I am not aware of any other relevant data that was not used.

7. Is the fre shwater chronic criterion scientifically defensible with mussels present and mussels absent?
As stated in question #2, EPA should determine whether simply the presence of mussels is important or the presence of specific species is important. The first step in determining whether mussels are present or absent is to define a standard for the conditions describing that a population exists at a site. This is not a straight

- forward exercise and can be quite difficult as seen in various site

- specific criteri a projects around the country. Guidance must be provided as to what parameters and measures of those parameters are to be used to define presence/absence of a population. Quantitative evidence of reproduction would be required at a minimum, as well as th e presence of various developmental stages. The normal abiotic conditions documented for a species should also be compared to that of a particularJames Pletl

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- 79 surface water to determine whether a conclusion of presence or absence is supported. Certainly the
availabi lity of host species could influence the determination of absence/presence.

Once absence/presence is established EPA should only use mussel data to develop water quality criteria for waters where mussels are deemed present. EPA should ensure that condi tions of the site where absence has been concluded have not been anthropogenically changed to a state where mussels cannot reproduce and biological records have documented the presence of previous populations. Historic records of water quality and biologi cal surveys would help support this determination. It is clear that EPA and the states will share the burden of the presence/absence determination and that there will be differences of opinion as to whether, when absence seems apparent, conditions changed due to human activity that could have been reasonably precluded or the species simply never successfully established a population at the site due to species

- specific factors. Given that the chronic sensitivity of mussel species drives the CCC approximate ly 44% lower than when mussel data is not used the management actions and costs based on criteria without mussel data could be significantly different than when mussel data is used to develop the CCC.

The presence/absence of species is usually a question that is asked when criteria are recalculated
or a site

- specific criterion is being developed. In these cases the issue of removing species from a database is addressed by determining whether data for other species with similar taxonomy is available.
The taxonomy and reproductive cycle of the freshwater mussels is so unique that this will not possible. Therefore a decision must be made as to whether to apply a CCC with or without mussel data. Given that
there is so little chronic data available for these mussels and that there are outstanding issues that must still be addressed (use of shell measurements to represent growth, tests without sediment, irregular concentration response curves, etc.) a CCC with mussel data will result in water quality standards with uncertain consequences. This uncertainty demands that criteria without mussel data be available for states to use when mussels are not resident (reproducing population found year to year). At this point in
time a CCC without mussel data would be mo re scientifically defensible than one with mussel data.

Use of 28

- day Juvenile Test Data (see Appendix C):

8. Given that the juvenile life stage of freshwater mussels is relatively long (2

- 6 years) are 28

- day exposure tests with juvenile mussels scienti fically defensible as "chronic" test data for criteria development?
This question is not only a function of the duration of exposure in the test but whether the test meets the requirements of the Guidelines. Clearly the test does not meet the Guideli nes requirementsJames Pletl

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because it does not address either the entire life cycle or, for partial life

- cycle tests, the appropriate life stages and test endpoints defined by the Guidelines. The reliability of this test is also clouded by the apparent impact of te sting juveniles in the absence of sediment and the use of shell length as a chronic test endpoint for freshwater mussels (see comments below). Perhaps the Guidelines require updating (has not been formally revised since its first release in 1985), but unt il this is done the 28

- day juvenile test does not meet the standard for scientific defensibility that EPA has established.

One might suggest using the data from these tests as an estimate of freshwater mussel chronic toxicity because the tests did not in clude the glochidia life stage and it is more appropriate than not using the data for criteria development. Unfortunately there is enough concern regarding the reliability of this test even to accurately represent the sensitivity of juveniles that it woul d not be appropriate to use the data in any water quality criteria capacity.

Independent of the issues raised above, it would seem that a 28

- day exposure test with juvenile mussels would be representative of the sensitivity of juveniles. However, chroni c tests used for developing a CCC are intended to evaluate or extrapolate the sensitivity of a population to a stressor. This requires reliable knowledge of the sensitivity of the most sensitive life stages of that population. It is
unknown how the sensi tivity of glochidia compares to juveniles because of the issues associated with both acute and chronic test methods. Without this knowledge and reasonable certainty it cannot be
determined that the 28

- day juvenile test with freshwater mussels is scientifi cally defensible relative to development of a CCC.

9. Should toxicity studies on the growth rates of mussel shells during 28

- day tests be considered quantitatively when developing water quality criteria?

Review of Wang et al. (2007) for both the copper and ammonia 28

- day tests shows that there is a very small difference between the length of the shells at the beginning and at the end of the test based on control responses. In fact it appears that some of the tests show no statistical difference i n shell length between the beginning and end of a test at alpha=0.05 and assuming a normal distribution. This does not support the use of shell length to represent growth in the 28

- day mussel tests. Further this paper shows that using shell length to rep resent growth does not provide reliable dose response curves in most cases. Although the authors only conducted 21

- day tests both Bringolf et al. (2007a) and Bringolf et al. (2007b) found that the concentration response curves for different chemicals were not what one would expect for a toxicant. Although there may be issues with organisms benefitting from certain concentrations of chemicals, this fact only complicates the interpretation of this data and increases the uncertainty of using toxicity test re sults based on shell length.James Pletl

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- 81 10. Regarding the position statement and rationale on use of juvenile mussel growth data

–
 Is it scientifically defensible to include the juvenile growth data from a 28

- day exposure period as “other data” that might influence the criteria however not be used directly in the derivation of the criteria value?
Based on the uncertainty associated with testing juveniles without sediment and the lack of reliability in the shell length endpoint it is not appropriate to use the chronic data in any way associated with development of a CCC.

 Should the statement also consider impaired growth of mussels which were affected at a 28

- day exposure could as likely continue to decline in longer exposures as another potential outcome (i.e., the chance they could recover or stabilize is one potential outcome only)?
The comments provided above on the reliability of the shell length endpoint for chronic tests of freshwater mussels indicate that it is not a reliable endpoint to represent the chronic sensitivity of these organisms. Without more data, it is not defensible to speculate on the outcome of tests with lon ger duration when the reliability of the endpoint being used is in question.

11. The values of the acute and chronic ammonia criteria have a strong dependence on pH. Juvenile and adult mussels, as sediment

- dwelling organisms, inhabit a medium that may ha ve vertical pH gradients, thereby creating some uncertainty about the appropriate pH to assign as their exposure conditions. For applying a criterion protecting mussels, do you have suggestions on how states and EPA might determine the pH applicable to th e sediment micro

- environment to which mussels are typically exposed?
It will be necessary for states and EPA to directly measure the pH of the environment where each life stage exists. For juvenile and adult mussels this will require measurements of the sediment in each location where the criteria are applied as water quality standards. Guidance from EPA on how to appropriately represent the pH of sediments where ammonia criteria are applied to protect freshwater mussels will be necessary. Measurements in the field will also be necessary because the pH of a discharge regulated by a NPDES permit cannot be used to estimate the pH of the sediment. The pH of the sediment can only be assessed through direct measurement. This may be done in situ or by rapid ly measuring the pH of relevant sediments collected with sediment grab or core equipment.

12. In general, should the criteria include a consideration for the potential pH difference between sediment and the water? If so, what is the most scientifically d efensible way to account for these differences when deriving protective water quality criteria?
The freshwater ammonia criteria must appropriately address the pH of the juvenile freshwater mussel environment to be reliable. Although the criteria must be protective they must also be representative of aquatic life sensitivities and implemented in a way that does not overstate thoseJames Pletl

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sensitivities. Being protective is straight

- forward using conservative assumptions but criteria development and implementatio n already have multiple layers of conservatism. These layers include the return frequency (once in 3 years) of the criteria, the assumption that exposure is continuous instream for a time period equivalent to the duration of the tests, an acute criterion averaging period of one hour, the
use of the 90 th percentile of temperature and pH to apply the ammonia criteria, the reasonable potential process of EPA’s Technical Support Document for Water Quality

- based Toxics Control which relies on a very high percen tile (95 th or 99 th ) of the distribution of the highest concentration measured for an effluent to issue permit limits, the 303(d) listing process which is dependent on the vast minority of data exceeding the criteria to conclude that a water body is not in compliance with standards, etc. The conservatism built into criteria development and implementation demands that pH instream be accurately assessed and applied to the criteria to reliably determine compliance with water quality standards.

EPA has not a dopted data quality objectives to limit the amount of uncertainty that can exist when applying water quality criteria instream, therefore a consensus standard to determine if water based data can be used to estimate sediment based responses is not availabl e and scientific defensibility cannot be assessed. However, there is an approach that will likely provide consensus agreement in scientific defensibility: to directly address the pH of the instream sediment within the context of a toxicity test
rather tha n trying to compensate for pH differences between water tests and the instream sediment. This approach will directly address concerns that juvenile test results are a function of the presence/absence of sediment and avoid the uncertainty of mechanisms att empting to extrapolate response from water only exposures. It is recognized that this recommendation would not allow the use of current data to derive ammonia water quality criteria inclusive of freshwater mussel sensitivities. It would ensure that the

u nique ecology of these mussels’ life stages is accurately captured (within the constraints of conventional testing methods) and that when the resulting criteria are implemented management actions will be
reliable.

13. Should exposure tests on juvenile mussels be conducted with or without sediment in the test chamber?
It is clear from Newton and Bartsch (2007) that the potential for juvenile freshwater mussels to be stressed in the absence of sediment exists. Wang e t al. (2007) found that sediment did not appear to impact shell growth and may have slightly reduced survival, but only one test using sediment was conducted and this test only consisted of two replicates per treatment. As stated previously toxicity tests must represent the stress experienced by a single stressor to be used in developing water quality criteria. Therefore for this particular use, until more information is available, juvenile tests of any duration must
include sediment in the test vessels i n order to be used in deriving water quality criteria.James Pletl

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- 83 LITERATURE CITED

1. R.B Bringolf, W.G. Cope, S. Mosher, M.C. Barnhart, D. Shea. 2007a. Acute and Chronic Toxicity of Glyphosate Compounds to Glochidia and Juveniles of Lampsilis siliquoidea (Unionidae). Env. Tox. Chem.26(10), p.2094

2. R.B Bringolf, W.G. Cope, M.C. Barnhart, S. Mosher, M.C. Barnhart, P. Lazaro, D. Shea. 2007b. Acute and Chronic Toxicity of Pesticide Formulations (Atrazine, Choropyrifos and Permethrin) to Glochidia and Juveni les of Lampsilis siliquoidea (Unionidae). Env. Tox. Chem.26(10), p.2101

3. T.J Newton and M.R. Bartsch. 2007. Lethal and Sublethal Effects of Ammonia to Juvenile Lampsilis Mussels (Unionidae) in Sediment and Water Only Exposures. Env. Tox. Chem. (26(10), p.2057

4. N. Wang, C.G. Ingersoll, I.E. Greer, D.K. Hardesty, C.D. Ivey, J.L. Kunz, W.G. Brumbaugh, F.J. Dwyer, A.D. Roberts, T. Augspurger, C.M. Kane, R.J. Neves, M.C. Barnhart. 2007. Chronic Toxicity of Copper and Ammonia to Juvenile Freshwater Mussels ( Unionidae). Env. Tox. Chem. 26(10), p. 2048