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

September 21, 2009

Printed on Recycled Paper

QUALITY 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 each 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 data 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 scientifically 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 defensible as "chronic" test data for 

criteria development?................................................................................................................. B-29

9. Should toxicity studies on the growth rates of mussel shells during 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 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?.................................................... B-39
ii

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?......................... 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

William L. Goodfellow Jr., MS .................................................................................................C-63

James J. Pletl, Ph.D....................................................................................................................C-71
A-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 Act.

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 the 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 criteria 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 scientifically 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 

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 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? 

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 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? 

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 chance 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 

these 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-1

SECTION B

Peer Review Comments Organized by Charge Question

B-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 deriving 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 reevaluation:

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. 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 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 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 evaluation 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 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 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 literature 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 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 follow 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 and 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 
B-5

test organisms and culturing (either reported or unreported) and lack of reference 

testing 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. 

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 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.

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 quality 

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 and/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 National 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; however 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 tests 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 Guidelines 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 this 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 intratreatment variability that commonly occurs in these tests; 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 
B-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 particular 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 towards 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 stress 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 update 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 however. 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 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 % 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 precedents 

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 fact 

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 specifically targets this issue. Most monitoring techniques for collecting 

benthic macroinvertebrates 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 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 survey 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. 

Steven Canton 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 reference 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 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 greater 

than the natural occurrence of decomposition due to heterotrophic bacteria?

 What is the within-site spatial and temporal variability in NH3 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 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, 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 halogenated 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 quality 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 be 
B-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 site 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 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 aquatic 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 would be given to a 

method that 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” 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 considerations coupled with only a recent attempt to describe 

how certain stream characteristics may serve to structure mussel communities, 

will need 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 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 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 number 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 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. 
B-10

Relative use of reference sites in determining relationships between areas of reduced 

freshwater 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 considerations 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 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 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 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 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 

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. 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 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 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 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 taxonomic group the differences in life 

cycles may also impact criteria implementation. 
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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 seems 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 difficult 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 values 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 data 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 tests 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 hour 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 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 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 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 different 

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 
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criteria development purposes.

 Second, concerning 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 when 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 

dramatically 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, there 

appear to be two 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 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 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 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 broadcast 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 (even 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 today (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-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 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 and 
B-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 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 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 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 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 test 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 and 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 better 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 

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 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 abundance. 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 model 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, 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 

Jerry Farris Information from tests with glochidia can be supportive of the consideration for site 
B-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 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. Therefore 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 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 glochidia 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 logical 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 compromises 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 quality the magnitude of 

uncertainty is unknown and probability of erroneous actions is elevated.
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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?

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 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 important 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 avoid continued uncertainty. Additional information as discussed in appendix 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 

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 it is a difficult to task to structure a criterion that includes 

population 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 conglomerates, 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 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 Diamond 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 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 

duration 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 species (e.g., Dodd et al. 2006). In other words, the number of glochidia that 
B-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 implications for further 

research 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 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 would 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 relevant 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 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 freshwater 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 same 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 is 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 speciesspecific 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 responses not 
B-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 

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 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 

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.

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 recommended 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 toxicity 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. Thus, 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 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.

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 chloride 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 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.

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 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 process 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.
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 toxicity of ammonia to aquatic organisms. The use 

of EC20 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, 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 

document), 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 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 serious 

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 Musculium 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 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 temperature 

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 fish 

species), to incorporate temperature dependence into the chronic equations. There are 

three problems with incorporating temperature in the chronic relationship, but not 
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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 rationale 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 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 

concentration 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 that 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 abundance 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.

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 Ozark 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 reason. 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 the 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 aware 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 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 
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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 

these 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 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 

(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 

early 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 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 

reasonable. 

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 have 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 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 requests 

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 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)

Steven 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 historically. Guidance on such data needs questions 

would be useful to include in the criteria document to support a with/without mussel 
B-26

criterion. 

Additionally, even when the “mussel present” option is applied, a whole other series 

of issues arise, because there is often a poor correlation between the water column 

NH3 concentration and the pore water NH3 concentration where most juvenile mussels 

reside. Site-specific sediment conditions can greatly influence the availability of NH3, 

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 NH3, or other confounding affects on mussel 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 question 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 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. 

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 mussels 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 

conditions describing that a population exists at a site. This is not a straight-forward 
B-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 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 

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 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 approximately 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 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. 

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. 

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 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 

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 exposure (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 stress 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 

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 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 

calculations.
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 collected 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 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 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 document 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 believe 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 EPA 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 conditions.

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 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 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 28-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 the 
B-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 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 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 than 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 effects 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 stage 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 

documentation. 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 meet 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 apparent 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 juveniles 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 with 
B-32

juvenile mussels would be representative of the sensitivity of juveniles. However, 

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 mussels 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 

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. 

Steven Canton 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 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 complicated, 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 standardization 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 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.
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 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 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 from 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 chance 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 magnitude 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 would 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 difference 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 for 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 chance they could recover 
B-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 addition, 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 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 meaningful. For this reason, I don’t think it 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 methodological 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 duration to a toxicant.

George Dixon With respect to the first point 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 question, 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 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. Studies that include recovery and or stability in 

growth or even degrowth with 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 potential outcome only)?

As stated, the above consideration seems very confusing and should be clarified 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 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 longer duration when the 

reliability of the endpoint being used is in question.

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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?

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

juveniles. The exposure 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. 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 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 water at depths where 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 2009 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 burial 

zone, per se. In addition, it is likely any measures of pH in the mussel microenvironment (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 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 sampling 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 
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factor affecting water 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 

criterion.

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 resembling 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.

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 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., sediment 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.

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

perspective is the pH of the respiratory 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 information 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 effect 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 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 
B-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 benefit 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 that 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 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 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 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 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 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 criteria 

can be adjusted to the pH in the interstitial 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 

typically 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, sitespecific variance procedures inclusive of sediment characteristics for a “mussels 

present “ 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 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 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 90th 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 percentile (95th or 

99th) 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 adopted 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 

available 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 than 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 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 

water-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 sensitivity 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 well as copper and chlorine. This lends 

confidence in these data for use in criteria development. Toxicity data from wateronly exposures are most relevant to the purposes that water quality criteria are most 

often employed; assessments of natural waters by 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 designed 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 more 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 “clean” 

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 

providing 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 
B-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 water-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 toxicity 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 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 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 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 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 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.
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 opportunity.

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 

mussel 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 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 juvenile mussels in an experimental setting. This will have to 

be determined experimentally. 

Jerry Farris 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 ensure 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 comparable 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 longer 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 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 complex 
B-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 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 should try to stay with water 

column only whenever 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 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 in order to be used in deriving 

water quality criteria.
B-49

Additional Comments

Steven Canton

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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.

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 macroinvertebrates. 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 commercially 
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valuable species (e.g., trout) but not an ecologically critical species? Point #3: I agree 

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 

absorption 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 

difference....”

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 temperature correction for 

invertebrates in the Reassessment Document, Daphnia couldn’t possibly go from 

Rank 19 in acute sensitivity to 8th in the draft document. This error has other 

ramifications as well: assuming the Daphnia value is incorrect in the draft, 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 River 

sucker) becomes the 4th 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.

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, 

based 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, 

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 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. For example, for the snail species Potamopyrgus
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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, 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 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 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 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 Glochidia in Stream Drift on 
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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 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). 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 

Society 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.

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 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 Quality 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, 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 

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. 
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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. 

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.

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 find 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 scientifically 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. 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 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 

pore-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.
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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 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. 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 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, 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 Ecotoxicology. 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. 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.

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.
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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 relevant. 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 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.

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 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 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."

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:

In my opinion the Asiatic clam (an exotic) should not be included in the database used 
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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. 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 

Juveniles 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

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SECTION C

Individual Peer Reviewer Comments

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PEER REVIEW COMMENTS FROM

Alex Barron

Virginia Department 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.gov

Alex Barron

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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 relevant 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) and 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 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 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 % 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 

precedents 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 
Alex Barron

C-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 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 specifically targets this issue. Most monitoring 

techniques for collecting benthic macroinvertibrates 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 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 survey 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 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 seems 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 difficult 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.. 
Alex Barron

C-7

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 values 

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 data 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 tests 

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 hour 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.

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 

concerning 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 important 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 avoid continued 

uncertainty. Additional information as discussed in appendix B would be needed to provide sufficient 

confidence in utilizing glochidia data to derive water quality criteria. 
Alex Barron

C-8

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 derivation, 

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. 

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 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 

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 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 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

C-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 EC20 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. 

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 showing 

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 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 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 reasonable. 

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 have 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 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 requests for consideration of developing site-specific 

criteria based on the recalculation procedure, which can require significant resources to pursue. 
Alex Barron

C-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 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 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 impacts. 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

C-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 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 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 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 magnitude 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.

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

C-12

Review Comment:

I would 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 difference 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. 

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 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?

Review Comment:

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

exposure 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. 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 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 water at depths where mussels are 

likely to be found at the site.
Alex Barron

C-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 where 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 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 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 criteria 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. 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 water-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 sensitivity 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 
Alex Barron

C-14

of 10. Water-only toxicity tests methods have shown good intra and inter laboratory variability for 

toxicity tests with ammonia, as well 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 by 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 designed 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 more 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 “clean” 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 providing 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 the 

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 quantitative 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 ammonia 
Alex Barron

C-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 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. 

C-17

PEER REVIEW 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.com

Steven Canton

C-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. 

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, 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. 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 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 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 evaluation to determine if they provide toxicity data 

meeting EPA data quality guidelines (Stephan et al. 1985). 
Steven Canton

C-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 reference 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 indepth technical 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 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 greater than the natural 

occurrence of decomposition due to heterotrophic bacteria?

 What is the within-site spatial and temporal variability in NH3 of the pore water and is this 

variability linked to season, storm events, temperature, or flow conditions.
Steven Canton

C-21

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

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?

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 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 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 different 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, concerning 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 when 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 dramatically 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, there appear to be two 

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 would 
Steven Canton

C-22

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 and 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 

exposure 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 “nonprotective” 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 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 conglomerates, 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 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.

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 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 recommended removal of Hyalella from the ammonia 

database, primarily because of the poor control performance (AWWQRP, 2006), which it now appears 
Steven Canton

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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 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 sitespecific 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 established 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 document), 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 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 serious 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 Musculium 
Steven Canton

C-24

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 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 reanalyze 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 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 fish 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 invertebrate 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 that 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 concentration 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 that 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 abundance 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 mussels 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 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 
Steven Canton

C-25

mussels have ever occurred within the site historically. Guidance 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 often a poor correlation between the water column NH3 concentration and the pore water 

NH3 concentration where most juvenile mussels reside. Site-specific sediment conditions can greatly 

influence the availability of NH3, 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 NH3, or other confounding affects on mussel 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 question 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. 

Addressing the “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 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 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 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 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 exposure (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 stress 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 
Steven Canton

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(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 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 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 calculations.

9. Should toxicity 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 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 complicated, and 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 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 for the reasons outlined in 

the previous two questions. 
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 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 longterm 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 addition, 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.

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?

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 2009 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 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 

differences 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 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 
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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 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 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 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 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 provide. 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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Arid West Water Quality Research Project (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., Pima 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. 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.

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 Salmo 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.

Dankers 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 together, 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 Responsible 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 
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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-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 certain 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 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.com

Jerome Diamond

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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). 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.

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, 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 halogenated 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 quality 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 
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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 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 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 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 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 broadcast 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 

(even 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 today (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-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 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 
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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 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 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 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 test 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 and 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 better 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 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 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 abundance. This would not be unlike the larval 

recruitment model EPA has developed for dissolved oxygen with the exception that the abundance of 
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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 malacologist 

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 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 duration 

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 species (e.g., 

Dodd et al. 2006). In other words, the number of glochidia 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 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.

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 agree 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 absorption 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 difference....”
Jerome Diamond

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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 

temperature correction for invertebrates in the Reassessment Document, Daphnia couldn’t possibly go 

from Rank 19 in acute sensitivity to 8th in the draft document. This error has other ramifications as well: 

assuming the Daphnia value is incorrect in the draft, 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 River 

sucker) becomes the 4th 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.

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, based 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, 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 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. For 
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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 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, 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 acute 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 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 derivation, 

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? 

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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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?

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 Ozark 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 

reason. 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 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?

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.

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 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 
Jerome 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 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 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 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 

document 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 

believe 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 EPA 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 conditions.

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 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 dealing with now for Hyalella.
Jerome Diamond

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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 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 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 

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.

9. Should toxicity studies on the growth rates of mussel shells during 28-day tests be considered 

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 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.

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 with 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 meaningful. For this reason, I don’t think it 
Jerome 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 methodological 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 duration 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 sampling 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 water 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 criterion.

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 resembling 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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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 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., sediment 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 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?

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. 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 mussel 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.
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 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 Glochidia in Stream Drift on 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 unionized 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 

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). 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 Society 26(2):261–272.
Jerome Diamond

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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 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 Quality 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, 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 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. 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. 

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.

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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.ca

George Dixon

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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 document. 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 questions 

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 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 literature closely, and I am unaware of any additional 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 determining 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 aquatic 

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 would 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 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?

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 the 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 research 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 will 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 derivation, 

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 

toxicity. 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 use? 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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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, 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? 

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 than 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. 

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 nonlife-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 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 

consistent with the practice for short-term growth results with other species, and I see no reason why 
George 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 uncertainty 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-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?

While mussels live in sediment, the pH of importance from an ammonia criteria perspective is the pH of 

the respiratory 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. 

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 sediment 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 

juvenile 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.edu

Jerry Farris

C-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 

feasibility 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 follow 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 and 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 testing 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 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” 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 considerations 
Jerry Farris

C-56

coupled with only a recent attempt to describe how certain stream characteristics may 

serve to structure mussel communities, will need 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 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 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 number 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 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 

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 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 there 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 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. 
Jerry Farris

C-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 and 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 as 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 composition. 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 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? 

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 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?

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? 

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 
Jerry Farris

C-58

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 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? 

Research that validates effective endpoints related to the range of pollutant effects 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 stage 

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. 

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. Studies that include recovery and or stability in growth or even degrowth with 
Jerry Farris

C-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 potential 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. 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?

Even if an adequate amount of information 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 effect 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 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 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. 
Jerry Farris

C-60

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?

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 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 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). 

13. 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 ensure 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 

comparable 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 longer 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 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 complex 

interactions between mussel, water, and sediment that influence their responses has been cited to 

reinforce those considerations (Thorsen et al., 2007). 
Jerry Farris

C-61

References:

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 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 pore-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.

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 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. 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 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, 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 Ecotoxicology. SETAC, 

Pensacola, FL, USA, pp 169-207. 
Jerry Farris

C-62

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.com

William Goodfellow

C-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 AmmoniaFreshwater.

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 to 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 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 relevant. 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 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

C-66

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 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 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 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 

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 everyone.
William Goodfellow

C-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 scientifically 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 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 derivation, 

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? 

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

C-68

7. Is the freshwater chronic criterion scientifically defensible 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 

the 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, 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? 

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 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 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.

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 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. 
William Goodfellow

C-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 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?

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 that 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.

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?

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 the 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 should try to stay with water 

column only whenever possible.
William Goodfellow

C-70

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 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 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."

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:

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.com

James Pletl

C-73

EPA’s Draft Reassessment of the 1999 Ambient Water Quality Criteria for Ammonia – 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 quality 

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 and/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 National 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; however 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 tests 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 Guidelines 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 this 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 tests; 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. 
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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 particular 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 towards 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 stress 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 update 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 acute 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 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 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 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 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. 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 
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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 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 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 taxonomic 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. Therefore 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 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 
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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 of 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 glochidia. 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 stressed 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 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?

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 freshwater 

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 same 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 is usually 

dependant on concentration, which may also be a function of other factors like pH, alkalinity, DOC, etc. 
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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 responses 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 necessary 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 water 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 process 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.
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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 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 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 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 these 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 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 (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.

7. Is the freshwater 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 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 species should also be compared to that of a particular 
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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 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 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 approximately 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 more 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 scientifically 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 Guidelines requirements 
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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 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 juveniles 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 with juvenile 

mussels would be representative of the sensitivity of juveniles. However, 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 mussels is scientifically 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 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 

from 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.
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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 longer 

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 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?

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 rapidly 

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 defensible 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 those 
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sensitivities. Being protective is straight-forward using conservative assumptions but criteria 

development and implementation 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 90th 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 percentile (95th or 99th) 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 adopted 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 available 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 than 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 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. 

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 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 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 in order to be used in deriving water quality criteria.
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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 Juveniles 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