Background
==========

One of the strategic objectives of the WHO is the eradication of poliomyelitis in all regions of the world within the next years. According to the WHO plans the transmission of wild poliovirus has to be interrupted worldwide by the end of 2002. The global certification will be held in 2005.

Although the European region is actually polio -- free, it remains subject to the risk of importation from endemic regions. The most recent cases of poliomyelitis due to an importation of wild polio type 1 occurred in unvaccinated Bulgarian children \[[@B1]\]. To prevent such importations it is necessary to keep a high level of population immunity.

The last indigenous case of poliomyelitis was diagnosed in Germany in 1990. The last imported wild viruses were detected in patients with travel history to India and Egypt in 1992. Since that period of time there were found only 0 -- 3 cases of vaccine-associated paralytic poliomyelitis (VAPP) per year. In order to prevent these VAPP -- cases the vaccination strategy has been changed in Germany. Since 1998 it is recommended to use only the inactivated vaccine (IPV).

The neutralizing antibody test is the method of choice in conducting serological surveys to identify epidemiologically important immunity gaps in the population. It is considered essential to provide the precise procedure for carrying out polio neutralizing antibody tests. Use of standard reagents, the International Standard Serum (ISS), standard Sabin strains, and expression of the results in International Units (IU) is a feature aimed at obtaining consistent results. This will assure that comparison between studies can be made with confidence. An International Standard Serum does exist, but WHO recommendations do not indicate a protective level in IU \[[@B2],[@B3]\].

Materials and methods
=====================

Serum samples
-------------

The first German Health Survey 1997/98 was a representative study of the health status of the population in unified Germany. In this project which has been carried out by the Robert Koch Institute on behalf of the Ministry of Health about 7200 study participants aged between 18 and 79 years were going through a medical check-up and interviewed as to health -- relevant issues. Germans as well as foreigners, who lived in Germany, were included in the survey. As a result of various selection criteria (age, sex, and size of town) a representative collection of study participants was obtained \[[@B4]\].

A representative panel of 2564 serum samples from the total amount of sera from the Health Survey was investigated for the presence of antibodies to all three poliovirus serotypes.

Additionally, 881 sera from children less than 18 years obtained from six large laboratories in Germany were included in this study. These laboratories performed wide-range diagnostics without any visible bias within the group of patients. The sera were collected in 1996/97 and stored at -20°C until testing.

Neutralization test
-------------------

The microneutralization test using 100 ID~50~ of challenge virus (Sabin strains) was performed according to the WHO-guidelines \[[@B5]\]\[[@B6]\]. An In-House Reference serum (IHR) of known neutralizing activity was included in each test to control reproducibility of results. Each test serum was investigated in duplicate. If the final serum dilutions of these duplicates differed more than one stage the test was rejected.

The International Standard Serum (ISS, a preparation of pooled human sera) containing 25 IU, 50 IU, and 5 IU for Poliovirus 1, 2 and 3, respectively has been used to calibrate the potency of the IHR. ISS and IHR were titrated in parallel on ten separate occasions using eight replicates per serum dilution (starting from serum dilution of 1/4).

Calculation of the antibody concentration in International Units (IU/ml)
------------------------------------------------------------------------

The calculation of the concentration with

![](1471-2334-2-2-i1.gif)

resulted in C~IHR1~ = 3.125 IU/ml, C~IHR2~ = 6.25 IU/ml, C~IHR3~ = 0,625 IU/ml for Polio 1, 2, 3, respectively for the used IHR (example for Polio 1: a serum dilution Val~ISS~ = 1/256, and Val~IHR~ = 1/32 would result in C~IHR~ = 3.125 IU/ml).

The calculation of the antibody concentration in a given test serum sample was done analogously.

![](1471-2334-2-2-i2.gif)

In contrast to WHO -- recommendations not the geometric mean (GMT) of all measured IHR\'s was used but the concentration of the IHR in the given test only. Thus each test was calibrated in respect to the appropriate IHR.

A given test was only accepted if the final serum dilution did not exceed a one-stage range of the GMT of all tested and accepted IHR\'s. For an accepted test the following condition holds:

![](1471-2334-2-2-i3.gif)

The threshold values Co were calculated as follows:

![](1471-2334-2-2-i4.gif)

with

![](1471-2334-2-2-i5.gif)

*SDev(C)* = StandardDeviation{C, with serumdil. \< 1/4}.

Results
=======

In the past the cut-off value for \"sufficiently protected\" against poliomyelitis was set if the final serum dilution giving protection against 100 ID~50~ of challenge virus was greater or equal to 1/4 (titer 8). This condition should be substituted by expressing results in IU/ml.

The cut-off values (Co) for the protective level were calculated as follows:

for Polio 1: C~0~ = 0.080 IU/ml

for Polio 2: C~0~ = 0.180 IU/ml

for Polio 3: C~0~ = 0.075 IU/ml

Neutralizing antibodies against poliovirus type 1, 2 and 3 were detected in 96.2% (95% confidence interval CI: 95.5%-96.9%), 96.8% (CI: 96.1%-97.5%) and 89.6% (CI: 88.4%-90.8%) of samples, respectively (Fig. [1](#F1){ref-type="fig"}). 85% of all tested persons had antibodies against all three virus types. Only 0.3% of tested persons had no detectable antibodies against all polioviruses.

![Prevalence of antibodies to polio viruses in Germany, n = 3445](1471-2334-2-2-1){#F1}

Statistically significant regional differences in the immunity level between the adult populations in the East and West of Germany have been detected for Polio 3 (86,6% / 90,9%; p = 0,001).

Within the given data set the decision \"sufficiently protected\" slightly changes according to Tab. [1](#T1){ref-type="table"} (expressing results in IU vs. serumdilution). Thus for Polio 3 about 3% (82/2564) of the samples were additionally classified as \"not protected sufficiently \" against disease.

::: {#T1 .table-wrap}
::: {.caption}
######

Differences in numbers of samples classified as \"sufficiently protected\" (serumdilution vs. concentration C in IU/ml), n = 2564
:::

  **Polio 1**        C\<C~0~   C\>=C~0~
  ------------------ --------- ----------
  Serum dil.\< 1/4   90        11
  Serum dil.\>=1/4   12        2451
  **Polio 2**
  Serum dil.\< 1/4   97        17
  Serum dil.\>=1/4   1         2449
  **Polio 3**
  Serum dil.\< 1/4   182       0
  Serum dil.\>=1/4   82        2300
:::

Discussion / Conclusions
========================

The goal of eradication of poliomyelitis has been complicated by some events in 2000--2001. First, the discovery of circulation of a vaccine virus strain that mutated to regain its disease-causing ability in Hispaniola. This outbreak in the Dominican Republic and Haiti was the first in the Americas since 1991 and occurred in areas of very low vaccination coverage \[[@B7]\]. Second, the polio cases in Bulgaria. There had been no reported cases of polio in Europa since November 1998. In spring 2001 two gypsy unvaccinated children were paralyzed by a wild poliovirus in Bulgaria, which was imported from India \[[@B1]\]. These outbreaks send a warning to all countries to maintain their guard against polio, despite the rapid decline in its incidence. Sufficient surveillance systems and high levels of population immunity can prevent polio outbreaks.

The level of immunity to poliomyelitis has been checked in Germany at regular intervals.

For the first time in Germany the results were expressed in International Units. A representative serum panel from the German Health Survey was investigated. Additionally, sera from children less than 18 years old were included in this study. These sera were not representative for all selection criteria because they originated from diagnostic laboratories. A representative study of the health status of persons under an age of 18 years is prepared by the Robert Koch Institute. The pilot phase has started in 2001. Within the next years valid data will be obtained for the younger population of Germany.

The given threshold values of 0.075 lU/ml, 0.180 IU/ml, and 0.080 IU/ml were calculated for Polio 1,2,3 respectively. The calculation was based on the assumption that the new criteria (threshold in IU/ml) should give comparable results with respect to the old one (threshold of serum dilution).

Overall, neutralizing antibodies against poliovirus type 1, 2 and 3 were detected in 96.2%, 96.8% and 89.6% of samples, respectively. This seroprevalence indicates a very high level of immunity of the German population. Although the prevalence of antibodies against poliovirus was generally somewhat lower for type 3 than for types 1 and 2, it was still close to 90%.

Data presented in this study are concordant to those obtained in other countries \[[@B8],[@B9]\].

A previous seroepidemiological study in Germany as well as this one revealed substantial regional variations of immunity levels to Polio 3 \[[@B10]\]. Compared to the results of this study other German studies have shown remarkable lower antibody prevalence to Polio 3 among blood donors in Berlin and in hospitalized patients \[[@B11],[@B12]\].

Limitations in some of these serological studies due to non-representative samples or methodological reasons resulted in different conclusions regarding immunity status. Standardization of the neutralization test as well as expressing results of serological studies in International Units will permit better comparability of polio immune status of populations in different studies, and in different countries.

Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural infection and after vaccination. It is unclear at present whether all those persons with a low level or no detectable antibodies are susceptible to infection; some, particularly the elderly, may be protected by memory immunity, an accelerated antibody response because the immune system has been primed previously. It has also been shown that enterovirus infections induce T-cell immunity \[[@B13]\]. The production of local secretory IgA antibodies in the gut mucosa may play a major role in protection \[[@B14],[@B15]\].

Although IPV offers excellent protection against disease, it is less effective in preventing poliovirus infection because of limited mucosal immunity. IPV recipients may contribute to the circulation of outbreak virus. Extensive circulation of Polio 3 was observed during 1984 outbreak in Finland. A study from the Netherlands has also shown that poliovirus circulation occurred during the early phase of the 1992--93 poliomyelitis outbreak. Most infected children had not been vaccinated \[[@B16]\]. A high level of immunity of the German population, including immigrants, must be monitored after the switch from attenuated to inactivated polio vaccine (OPV to IPV).

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

The authors are grateful to Helga Schickhoff for her excellent technical assistance.


Background
==========

The most common localized tumor of the head and neck is the hemangioma, and more than 50% of all congenital hemangiomas are located on the head and neck \[[@B1]\]. The hemangioma may present in various forms, for example capillary, cavernous, or mixed, the most common being a capillary hemangioma. Most authors dispute whether this classification can be based on clinical appearance only. In fact, most of these tumors may actually have mixed capillary and cavernous hemangiomas, and are better known as Capillaro-Venous malformations.

These capillaro-venous malformations may affect not only the cutaneous surfaces of head and neck but also the mucosal surfaces.

Because these capillaro-venous malformations in the upper aero-digestive tract may cause difficulty in swallowing and/or breathing, and may even threaten the life if the patient aspirates while bleeding, they are best treated surgically \[[@B2]-[@B4]\].

As these malformations have a marked propensity for bleeding during excision, the value of preoperative embolization is well established. The objective of embolization is to devascularise these malformations by catheterization and embolization of each vessel supplying the tumor, which is not always achieved because most of them are fed, by many small vessels that may be too small for the catheter to pass. We present two cases where we have used a devascularization technique for these malformations by direct injection of *n-butyl cyanoacrylate* (NBCA) into the capillaro-venous bed.

Case Presentation
=================

Case 1
------

A 24 years male had presented with slowly increasing bluish swelling over palate since birth that had occasionally bled. He had a capillaro-venous malformation over oral surface of the soft palate; the nasopharyngeal surface of the soft palate was not involved. The capillaro-venous malformation was punctured with a 22-guage needle and progression of the needle was monitored by 2-D fluoroscopy. After verifying the correct location of the needle by reflux of blood at the hub, the contrast agent was injected into the capillaro-venous malformation, revealing the local parenchymography draining to the regional veins without extravasation. NBCA and lipiodal then were mixed in equal proportions and injected slowly under fluoroscopy control with a 2 ml syringe to completely occupy the entire capillary bed (fig 1). Subsequently the capillaro-venous malformation was totally resected in the same sitting. A good plane of cleavage was obtained because of retraction of the malformation induced by devascularization. Peroperative blood loss was negligible (\<10 ml). The resected specimen was subjected to histopathological examination which confirmed the nature of capillaro-venous malformation and demonstrated that the embolization agent occupied almost whole of the capillaro-venous bed.

Case 2
------

A 55 years old male presented with repeated episodes of bleeding from mouth especially after taking meals. On detailed examination left tonsillar hemangioma (capillaro-venous malformation) was diagnosed to be the cause of this bleed. This hemangioma was also devascularised with direct puncture technique under general anesthesia (fig 2) as described above and subsequently left tonsillectomy along with excision of the hemangioma was done. Histological confirmation of the capillaro-venous malformation was done. Peroperative blood loss was negligible (\<10 ml) and a good plane of cleavage was obtained.

Discussion
==========

Preoperatively devascularization of the capillaro-venous malformation to decrease peroperative blood loss is desired by the surgeons. The conventional intravascular embolization technique has following limitations- (1) the devascularization is rarely complete as it is often not possible to embolize each and every feeding vessel, as some of the feeding vessels may be too small to negotiate the catheter, (2) a very experienced interventional radiologist team is required to carry out embolization, (3) time consuming, (4) expensive, (5) difficult to carry out surgical excision of the malformation in the same sitting.

Whereas, the direct puncture technique has following advantages- (1) total devascularization is easily achieved, (2) does not require a team of intervention radiologists and can be carried out by the surgeon, (3) can be done immediately before surgery in the same sitting, (4) less time consuming, (5) relatively inexpensive.

Although direct puncture technique is safe but sometimes anaphylactic shock \[[@B5]\] can occur. The embolizing agent getting transported into draining vessels causing complications e.g. pulmonary embolization \[[@B1]\] is reported. This technique can be safely carried out under fluoroscopic control by first doing local parenchymography and then keeping a close vigil on the slow progression of the embolization agent, suspending the injection when embolization agent penetrates into the draining vessels. This technique should be carried out under general anesthesia as direct injection of the embolization material into the capillaro-venous malformation in a conscious patient is extremely painful and also immediate surgery cannot be done.

We chose NBCA as the embolising agent because it solidifies immediately on coming in contact with moisture, has antimicrobial properties \[[@B6]\] and has minimal tissue toxicity. NBCA is mixed with Lipiodal to make it radio-opaque. Other embolising agents may be used, for example ethibloc, ethanolamine oleate, sodium tetradecyl sulfate, polidocanol but they have various demerits in their use like Ethanolamine oleate has high viscosity that makes injection difficult, it also has a tendency of renal failure at high doses, and allergic reactions are also known. The main disadvantage of Ethibloc as an embolising agent is its long solidification time (3--5 minutes) making it susceptible to flow out into systemic circulation \[[@B7]\]. Sodium tetradecyl sulfate has a significant incidence of epidermal necrosis, causes hyper-pigmentation, and occasional anaphylaxis is known. Telangiectatic matting is seen following injection of Polidocanol, it also causes hyper pigmentation and anaphylaxis.

Conclusion
==========

We believe that this technique is relatively simple, less time consuming, cheap and permits surgical excision of the capillaro-venous malformation with lesser extent of bleeding.

Competing interests
===================

None declared.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>


Background
==========

T wave alternans (TA) is a repolarisation phenomenon manifesting as a microvolt beat to beat change in amplitude of the T wave and ST segment. The mechanism responsible for TA is a dynamic (beat to beat) variation of the repolarisation sequence \[[@B1]\] due to either an alternation of action potential duration, frequently associated with the long QT syndrome \[[@B2]\] or an alternation of the time course of repolarisation \[[@B3]\] frequently associated with ischaemia \[[@B4]\]. The alternation of time course and multilevel distribution of electrical repolarisation activity during ischaemia \[[@B4]\] cause the electrocardiographic manifestation of TA in addition to alternating dispersion through islands of partially and fully refractory tissue which promote wavefront fractionation and vulnerability to reentrant malignant arrhythmias (MA)\[[@B5],[@B6]\].

The earliest description of TA was made in 1910 by Lewis \[[@B7]\]. Animal work by Hellerstein and Liebow showed a mechanistic link between TA and arrhythmogenesis \[[@B8]\] and recently the presence of TA has been recognised as being a harbinger of sudden death \[[@B9],[@B10]\]. Rosenbaum et al reported TA to be an independent prognosticator of vulnerability to arrhythmias predicted by electrophysiology testing\[[@B11]\] and these findings were repeated by Armoundas and co-workers showing TA to be superior to signal average electrocardiography as a predictor of malignant ventricular arrhythmias in high risk populations \[[@B12]\]. TA has demonstrated high sensitivities and specificities as a predictor of arrhythmic risk in unselected myocardial infarction populations \[[@B13]\] and idiopathic cardiomyopathy \[[@B14]\] but TA has not been used to differentiate risk in the ischaemic cardiomyopathy group.

The purpose of this pilot study was to use indices of absolute micro voltage TA and the frequency content of alternating T waves to assess the ability to differentiate post infarction heart failure patients with significant substrates for developing re-entrant MA but without arrhythmic events, from those post infarction patients at most risk from developing MA.

Materials and Methods
=====================

Approval was obtained from the local Ethics Committee prior to performing the described research. The research was conducted in accordance the Helsinki Declaration, written informed consent was obtained from each patient prior to collecting data. The study population consisted of two groups of stable treated patients: Group 1 comprised 7 patients with extensive scarring secondary to myocardial infarction and compensated treated left sided heart failure with mean ejection fraction of 35%. The patient with the most recent infarct event in this group occurred over 20 months prior to the study. No patient within group 1 sustained a symptomatic arrhythmic event. Group 2 comprised 9 patients with a history of myocardial infarction with a mean ejection fraction of 38%, a distant episode of sustained ventricular tachycardia or aborted sudden death, positive induction of sustained ventricular tachycardia during an electrical stimulation study and a history of prophylactic implantation of an automatic defibrillator.

20 sequential electrocardiographic complexes were recorded from X,Y and Z orthogonal leads on each resting supine patient during a held half expiration breath without straining. The ECG recordings were made using and analog-to-digital converter (Powerlab), sampling at 1000 Hz. All the recorded signals were not filtered, because it was intended to make inter-group statistical comparisons in the frequency domain between the higher frequency band of 80--500 Hertz, where there may exist representations of possible late potential repolarisation. As all significant non-noisy signals have been found to occur within a 0 to 40 Hertz pass-band \[[@B15]\], the recorded signals underwent 40 Hertz low pass filtering prior to analysis in the time domain.

For each lead on each patient, sequences of the alternate ST-T waves were ensemble averaged over a 400 millisecond duration using the positive or negative peak of the T wave as the fiducial central point of data collection over 400 milliseconds, see figure [1](#F1){ref-type="fig"}. In leads where the T wave was of low magnitude, the fiducial central point was taken to occur as the point at an average of the times from the peak QRS deflection to the fiducial T wave point in the other clearly demarcated orthogonal leads. The vectors of the squared magnitude of differences in ensemble averaged time aligned alternating ST-T sequences were calculated on each lead from each patient. For each patient, the resultant absolute difference vector (ATA) was calculated as the square root of the summed squared differences between alternate ensemble averaged T waves for each X, Y and Z vector. All calculations were performed using Mathcad Professional signal processing software on a Acer Pentium 2 PC. Comparisons were made between each mean group ATA as well as between the intra and inter-group means of vectors representing each third time interval of the respective mean ATAs. The spectral content was calculated for each group mean ATA by digital fast Fourier algorithm and cross spectral analysis was calculated between each group mean ATA to assess differences in the frequency components within each group. In order to assess group differences in the 80 to 500 Hertz frequency band, mean magnitude and standard deviation of the 80 to 500 Hz frequency band in the group with the smallest mean magnitude was considered noise and group differences between mean magnitudes in this band only considered significant if they exceeded three times the previously calculated standard deviation.

![Diagram showing methodology for sampling analog ECG signal in X, Y and Z leads in a rested, supine patient during breath holding. 400 milliseconds of alternate T wave sequences were ensemble averaged for each lead and collected in matrix bins represented by X1, Y1, Z1 and X2, Y2 and Z2.](1471-2261-2-6-1){#F1}

The following describes the method by which the digitised ensemble averaged alternate T wave vectors (X1, X2, Y1, Y2, Z1 and Z2) were used to calculate the mean ATAs for each group.

Time of T wave Analysis= t = 400 milliseconds

Subjects in groups 1 and 2 = 1 to N1 and 1 to N2

\[Vector **X1** - Vector **X2**\]^2^ = Vector (**DiffX**)^2^

\[Vector **Y1** - Vector **Y2**\]^2^ = Vector (**DiffY**)^2^

\[Vector **Z1** - Vector **Z2**\]^2^ = Vector (**DiffZ**)^2^

Resultant Absolute Difference Vector (ATA) = √ (**DiffX**^2^ + **DiffY**^2^ + **DiffZ**^2^)

![](1471-2261-2-6-i1.gif)

Mean value of Group (1 or 2) mean ATA= \[Group (1 or 2) mean ATA\]/t

Statistics
----------

Two tailed t test was used to assess group differences between mean ATAs and intra and inter-group differences between the first (0--133 milliseconds), second (134--237 milliseconds) and third mean ATA (238--400 milliseconds). The statistical group differences between the magnitude of components within the 80 to 500 Hz band have been described above.

Results
=======

An example of the time aligned alternate sequences of ST-T waves over 400 milliseconds duration is shown in figure [2](#F2){ref-type="fig"}. The mean value and standard deviation for each group member ATA are shown in Table [1](#T1){ref-type="table"}. There was no significant difference between the means for groups 1 and 2 mean ATAs. The mean value and standard deviation for group 1 *v* group 2 being 10.7 microvolts (standard deviation= 7.17 microvolts) *v* 11.74 microvolts (standard deviation= 8.48 microvolts) respectively. All the values for the difference between ensemble averaged T waves for each vector in each patient are shown in table [1](#T1){ref-type="table"}. There was no intra or inter-group significant difference in the mean values between the first, second or third time intervals for each groups mean ATA. The mean values for first, second and third intervals for Group 1 *v* Group 2 being 6.59 v 7.17, 6.37 *v* 7.96 and 6.06 v 6.18, respectively. The Power Spectrum for each groups resultant absolute difference vector between alternate sequences of ensemble averaged ST-T waves showed the largest values between 0 and 25 Hertz with maximum significant differences at the mean or DC frequency, see figures [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}. The cross spectral density function showed that both groups shared the same frequency components, see figure [5](#F5){ref-type="fig"}. There was no significant group differences between the mean magnitudes of frequency band 80--500 Hertz for Group 1 *v* Group 2. The standard deviation in this band for Group 1 (noise) = 0.562, the mean magnitudes for Group 1 v Group 2 in this band being 0.615 v 0.823.

![An example of two ensemble averaged, time aligned, alternate 400 milliseconds of T wave sequences for lead X in subject 2. The x axis is in milliseconds and the y axis in microvolts. The dashed and continuous tracings represent an inverted T wave in this subject.](1471-2261-2-6-2){#F2}

![Spectral analysis of the resultant vectored absolute mean TA for the ischaemic cardiomyopathy group (group 1). The y axis represents the magnitude of spectral components and the x axis represents frequency in Hertz. Only the range from 0 to 40 Hertz is shown for clarity.](1471-2261-2-6-3){#F3}

![Spectral analysis of the resultant vectored absolute mean TA for the implanted defibrillator group (group 2). The y axis represents the magnitude of spectral components and the x axis represents frequency in Hertz. Only the range from 0 to 40 Hertz is shown for clarity.](1471-2261-2-6-4){#F4}

![Cross-Spectral analysis for the resultant vectored absolute mean TA for both groups. The y axis is the magnitude of cross-spectral frequency components and the x axis is in Hertz. Note that all positive values indicate the presence of a common frequency component between both groups with expected large magnitudes in the frequency range between 0 to 40 Hertz.](1471-2261-2-6-5){#F5}

::: {#T1 .table-wrap}
::: {.caption}
######

Values of the squared microvolt differences between ensemble averaged T waves meaned over time, in each patients X, Y and Z leads and values of each patients ATA. Patient numbers 1 to 7 represent Group 1 and patient numbers 8 to 16 represent Group 2. Values \< 0.015 rounded to 0.
:::

       X        Y        Z       ATA
  ---- -------- -------- ------- -------
  1    304.85   0        72.76   19.43
  2    0        0        0       0
  3    144.96   0        94.67   15.48
  4    24.6     0        0       4.96
  5    38.8     55.65    0       9.72
  6    4.6      44.62    7.07    7.5
  7    192.93   32.26    91.58   17.8
  8    143      278.9    0       20.05
  9    2.16     2.16     17.5    4.67
  10   40.57    0        6.66    6.87
  11   186.6    0        0       13.66
  12   57.5     31.92    33.9    11.1
  13   0        0        0       0
  14   0        17.3     0       4.16
  15   225.6    291.7    9.36    22.9
  16   19.45    320.41   133.9   21.8
:::

Discussion
==========

The purpose of this pilot study was to assess the ability of the absolute measurements for the microvolt magnitude of TA to differentiate between one group with an established high risk of MA (Group2) versus a group of patients with multiple physical substrates for developing MA but without a history of a significant arrhythmic event (Group 1). Group 1 was chosen to be representative of those patients whom make up a large proportion of the inexorably increasing coronary heart disease population and on clinical criteria would theoretically be at highest risk from developing a MA and therefore most likely to benefit from accurate risk stratification and defibrillator implantation. It is important to scrutinise the limitations of any index proffered to help risk stratification and aid the focussing of limited resources of invasive electrophysiological investigation with a view to device therapy.

Assuming a standard error of approximately 6% for the measurement of ejection fraction by acoustic quantification method, the two groups had similar ejections fractions and presumably similar degrees of post-infarction ventricular damage. The lower ejection fraction of Group 1 would be compatible with the intended selection bias and the greater degree of scarring and contractile dysfunction function in this group. Both groups were stable in terms of heart failure and the absence of dynamic symptomatic ischaemic syndromes, therefore the observed measurements of TA in time and frequency domains are expected to arise from mechanism associated with the post myocardial infarction scarred tissue and it\'s immediate periphery.

Conventional TA indices either measure the alternans ratio \[[@B11]\] which is the extent to which the measured alternans exceeds the uncertainty (noise) of the measurement and conveys the statistical degree of confidence in the alternans measurement, or the cumulative alternans voltage in microvolts, which is derived from the frequency power spectrum and analogous, but not equivalent to resultant vector of absolute difference in alternate T wave sequences (ATA) used as the index in this study. The spectral method is an indirect method which measures the magnitude of TA as the amplitude of spectral power at the 0.5 cycles per beat frequency and then subtracts the mean background noise.

This method will not compensate for the presence of superposed noise contamination of the 0.5 frequency component amplitude which is then squared to derive the power term. There is an assumption with the spectral measurement method, that the noise is all broad band but it may be non-linear or multiplicative and therefore not eliminated by the spectral method of subtracting the mean of the baseline noise. Perturbations to increase the heart rate have been used to improve the signal to noise ratio of the spectral method in order to improve the sensitivity to detect significant TA. This study describes a direct method which measures absolute TA which for reasons described in the next paragraph has a high signal to noise ratio. This may account for the higher mean values of ATA compared to the values of mean cumulative alternans voltage which is circa 1.2 microvolts. As the index ATA is an absolute measure of the alternating microvolt T wave amplitude, what it conveys is more in faith with the definition of TA. This pilot study demonstrates that the resultant vector of absolute differences in TA microvoltage measured as the index ATA, which was obtained from ensemble averaged alternate T wave sequences during breath holding, may demonstrate low specificity when applied to the risk stratification in those post-infarction patients with ischaemic cardiomyopathies. A recent study by Sakabe and colleagues have demonstrated a specificity of only 35% in a prospective study using TA to predict the development of ventricular tachycardia in non-ischaemic cardiomyopathy patients \[[@B16]\]. This paper supports our results and has significant implications for the specificity of TA when used as a discriminatory probe in the cardiomyopathy patient subset.

Our method of measuring absolute TA minimises inaccuracies due to noise by ensemble averaging the data collected under optimal conditions of stability of the electrocardiographic tracing by suspending respiration in a supine rested patient. The noise remaining after ensemble averaging is then largely eliminated by subtracting the alternate ensemble averaged T wave signal sequences from each other, thus subtracting the equal amounts of background noise within each of the sequences from each other. It is known that TA can be produced by motion of the heart within the thoracic cavity, previous studies have not addressed the augmentation of such motion with respiration. It has been suggested that using a resultant vector of orthogonal leads is sufficient to mitigate against the effects of mechanical rotation of the heart when measuring TA \[[@B11]\] but theoretically the resultant vector will oscillate with cardiac and respiratory motion. Data from long, noisy, non-stationary ECG recordings, of the type found in Holter recordings or during exercise, would add to inaccuracies resulting from cardiac motion. We attempted to correct for any such inaccuracy arising from respiration by suspending the respiration in half held breath. The number of recordings of complexes was limited due to the time over which the patients could comfortably breath-hold but the accuracy of the measured absolute resultant vector of microvolt TA in a limited number of complexes was considered an advantage over a TA index compiled from a more numerous but more variable and inaccurate data source.

For both groups the absolute difference in mean ATA was distributed equally in time throughout the ST-T wave, which corresponds to the finding that the maximum differences in frequency content for the ATAs in both groups occur at the mean or dc frequency component but there was also a significantly increased magnitude of ATA Within the 0--25 Hertz frequency band which would include the T wave peak frequency and subharmonics within the T wave. In the same way that spatial heterogeneities of depolarisation are responsible for the spectral component markers of late potentials within the QRS complexes, Fourier techniques were applied to the ST-T waves to determine the presence of any difference in the higher frequency bands between alternating ST-T sequences which may indicate the presence of alternating spatial heterogeneities of repolarisation. The finding of no statistically significant group differences in mean magnitude within the frequency band 80 to 500 Hertz is compatible with the findings of Steinbigler et al \[[@B15]\] and does not provide a probe to differentiate these two groups. This study only showed a significant difference in the mean frequency of alternating sequences, which suggests that the differences in measured alternating microvolt magnitudes of the ST-T wave were not due to a change in the morphology of the wave due to introduction of discrete frequency components. The ATAs in the time domain also showed homogenous distribution throughout the ST-T waves, which implies that the origin of TA is not an anatomically localised phenomena as it does not appear to arise from the repolarisation gradient generated between the epicardial, endocardial and M myocardial layers, which according to Antzelevitch and colleagues correspond to the epochs of the start of the peak T wave, the initial descending T wave and end of the descending T wave respectively \[[@B17]\].

There is controversy as to whether TA indices show significant specificity when identifying patients with idiopathic cardiomyopathy at high risk of ventricular arrhythmias \[[@B14],[@B16]\]. Alternatively, TA has been shown to be very useful in identifying arrhythmic risk in the non-specific post infarction population, where sensitivities and negative predictive values are 93% and 98% respectively \[[@B13]\]. However, the major arrhythmic risk lies in the large post infarction cardiomyopathy population but there has been little focus on the use of TA markers in this specific subgroup with coronary heart disease. On the basis of this pilot study, larger scale studies are indicated with electrophysiological ventricular stimulation as reference standard to further assess the sensitivity and specificity of absolute and spectral derived indices of TA to identify patients at high risk of MA within the population of post infarction ischaemic cardiomyopathic heart failure patients.

Conclusions
===========

This pilot study did not show any significant difference in the magnitude and distribution of absolute TA microvoltages or spectral components between Groups 1 and 2. Our results in ischaemic cardiomyopathy patients is supported by another study on cardiomyopathy subsets which has shown a low specificity for TA to identify arrhythmic risk. These results suggest larger scale studies are needed.

Abbreviations
=============

TA= T wave alternans

ATA = Resultant absolute difference vectors

MA= malignant arrhythmia

Competing interests
===================

None declared.


Background
==========

Nutrient cycling between geographically distinctive ecosystems can produce zones of major productivity and biodiversity. It is generally recognized that downstream transport of terrestrial nutrients into marine estuaries produces one of the world\'s most productive habitats, but recent investigations suggest that the reverse flow, from marine to terrestrial habitats, may also be exceptionally important in structuring highly diverse coastal ecosystems \[[@B1]\].

Every year in the Pacific Northwest anadromous salmon (*Oncorhynchus* spp.) transport marine-derived nutrients from the North Pacific Ocean into coastal ecosystems. This salmon nutrient subsidy extends from aquatic habitats into riparian forests, and is thought to be ecologically equivalent to the migration of the wildebeest on the Serengeti \[[@B2]\]. Stable isotope studies in aquatic and terrestrial ecosystems reveal that salmon contribute highly to yearly protein intake for many vertebrates \[[@B1],[@B3]-[@B5]\] and invertebrates \[[@B6],[@B7]\], and provide substantial nutrient inputs to limnetic food webs \[[@B6],[@B8]-[@B10]\], and riparian vegetation \[[@B7],[@B11]-[@B13]\], emphasizing the ecological magnitude of this keystone resource for coastal communities.

Transfer of salmon nutrients into terrestrial habitats occurs primarily through bear (*Ursus* spp.) mediated salmon carcass transfer \[[@B14]-[@B16]\] and urine deposition \[[@B12]\], but can also occur as a result of flooding events \[[@B11]\], hyporheic zone transfer \[[@B5]\], or the activities of other scavengers and predators \[[@B3],[@B5]\]. Since nitrogen is often limiting in coastal temperate rainforests of the Pacific Northwest \[[@B17]\], this salmon nutrient pulse to riparian forests can provide a significant proportion of plant total nitrogen \[[@B11]-[@B13]\], and is thought to increase riparian primary productivity, vegetation and litter quality, and soil nutrient capital \[[@B13]\].

Studies in forest ecosystems adjacent to salmon streams have so far been limited to vegetational use of salmon nutrients and have ignored other potential food web beneficiaries, particularly terrestrial invertebrates. Macro-invertebrates of coastal coniferous forests of the Pacific Northwest, including insects, arachnids, myriapods, annelid worms, isopods and gastropods, comprise the base of the myriad of nutrient and energy pathways from primary producers through to higher vertebrate consumers, and are highly important in many ecosystem processes including herbivory, litter decomposition, and nutrient cycling \[[@B18]-[@B20]\].

We use a dual isotope approach of δ^15^N and δ^13^C to assess: a) the extent of utilization of salmon-derived nitrogen and carbon by various trophic groups in a terrestrial invertebrate forest litter community and b) the mechanism of salmon nutrient utilization by invertebrates; either directly through salmon tissue consumption, or indirectly through utilization of salmon nitrogen sequestered into riparian vegetation or soil N pools. We compare the cycling of nutrients above and below waterfalls as a means of examining ecological discontinuities that may occur in litter-based macro-invertebrates between salmon and salmon-free forest sites, and speculate on possible implications to invertebrate community structure and higher vertebrate consumers. We also discuss components of invertebrate isotopic variability as it relates to microspatial variability in δ^15^N, invertebrate trophic structure, and invertebrate niche.

Results
=======

Invertebrate trophic groups varied predictably with respect to δ^15^N. The nested ANOVA analysis demonstrated that the majority of variance in δ^15^N was due to falls within watersheds (F = 9.191; p = 0.031; R^2^ = 0.819) and taxonomic group within all other factors (F = 13.71; p \< 0.001; R^2^ = 0.689). Variation in δ^15^N that occurred between watersheds or distance of collection from the stream contributed little to total variance and was insignificant in the model (See methods for violations). Invertebrates were enriched by 3--8‰ along salmon spawning reaches compared to similar groups collected above the falls, and showed a gradient of increasing values with increased trophic level at both salmon and non-salmon sites (Figure [1](#F1){ref-type="fig"}). There were highly significant differences in δ^15^N (t-tests: p \< 0.01) above and below waterfalls for all trophic groups at both watersheds. Multiple comparison tests (Tukey\'s post hoc) revealed distinct trophic separation in δ^15^N between at least two invertebrate groups depending on site of collection (Table [1](#T1){ref-type="table"}). Millipede detritivores had higher δ^15^N values than root feeding weevils on all sites but only on the Clatse above the falls was this trend significant. Carabid beetles demonstrated higher δ^15^N values than millipedes at all sites with significant differences on the Clatse River below and above the falls and on the Neekas River above the falls. Spider predators were significantly more enriched than carabid beetles on the Neekas River on both salmon and non-salmon sites, but demonstrated only marginally higher δ^15^N values than these beetles on the Clatse River. Carabid beetle omnivores and spider predators demonstrated significantly higher variance in δ^15^N below the falls than above on both watersheds (Carabidae Clatse: F~14,6~ = 14.61, p \< 0.005; Carabidae Neekas: F~21,6~= 21.94, p \< 0.001; Araneae Clatse: F~18,16~ = 5.41, p \< 0.002; Araneae Neekas: F~17,11~ = 4.94, p \< 0.02) (F-ratio tests).

![δ^15^N values in four trophic groupings of litter-based invertebrates collected above and below waterfall barriers to salmon migration on the Clatse and Neekas Rivers, British Columbia. Invertebrates are ranked (left to right) based on increasing consumption of animal protein (see methods). t-test results: ^\*\*^ denotes p \< 0.01; ^\*\*\*^ denotes p \< 0.001.](1472-6785-2-4-1){#F1}

::: {#T1 .table-wrap}
::: {.caption}
######

Tukey\'s multiple comparison post hoc tests for δ^15^N values in four invertebrate trophic groups collected above and below waterfalls on the Clatse and Neekas Rivers, British Columbia. N/A indicates post hoc tests not possible due to low sample sizes.
:::

                                                Clatse River
  ---------------------- ---------------------- ----------------------- -------------- ----------------------- --------------
  Predators              Root Feeders           5.93                    \<0.001        8.31                    \<0.001
  Predators              Detritivores           5.81                    \<0.001        4.39                    \<0.001
  Predators              Omnivores              1.28                    0.615          1.30                    0.123
  Omnivores              Root Feeders           4.65                    0.002          7.01                    \<0.001
  Omnivores              Detritivores           4.52                    0.005          3.09                    \<0.001
  Detritivores           Root Feeders           0.13                    1.000          3.92                    \<0.001
                                                Neekas River

                                                Below falls             Above falls

  Trophic grouping (1)   Trophic grouping (J)   Mean Difference (I-J)   Significance   Mean Difference (I-J)   Significance

  Predators              Root Feeders           8.55                    \<0.001        5.33                    N/A
  Predators              Detritivores           6.14                    \<0.001        5.32                    \<0.001
  Predators              Omnivores              5.84                    \<0.001        3.34                    \<0.001
  Omnivores              Root Feeders           2.7                     0.548          1.99                    N/A
  Omnivores              Detritivores           0.3                     0.999          1.98                    0.006
  Detritivores           Root Feeders           2.4                     0.693          0.01                    N/A
:::

Invertebrate groups varied in δ^13^C but did not always vary predictably with trophic level or habitat (Figure [2](#F2){ref-type="fig"}). Nested ANOVA analysis using δ^13^C indicated significant variability only in taxonomic groupings (F = 11.801; p \< 0.001; R^2^ = 0.657), with all other levels insignificant. Relatively high δ^13^C values were observed in millipedes from both watersheds in salmon and non-salmon sites, most likely a reflection of inorganic carbon content. Multiple comparisons revealed trophic separation for spiders over carabid beetles in all sites (Table [2](#T2){ref-type="table"}). Spiders were enriched over root feeders on the Clatse River above the falls and on the Neekas below the falls. Carabids and root feeders did not differ in their δ^13^C values. Carabid beetles collected on the Neekas River were the only group to demonstrate isotopic enrichment below the falls (p = 0.042). Spiders on the Clatse River were found to be higher in δ^13^C above the falls than below (p= 0.016).

![δ^13^C values in four trophic groupings of litter-based invertebrates collected above and below waterfall barriers to salmon migration on the Clatse and Neekas Rivers, British Columbia. Invertebrates are ranked (left to right) based on increasing consumption of animal protein (see methods). t-test results: ^\*^ denotes 0.01 \< p \< 0.05](1472-6785-2-4-2){#F2}

::: {#T2 .table-wrap}
::: {.caption}
######

Tukey\'s multiple comparison post hoc tests for δ^13^C values in four invertebrate trophic groups collected above and below waterfall barriers to salmon migration on the Clatse and Neekas Rivers, British Columbia. N/A indicates post hoc tests not possible due to low sample sizes.
:::

                                                Clatse River
  ---------------------- ---------------------- ----------------------- -------------- ----------------------- --------------
  Predators              Root Feeders           0.77                    0.555          1.17                    0.004
  Predators              Omnivores              1.27                    0.036          2.14                    \<0.001
  Omnivores              Root Feeders           -0.49                   0.847          -0.92                   0.058
                                                Neekas River

                                                Below falls             Above falls

  Trophic grouping (1)   Trophic grouping (J)   Mean Difference (I-J)   Significance   Mean Difference (I-J)   Significance

  Predators              Root Feeders           1.80                    0.033          0.90                    N/A
  Predators              Omnivores              2.02                    \<0.001        2.46                    \<0.001
  Omnivores              Root Feeders           -0.22                   0.984          -2.08                   N/A
:::

We examined isotopic levels in relation to distance upstream from the ocean. At Clatse River, δ^15^N declined with increased distance upstream with the lowest levels occurring above the waterfalls. However, at Neekas River, δ^15^N levels were high but variable throughout the stream channel below the waterfall, above which there was a striking reduction in δ^15^N over short distance delineated by the geological barrier to salmon (Figure [3](#F3){ref-type="fig"}).

![δ^15^N values in ground beetles (Carabidae) and spiders (Araneae) with distance of collection upstream from the estuary (m) on the Clatse and Neekas Rivers, British Columbia.](1472-6785-2-4-3){#F3}

In order to assess niche differences within and among groups, we examined the relationships between δ^15^N and δ^13^C. Below the falls, there were significant positive correlations between δ^15^N and δ^13^C in spiders on the Clatse (R = 0.562; p = 0.012) and on the Neekas (R = 0.741; p = 0.001), and in carabid beetles on the Clatse (R = 0.682; p = 0.005) and on the Neekas (R = 0.538; p = 0.010) (Figure [4](#F4){ref-type="fig"}). None of the remaining correlations were significant in groups collected below the falls, and there were no significant correlations between δ^15^N and δ^13^C for any group collected above the falls.

![δ^15^N and δ^13^C values in ground beetles (Carabidae) and spiders (Araneae) below and above waterfalls on the Clatse and Neekas Rivers, British Columbia.](1472-6785-2-4-4){#F4}

We estimated contribution of marine-derived nitrogen to the total nitrogen content among invertebrate groups on both watersheds (Table [3](#T3){ref-type="table"}). At Clatse River, assuming no fractionation, values ranged from 19% in millipedes to 49% in weevils (with fractionation: 28% in millipedes to 71% in weevils). At Neekas River, assuming no fractionation, values ranged from 35% in ground beetles to 51% in spiders (with fractionation 47% in ground beetles to 70% in spiders).

::: {#T3 .table-wrap}
::: {.caption}
######

\% Marine-derived nitrogen (MDN) estimates in invertebrates collected above and below waterfall barriers to salmon migration on the Clatse and Neekas Rivers, British Columbia. Estimates of % MDN were made under conditions of no fractionation from the source and maximum fractionation of 4‰ from the source nitrogen by primary producers.
:::

                              Clatse River
  --------------------------- ------------------------- --------------------------
  Curculionidae               49.1%                     71.5%
  Parajulidae                 19.4%                     28.2%
  Carabidae                   30.6%                     44.6%
  Agelenidae/Antrodiaetidae   30.4%                     44.3%
                              Neekas River

  Invertebrate family(ies)    %MDN (No Fractionation)   %MDN (Max Fractionation)

  Parajulidae                 45.8%                     62.3%
  Carabidae                   34.8%                     47.3%
  Agelenidae/Antrodiaetidae   51.2%                     69.6%
:::

Discussion
==========

We demonstrate isotopic evidence for substantive incorporation of salmon-derived nitrogen into multiple trophic levels of terrestrial litter-based invertebrates from two salmon bearing watersheds. Enrichment in δ^15^N in terrestrial invertebrates occurs through two possible pathways: 1) direct consumption of salmon tissue and/or predation off of direct salmon consumers such as larval blowflies; or 2) indirect enrichment through δ^15^N enriched soil and vegetation N pools. Here, the use of the dual isotope method provides insight into the mechanism of salmon nitrogen utilization by terrestrial invertebrates. Direct consumption of salmon, with approximate δ^15^N and δ^13^C values of +11.2‰ \[[@B21]\] and -21‰ \[[@B9]\] respectively, would lead to enriched signatures of δ^15^N and δ^13^C in animal tissues. For example, consumption of salmon carcasses by larval blowflies (Calliphoridae) has been documented through the dual isotope method \[[@B7]\]. However, terrestrially derived carbon through C~3~ photosynthesis dominates δ^13^C pools in coniferous forest soils and salmon-derived carbon is assumed to contribute little to total carbon in litter and soil. The process of indirect utilization of salmon-derived nitrogen by animals has been observed previously in small mammals \[[@B11]\], whereby individuals were enriched in δ^15^N but not δ^13^C. Because we found little differences in δ^13^C in all trophic groups collected above versus below the waterfalls, this suggests that the primary mechanism of δ^15^N enrichment is by indirect processes through salmon-derived nitrogen subsidies to soil and vegetation N pools.

δ^15^N / δ^14^N ratios of forest nitrogen pools are influenced by the isotopic values of nitrogen inputs and outputs and fractionation that occurs during nitrogen transformations within ecosystems \[[@B22]\]. Nitrogen inputs to typical Pacific coast forest ecosystems include atmospheric deposition and biological nitrogen fixation. In the case of forests adjacent to salmon streams there is substantial evidence that marine-derived nitrogen from salmon is transferred to forest ecosystems through predator activity \[[@B11],[@B12],[@B14]-[@B16]\], flooding events \[[@B11]\] and hyporheic zone transfer \[[@B5]\], and is incorporated into soil N pools through uptake by vegetation \[[@B6],[@B7],[@B11]-[@B13]\].

Vegetation δ^15^N values tend to parallel those in the soil and litter across multiple sites and are typically slightly depleted in δ^15^N relative to the soil source \[[@B22],[@B23]\]. Recent estimates for the contribution of marine-derived nitrogen from salmon in riparian ecosystems to total plant nitrogen have ranged from 15.5--24% \[[@B6],[@B12],[@B13]\]. These values may be conservative as they are based on the assumption of no plant fractionation from the original source nitrogen. In the case of high nitrogen inputs from salmon, vegetation may preferentially assimilate isotopically light nitrogen (even though it is also originally from salmon). However, in nutrient rich habitats fractionation from the source is potentially not as marked compared with nutrient poor soils \[[@B23],[@B24]\], making %MDN estimates challenging. %MDN estimates from hemlock (Mathewson & Reimchen unpublished data), possibly constituting a large percentage of litter biomass, vary from 23--34% on the Clatse River and 49--66% on the Neekas River depending on degree of fractionation from the source. These estimates are higher than previously reported, yet remain the baseline for comparison with %MDN estimates in our litter-based invertebrate community.

Ponsard and Arditi \[[@B25]\] observed substantial site variation in litter and soil δ^15^N due to variations in soil processes and nitrogen sources across small scales (\< 1 km). Soil and litter δ^15^N and δ^13^C values are not yet available for our sites. However, δ^15^N values in litter-based terrestrial invertebrates are known to parallel the δ^15^N values in the litter and soil \[[@B25],[@B26]\]. We suspect that because vegetation and all invertebrates collected below the waterfall barrier to salmon migration are enriched in δ^15^N, that soil and litter δ^15^N are also enriched at these sites. Our data demonstrates that terrestrial invertebrates exhibit a substantial shift in δ^15^N over a sharp ecological discontinuity (ca. 250 m) in the source of nitrogen to the forest community, as a consequence of a distinct salmon-derived nitrogen subsidy to litter, soil and vegetation N pools. We estimate that %MDN to multiple trophic levels of litter-based invertebrates ranges from 19--71% on the Clatse River and 34--70% on the Neekas River depending on trophic grouping, and on the extent of fractionation from the original source nitrogen. These values are similar to %MDN estimates of hemlock and indicate that salmon-derived nitrogen is cycled from primary producers through multiple trophic levels of litter-based terrestrial invertebrates.

Grouping all invertebrate samples over the entire 100 m riparian zone may have reduced the extent of statistical differences for δ^15^N in our comparisons above and below falls. This occurs because of a potential isotopic gradient of decreasing δ^15^N from salmon in terrestrial vegetation with increasing distance from the stream over a relatively small scale (\< 100 meters) \[[@B11]-[@B13]\]. Nevertheless, our %MDN estimates are higher than any other study investigating salmon nutrient transfer into terrestrial ecosystems and emphasizes the magnitude of the discontinuity that occurs across the waterfall barrier to salmon migration in these watersheds.

These %MDN estimates assume salmon tissue δ^15^N as the marine end-member in the model. However, there are other factors that can influence these estimates. Vertebrate urine, particularly from bears (*Ursus* spp.) \[[@B12]\], faeces and guano deposition may contribute highly to nitrogen inputs during the salmon spawning season. Despite the fact that these inputs are ultimately from salmon tissue consumption, high fractionation during multiple transformation steps prior to nitrogen availability, such as ammonia volatilization \[[@B22]\], may lead to unknown shifts in the δ^15^N levels of the source nitrogen. This may increase the microspatial variability in δ^15^N in litter, soil, and vegetation, and subsequently invertebrates, along the salmon spawning channel.

Variation in δ^15^N in carabid beetles and spiders collected below the waterfall barrier was substantially greater than above the falls. It was only marginally higher (non-significant) in root feeding weevils and millipede detritivores, possibly due to low sample sizes. This may indicate higher microspatial variability in δ^15^N in soil, litter and vegetation N pools, increased range of prey resources below the falls, and/ or invertebrate dispersal from other habitats into the zone of substantial salmon transfer.

We detected variation in δ^15^N at different stream reaches, most likely as a function of abundance and species of spawning salmon. On the Clatse River, δ^15^N values decreased with increasing distance upstream. Potentially, this might result from a gradient in marine subsidies other than salmon as a function of distance from the estuary \[[@B27]\]. However, this trend was not observed on the Neekas River where δ^15^N values remain high, even at 2 km upstream. The difference between these two watersheds in the distribution of marine-derived nitrogen appears to be due to topography and the species and distribution spawning salmon. Clatse River is pink salmon dominated, with the majority of spawning, and subsequent predator activity, occurring in the lower 500 meters of the spawning channel \[[@B28]\] (personal observations). Above 600 meters the stream narrows and the riparian profile becomes increasingly steep on both sides. The Neekas River has high density chum spawning to the base of the falls with high salmon nutrient transfer and predator activity occurring in this region \[[@B28]\] (personal observations). Chum salmon contain twice the biomass of nitrogen than pink salmon, and this may partly explain the higher %MDN estimates obtained on the Neekas River compared to the Clatse. The distribution of δ^15^N in these terrestrial invertebrate groups thus appears to be directly correlated to salmon spawning density and biomass, and subsequent predator activity, a pattern that has been observed for δ^15^N in ground beetles (Carabidae) occurring between watersheds on Vancouver Island \[[@B7]\].

Differences in the variance of isotopic signatures within a population provide insight as to the range of diet available to the individual. For example, this has been found in stable isotope studies of marine mammals and chimpanzees \[[@B29],[@B30]\]. In the case of carabid beetles and spiders, high variability in δ^15^N along the salmon-spawning channel compared to above the falls, may indicate higher prey variability in this region. Variance in isotopic signatures can also indicate mobility between habitats \[[@B31],[@B32]\]. Carabid beetles, particularly on the Neekas River, exhibited high variance in signatures. The carabid beetle species collected, although brachypterous, can move freely between habitats \[[@B33]\], and captured individuals may not have obtained their nutrition along the salmon spawning channel for their entire life history.

Correlations between δ^15^N and δ^13^C values provide further resolution into individual niche variability. We observed a significant positive correlation between δ^15^N and δ^13^C values in carabid beetles and spiders below waterfalls, with access to salmon nutrients, but not above falls. Both groups feed on a diverse array of prey including primary and secondary consumers, and in the case of the ground beetles, vegetative matter as well. Individuals within each group that fed at a higher average trophic level would be expected to exhibit more enrichment for δ^15^N and δ^13^C \[[@B34],[@B35]\]. Alternatively, individuals that fed on salmon directly or on prey that fed on salmon would also demonstrate isotopic enrichment in both isotopes \[[@B3]-[@B7]\]. Positive relationships in δ^15^N and δ^13^C below the falls and the absence of that relationship above the falls hints that direct consumption of salmon or salmon consumers below the falls may be a factor for some individuals of these species. However, increased range of food resources below the falls would also be consistent with this finding. Furthermore, smaller sample sizes above the falls may have reduced our ability to detect relationships. For the majority of the spiders and ground beetles, direct uptake of the marine isotopes most likely contributes only a minor component to yearly protein intake, as uptake of marine-derived nitrogen occurs by indirect means. The use of dual isotope model becomes most relevant when investigating terrestrial organisms that use salmon protein as a major contributor to diet. This is the case for several terrestrial necrophages including flies (Diptera: Calliphoridae, Scathophagidae, Anthomyiidae), and beetles (Coleoptera: Silphidae, Leiodidae, Staphylinidae) \[[@B7]\] (Hocking unpublished data).

Animals are isotopically enriched in δ^15^N and δ^13^C relative to their dietary intake as a consequence of preferential excretion of the lighter isotope in metabolism \[[@B36]\], and this allows insight into relative trophic position within a community. Isotopic enrichment varies widely by body tissue, but there is an approximate stepwise enrichment of 3.4 ± 1.1‰ for δ^15^N \[[@B35],[@B37]\] and 0.4 ± 1.4‰ for δ^13^C \[[@B34],[@B38]\] for each sequential trophic level. Ponsard & Arditi \[[@B25]\] suggest that there are on average two trophic levels within litter-based invertebrate communities. We also find general evidence for two general trophic levels within the litter-based community at Clatse and Neekas Rivers usually consisting of: 1) root feeders and detritivores (weevils and millipedes) as primary consumers of plant material, and 2) predators (carabid beetles and spiders) that feed on these and other presumed plant feeders within the litter community. Our data, however, provides substantial evidence for a gradient in trophic level among our litter-based invertebrates rather than two distinct trophic groupings, a finding that coincides with that of Scheu & Faica \[[@B26]\]. Millipedes, for instance, were often found to be enriched in δ^15^N compared to root feeders, a finding that suggests that either weevils (Curculionidae) feed on roots that are somewhat depleted in δ^15^N compared to litter, or that millipede detritivores utilize some δ^15^N enriched protein food sources such as bacteria in their guts, or both \[[@B25]\]. Spiders were enriched in δ^15^N in all cases over those in carabid beetles, and below the falls on the Neekas this constituted a mean difference greater than a single trophic level. Evidence for omnivory is emerging in the carabid beetles \[[@B33],[@B39]-[@B42]\] and the observed discrepancy between spiders and carabid beetles is most likely a result of the purely predaceous versus omnivorous life histories of these groups. Spiders also demonstrated trophic enrichment in δ^13^C over carabid beetles at all sites. However, spiders were not consistently enriched over root feeders at each site and carabid beetles exhibited the lowest δ^13^C values. We conclude that, in general, carbon is a poor trophic level indicator \[[@B25]\]. Overall, this suggests that increased trophic and individual niche resolution in stable isotope studies will more likely extend from a detailed taxonomic separation rather than with guild analyses \[[@B26]\].

Implications
============

With the use of stable isotopes (δ^15^N and δ^13^C), spawning salmon have been shown to provide substantial nutrient inputs to limnetic food webs \[[@B6],[@B8]-[@B10]\], with implications for stream primary productivity and subsequent juvenile salmonid survivorship. Young salmon may in fact derive a large proportion of their required nitrogen and carbon from the death and decomposition of their parents, through food web utilization of salmon nutrients by algae and aquatic invertebrates.

Other than inputs to terrestrial vegetation, salmon nutrient effects in forest food webs are poorly known. Input of salmon-derived nitrogen contributes to total available N in the soil and thereby increases forest primary productivity and vegetation and litter quality \[[@B11]-[@B13]\]. Nutrient subsidies (other than salmon) to terrestrial invertebrate communities can result in shifts in invertebrate community structure and abundance as a consequence of bottom-up ecosystem effects \[[@B27],[@B43],[@B44]\]. Soils in coniferous forests of low nutrient status are typically dominated by fungi as the primary decomposers of organic material, and thick humus layers quickly accumulate due to slow rates of nutrient turnover \[[@B45]\]. In nutrient-rich conditions, fungi are replaced by bacteria and invertebrates as the dominant decomposers, resulting in higher net rates of nitrogen mineralization and total available nitrogen \[[@B44],[@B45]\]. Shifts in invertebrate community structure and abundance due to a nutrient subsidy may have further implications for higher invertebrate and vertebrate consumers such as predaceous beetles, spiders, hymenopteran parasitoids, small mammals, amphibians and passerines. For example, in another form of marine subsidy, spider densities have been reported to be 4--5 times higher on islands with marine bird colonies than those without \[[@B46]\]. Furthermore, avian populations in boreal forests have been observed to respond to experimental nitrogen fertilization \[[@B47]\], a pattern that also may well be true in the case of nutrient inputs to forest communities along salmon streams \[[@B48]\]. Shifts in litter-based invertebrate community structure and abundance could have particular benefits for ground foraging birds such as the resident and migratory sparrows, thrushes and wrens. The widespread enrichment in salmon derived nitrogen among multiple trophic levels also hints at an ecosystem level effect that has further implications for shrub and canopy level invertebrate communities and their various vertebrate consumers \[[@B1],[@B5],[@B48]\].

Conclusions
===========

The increasing evidence for the coast-wide decline in salmon abundance on the Pacific coast of North America \[[@B49]\] may have substantially more ecological implications to terrestrial forest food webs than previously recognized \[[@B5]\]. We present evidence for major uptake of salmon-derived nitrogen into a terrestrial invertebrate food web, with a sharp reduction in uptake across a waterfall barrier to salmon migration. These results supplement the conclusions of a diversity of recent contributions that have focused on the ecological consequences of the decline of salmon on the west coast of North America \[[@B1],[@B2],[@B5]-[@B13],[@B48]\].

Methods
=======

Site Description
----------------

Two salmon bearing streams were investigated -- the Clatse (52° 20.6\'N; 127° 50.3\'W) and Neekas Rivers (52° 28.4\'N; 128° 8.0\'W), on the mid-coast of British Columbia, near Bella Bella, Canada. Both watersheds occur in the Coastal Western Hemlock Biogeoclimatic Zone along the boundary between the central very wet hyper-maritime (CWHvh2) and sub-montane very-wet maritime (CWHvm1) subzones \[[@B50]\]. Climate is considered cool and wet with mean annual temperature of approximately 8°C, and mean annual precipitation above 4000 mm (Environment Canada 2001). Dominant tree species include Western Hemlock (*Tsuga heterophylla*), Sitka spruce (*Picea sitchensis*), Amabilis fir (*Abies amabilis*), Western redcedar (*Thuja plicata*), and Red alder (*Alnus rubra*). Common understory species include Alaskan blueberry (*Yaccinium alaskaense*), red huckleberry (*V. parvifolium*), false azalea (*Menziesia feruginea*), deer fern (*Blechnum spicant*), bunchberry (*Cornus canadensis*), lanky moss (*Rhytidiadelphus loreus*), step moss (*Hylocomium splendens*), and common green sphagnum (*Sphagnum girgensohnii*) on zonal sites, and salmonberry (*Rubus spectabilis*), red elderberry (*Sambucus racemosa*), stink current (*Ribies bracteosum*), and spiny-wood fern (*Dryopteris expansa*) on nutrient rich sites. Deep acidic soils predominate with high organic matter content due to low rates of decomposition. Soil deposits are typically alluvial or glacial in origin, are heavily leached, and often contain iron deposits in the B layer. Mor humus types are most common with a thick layer of moss, but moder/mull humus forms occur in nutrient rich sites along the salmon spawning channel.

Both the Clatse and Neekas watersheds are dominated by high-density returns of pink (*Oncorhynchus gorbuscha*) and chum (*O. keta*) salmon, with minor runs of coho (*O. kisutch*) and the occasional sockeye (*O. nerka*). In the last ten years, pink and chum salmon returns on the Clatse River average 17000 and 5000 individuals respectively. Chum salmon constitute the majority of spawning biomass on the Neekas (mean = 30000). Mean pink salmon returns on the Neekas River vary from an average of 33000 on even years to an average of 2700 on odd years (Department of Fisheries and Oceans Escapement data: 1990--1999). Suitable spawning habitat extends for 2.1 km on the Neekas River, roughly twice that of the Clatse (1 km), whereby both are interrupted by waterfalls that act as a barrier to salmon migration \[[@B28]\].

Invertebrate samples
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In August of 2000 terrestrial macro-invertebrates were collected in each watershed through passive pitfall trapping and hand collection from the soil and course woody debris. Invertebrate sampling occurred above and below the waterfall barrier and up to 100 meters from the stream. On the Clatse River, main invertebrate sampling occurred from 200 to 800 meters upstream from the mouth, and again above the falls at 1200 and 1600 meters. The majority of invertebrate trapping on the Neekas occurred at 1 km, and again at 2 km, just below the falls. Control samples from the Neekas were collected just above the falls from 2250 to 2400 meters upstream from the mouth.

Pitfall arrays were arranged in a three-way branching fashion. This included a central 10 cm diameter pitfall connected via three 24-inch by 6-inch aluminium drift fences (separated by 120°) to a perimeter pitfall at the end of each fence \[[@B7]\]. Pitfall arrays were cleared from four to five days after initial set-up, and to prevent rotting of invertebrate tissue 70% ethanol was used as a field preservative within each pitfall cup. Hand collection of invertebrates occurred more randomly as individuals were discovered in the riparian area. All specimens were stored in 70% ethanol prior to identification and isotopic analysis.

Stable Isotope Analysis
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Whole invertebrate specimens were dried at 60°C for at least 48 hours and ground into a fine powder with a Wig-L-Bug grinder (Crescent Dental Co., Chicago, 111). Approximately 1 mg dry weight per ground specimen was then sub-sampled for continuous-flow isotope ratio mass spectrometry (CF-IRMS) analysis of nitrogen and carbon. Mass spectrometry analysis of δ^15^N and δ^13^C was conducted at the stable isotope facility, University of Saskatchewan, Saskatoon, Canada using a Europa Scientific ANCA NT gas/solid/liquid preparation module coupled to a Europa Scientific Tracer/ 20 mass spectrometer.

Isotopic contents are expressed in \'δ\' (delta) notation representing the difference between the isotopic content of the sample and known isotopic standards (atmospheric N~2~ for nitrogen and PeeDee Belemnite (PDB) limestone for carbon). This is expressed in parts per thousand (‰) according to the formula (1):

1\) δ^15^N Or δ^13^C (‰) = (R~sample~ / R~standard~ - 1) \* 1000

where R is the ratio of the heavy isotope (^15^N or ^13^C) / light isotope (^14^N or ^12^C).

Data Analysis
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Individual terrestrial macro-invertebrates processed for δ^15^N or δ^13^C were separated into four main groups based on taxonomic similarity and ranked according to degree of animal protein consumption, thus providing a proxy for relative trophic level within the litter based food chain: 1) Root feeders (Coleoptera: Curculionidae) \[[@B18],[@B51]\]; 2) Detritivores (Julida: Parajulidae) \[[@B25],[@B52]\]; 3) Omnivores (Coleoptera: Carabidae) \[[@B33],[@B39]-[@B42]\]; 4) Predators (Araneae: Agelenidae, Antrodiaetidae) \[[@B53],[@B54]\] (Table [4](#T4){ref-type="table"}).

::: {#T4 .table-wrap}
::: {.caption}
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Family and species level designations by trophic grouping for invertebrates collected on the Clatse and Neekas Rivers, British Columbia, in August 2000.
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  Trophic grouping   Family                      Species
  ------------------ --------------------------- ---------------------------------------------------------------------------------------------
  1\) Root Feeders   Curculionidae               *Steremnius carinatus* Boh. *Steremnius tuberosus* Gyll.
  2\) Detritivores   Parajulidae                 Unknown
  3\) Omnivores      Carabidae                   *Pterostichus crenicollis* LeC. *Scaphinotus angusticollis* Mann. *Zacotus matthewsii* LeC.
  4\) Predators      Agelenidae Antrodiaetidae   *Cybaeus reticulatus* Simon *Antrodiaetus pacificus* Simon
:::

Curculionid beetles of the genus *Steremnius* feed as larvae and adults on the roots and slash of conifers and are assigned the lowest trophic rank, as there is no current evidence that these beetles utilize animal protein \[[@B18],[@B51]\]. Millipedes are detritivores, feeding primarily on dead plant material and fragments of organic matter. This potentially includes small amounts of animal protein from faeces, dead animals or microorganisms that occur on the litter material \[[@B25],[@B52]\]. The Parajulidae are indigenous to the forest ecosystems of the Pacific Northwest but are poorly known at the species level \[[@B55]\]. A priori, we assume here that the parajulid millipedes include minor contributions of organic matter derived from animal protein in diet. Carabid beetles of the genera *Pterostichus, Scaphinotus* and *Zacotus* are generalist forest floor predators on a variety of soil invertebrates including snails and slugs (Gastropoda), millipedes (Diplopoda), isopods (Isopoda), worms (Oligochaeta) and springtails (Collembola) \[[@B33],[@B40],[@B56]\]. However, documented observations of carabids feeding on plant material including seeds and fruit suggest that these beetles may be omnivorous rather than purely predaceous \[[@B39],[@B41],[@B42]\]. Arachnids of the genera *Cybaeus* and *Antrodiaetus* are known to be funnel-web \[[@B54]\] and trap-door spiders \[[@B53]\] respectively, feeding exclusively on animals including various insects, myriapods, isopods, other spiders and even small vertebrates \[[@B57]\].

Independent sample t-tests (two-tailed) were used to test for differences between invertebrate groups collected above and below the falls for δ^15^N and δ^13^C on each watershed (equal variances not assumed in all tests). All invertebrates collected within 100 meters of the stream were pooled for the analysis, and those collected less than 200 meters from the estuary were removed since these were assumed to possess ambiguous isotopic signatures where marine incursions other than salmon input may particularly obscure soil N pools \[[@B27]\]. F-ratio tests (two-tailed) were conducted for δ^15^N between invertebrate groups collected above versus below the falls under the null hypothesis of equal variances. We also performed separate Nested ANOVA\'s on δ^15^N and δ^13^C to examine the effects of trophic group, distance from the stream, above and below falls and watershed \[model: watershed, watershed(falls), watershed(falls(distance)), watershed(falls(distance (invertebrate group)))\]. However, assumptions of normality and homoscedasticity were not met and as such, we place more emphasis on the t-test comparisons. Tukey HSD multiple comparison post hoc tests were performed for δ^15^N and δ^13^C within sites under the null hypothesis that all invertebrate groups were isotopically indistinct. Since inorganic carbon in the form of CaCO~3~, present in the exoskeleton of our millipedes \[[@B52]\], is enriched in δ^13^C relative to organic forms \[[@B25]\], we removed millipedes from the post hoc analysis of δ^13^C among feeding groups. Pearson\'s Correlation Coefficients were used to examine the relationships between δ^15^N and δ^13^C within trophic groups at different sites to investigate the individual niche variability.

Estimating % MDN
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δ^15^N values in animals are influenced by the δ^15^N value of the principal N sources, and fractionation during nitrogen transformations within ecosystems. Principal N sources to riparian ecosystems include atmospheric N~2~ with a δ^15^N value of 0‰ \[[@B36]\], and salmon N with a δ^15^N value of approximately 11.2 ± 1.0‰ \[[@B21]\]. Variations in δ^15^N with trophic level appear to be relatively predictable such that biota are enriched by 3.4 ± 1.1‰ more than their food \[[@B37]\], a pattern that seems to hold true for soil macro-invertebrates \[[@B25],[@B26]\]. Estimates for % marine-derived nitrogen (MDN) in our litter-based macro-invertebrate food chain were obtained based on a combination of a limnetic trophic model proposed by Kline *et al.*\[[@B8]\] and a terrestrial vegetation model utilized by Helfield and Naiman \[[@B13]\] and is expressed mathematically by (2):

2\) %MDN = \[(Obs - TEM) / (MEM~TL~ - TEM) \] \* 100%

where Obs is the observed δ^15^N value of a particular taxa below the waterfall barrier to salmon, TEM is the terrestrial end-member (the isotopic value obtained for the same taxa above the falls in absence of salmon input), MEM is the marine end-member (δ^15^N value of salmon of 11.2‰ \[[@B21]\] which should equal maximum vegetation δ^15^N values), and TL refers to the trophic level correction factor that applies to the marine end-member in the model. Since variability in utilization of MDN by the various invertebrate groups below the falls might obscure relative trophic level, we used invertebrate δ^15^N values above the falls on each watershed to provide an indication of relative trophic position. The trophic level correction factor was thus calculated by subtracting mean δ^15^N values in hemlock above the falls, (Mathewson & Reimchen unpublished data: Clatse mean δ^15^N = -1.55‰; Neekas mean δ^15^N = -3.93‰) from mean δ^15^N values in each invertebrate group above the falls on each watershed. This simplifies the above equation to (3):

3\) % MDN = \[(Obs - TEM) / (MEM - VEGabv) \] \* 100%

where MEM equals salmon tissue \[[@B21]\] and VEGabv equals mean vegetation δ^15^N values above the falls. We also calculated %MDN for vegetation below the falls (Mathewson & Reimchen unpublished data: Clatse mean δ^15^N = +1.43‰; Neekas mean δ^15^N = +3.44‰) as a benchmark comparison to our invertebrate estimates. We were not able to assess the extent of fractionation occurring in the situation of 100% MDN at the level of primary producers (See assumptions in \[[@B13]\]). As such, we calculated two %MDN estimates based on no fractionation (MEM = 11.2‰) and maximum fractionation of 4‰ (MEM = 7.2‰), which is a typical maximum level of fractionation in vegetation from atmospheric N~2~ that is observed in the Clatse-Neekas non-salmon habitats (Mathewson & Reimchen unpublished data). This model assumes that invertebrate trophic level does not differ above and below the falls and that the marine end-member for vegetation δ^15^N values is represented by salmon tissue.

Author\'s contributions
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MDH conducted the field research, sorted and processed the invertebrate samples, performed the statistical analyses, and drafted the manuscript. TER conceived of the study, participated in its design and coordination, and contributed to the manuscript preparation. All authors read and approved the final draft.

Acknowledgements
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We would like to thank Carsten Brinkmeier, Mike Windsor, Dan Windsor and Chester Starr for field assistance, Dr. Richard Ring for laboratory support and discussion, Myles Stocki for stable isotope analysis Dr. Bristol Foster for the zodiac, Larry Jorgenson for logistical assistance, and Rob Bennett for identification of the Arachnids. Thanks to the Heiltsuk First Nations and the Raincoast Conservation Society for local project support. This work was supported by funds from the David Suzuki Foundation and the Natural Sciences and Engineering Research operating grant (NRC2354) to TER, and from an NSERC -- Industrial Post-graduate Scholarship to MDH.