Fabrication and Validation of Rapid Test for MonitoringAmmonium Water Quality.

Hossam E.M. SayourBiomedical chemistry unit, Dept. of Chemistry and Nutritional

Deficiency Disorders, Animal Health Research Institute,12618 Dokki,Gizza, Cairo, Egypt

Abstract:

A rapid, field, equipment-free and micro-

colorimetric assay that are based on a standard 96-well

micro-titer plate format and color matching that simplify

the quantification of total ammonium/ammonia was

proposed. Rapid analysis of such parameters in aquatic

systems is essential for monitoring and maintaining both

healthy aquatic environments and fish stocks in

aquacultures. Monitoring of municipal and industrial

wastewater facilities to comply with local, state or

federal water quality effluent regulations for total

ammonium/ammonia of certain threshold limits are highly

recommended. Traditional techniques for quantifying

ammonium/ammonia concentrations often require laborious

or expensive specialized equipment making these analyses

difficult. An alternative colorimetric method to those

traditional techniques that based on Berthelot’s reaction

was investigated to optimize the reaction conditions for

better performance characteristics. Both the reaction

time (7-minute per sample) and the test cost were

1

minimized (less than 0.5 L.E per sample). The proposed

method allow parallel multi-determination of small

sample volumes (50 µL), working range 0.5-10 ppm,

detection limit ≤0.1 ppm of total ammonium/ammonia in

both fresh water and brackish water of maximum salinity

of 40 parts per thousand, precision of at least 10% and

compares favorably with standard reference analytical

procedures upon performing test of validation. Routine

use of this method in the laboratory, aquaculture

associated with microalgae growth or wastewater streams

for monitoring total ammonium/ammonia concentrations

demonstrates feasibility, accuracy and highly

reproducibility among different users.

Keywords: field rapid test, micro-titer plate, visual,

ammonium kits, equipment free, aquaculture, water

quality.

Introduction:

Ammonia is one of the most typical of spoilage

products since it arises from a variety of sources common

to all biological tissues. It is produced by the action

of microorganism as well as endogenous enzymes through

de-amination of proteins and amino acids and though

breakdown of purines and various amines. Ammonia plays an

important part in the nitrogen cycle of many biological

and industrial processes as well as in shallow and

2

eutrophicated waters (. Ivančič and Degobbis, 1984;

Clinch et al, 1988; van Staden and Taljaard, 1997; Strauss et

al, 1991) and is one of the main components in many

industrial effluent streams.

The introduction of increasingly stringent

legislation on water quality has enhanced the need for

on-line analytical monitors (van Staden and Taljaard,

1997). In aquaculture control water quality includes all

physical, chemical and biological factors that influence

the beneficial use of water. Fish influence water quality

through processes like nitrogen metabolism and

respiration. Some water quality parameters are more

likely to be involved with fish losses such as dissolved

oxygen, temperature, salinity, ammonia, nitrite and

nitrate. Others, such as pH, alkalinity, hardness and

clarity affect fish, but usually are not directly toxic.

High concentrations of total ammonium/ammonia can

adversely affect growth and stocking density in a variety

of species. The toxic levels of un-ionised ammonia (UIA)

for short-term exposure usually are reported to lie

between 0.6 and 2 mg/l (Pillay, 1992; Gijzen et. al., 2004).

The lethal ammonia concentration for most warm water fish

is between 0.6–2.0 mg/L ammonia nitrogen (NH3-N). Tilapia

begins to die when unionized ammonia concentrations are

higher than 2.0 mg/L NH3-N. However, unionized ammonia

concentrations as low as 1.0 mg/L NH3-N will decrease

3

growth and performance in tilapia as reported before

(Riche and Garling, 2003). The acute toxicity of UIA to

Nile tilapia was measured in a 96-h static test. The

median lethal concentration (LC50) was 1.46 mg/L UIA at

24 and 48 h post-exposure, 1.33 mg/L at 72 h post-

exposure, and 0.98 mg/L at 96 h post-exposure (Evans et.

al., 2006). In general, NH3-N concentrations should be

held below 0.05 mg /L and total ammonia nitrogen (TAN)

concentrations below 1.0 mg/ L for long-term exposure

(Timmons et al. 2002; Isla, et. al., 2008). Consequently,

strategies have been developed to monitor and remove this

potentially harmful chemical.

Several procedures have been proposed for the

determination of the ammonium ion /ammonia (Ivančič and

Degobbis, 1984; Clinch et al, 1988; van Son et al, 1983). The

three spectrophotometric methods that appear to be used

almost universally are the indophenol blue method based

on Berthelot reaction (Babko and Pilipenko, 1976; Krug et

al, 1979; Aminot et al, 1997; Monliner-Martínez et al ,2005),

the gas diffusion/acid-base indicator procedure (Standing

Committee of Analysts, 1981) and the use of Nessler’s

reagent (Schmidt et al, 1984). Other methods include

titrations, potentiometric determinations (Yost, 2007),

the use of ammonia selective electrodes (Perry and

Phillips, 1995) and determination of ammonia using carbon

dioxide laser photocoustic spectroscopy.

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Ammonia determination based on Berthelot reaction is

known to be a standard method in many institutions

(Swedish Standard Methods, 1976; Krug et al, 1979; United

States Environmental Protection Agency, 1983; American

Public Health Association, 2005) until today. Traditional

methods for quantifying ammonium ion /ammonia

concentrations typically use colorimetric based

techniques in conjunction with standard

spectrophotometers, which although precise and reliable,

often require laborious procedures or specialized

equipment making these analyses difficult. For example,

many of these classical manual techniques require large

sample and reagent volumes and each sample must be

processed separately (Strickl and Parsons, 1972, American

Public Health Association, 2005). Alternatively, more

modern techniques use flow injection or segmented flow

auto-analyzers to automate ammonium/ammonia analysis

process (Hager, et al, 1972; Woodward and Rees, 2001;

Yebra-Biurrun, 2009). But these instruments are expensive

and require specialized training to successfully operate.

Towards developing more streamlined alternatives, several

researchers have advanced 96-well microplate-based

ammonium/ammonia analyses techniques (Baudinet and

Galgani, 1991; Hernandez-Lopez and Vargas-Albores, 2003;

Laskov, et al, 2007; Poulin and Pelletier, 2007; Johnson,

et al, 2011). These techniques can greatly reduce sample

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volume, have proven to be a quick, of relatively low-cost

alternative to conventional auto-analyzer techniques

because they speed up the analysis and do not require

expensive specialized equipment. But when small-scale

farmers control their aquacultures the cost of these

laboratory-based monitoring techniques that requiring

micro-titer plate reader will be still high. Moreover, in

critical situations where decisions have to be made in

the shortest possible time frame, the conventional way of

doing laboratory analysis is often unsatisfactory due to

the fact that by the time the analysis result is

available, the problem might have disappeared

[ammonium/ammonia within an aquaculture pond is

continuously changing “Pond Dynamics” and must be

understood and monitored if a pond system is to be

controlled effectively (Boyed, 1998)]

In such cases rapid field test kits can provide

waning alarm and immediate answers to the cause and

corrective control action can be made. A commercial lab-

fabricated rapid test colorimetric micro-titer plate

based, user-friendly, on-site, robust, equipment-free,

specific and semi-quantitative will be proposed,

fabricated and validated for screening water quality of

different sources. This proposed rapid field test for

monitoring ammonium/ammonia to judge the waters quality

(Fresh/brackish ponds in aquacultures and

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surface/underground waters streams from different

sources) will be validated according to EURACHEM

Recommendation, United States Environmental Verification

Program and AOAC Performance Tested Methods Program

(EURACHEM, 1998; Unger-Heumann, 1996; Soler, 2006;

U.S.EPA ETV, 2008; AOAC, 2009).

Experimental:Materials:

Phenol, acetone, methyl alcohol and ammonium sulphate

supported by Merck. Sodium hypochlorite not less than 5%

chlorine of commercial grade was supported from local

markets. Sodium nitroprusside was supported by Prolabo,

France. Micro-titer flat bottomed plates were purchased

from bio-gene, Germany.

Reagents:

All regents are prepared according to previously reported

methods before (Koroleff, 1969; Babko and Pilipenko,

1976)

Instruments:

All pH measurements were performed on Jenway 3510

(UK). All colorimetric measurements were made with micro-

titer plate reader Dynatech MR5000 (USA) absorbance wasmeasured at 630 nm.

Technology Description:

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The test kit is a field technology for determining

total NH4+ content in water samples. The test kit is self-

contained and does not require additional analysis

instruments. Analysis results are obtained in 7 minutes,

and used equipment is recyclable. The complete ammonia

NH4+/NH3 rapid test kit contains 96-well micro-titer plate

of 350 µL-reaction well, three reaction reagents droppers

(R1, R2, R3), 1-3 ml Pasteur- plastic dropper for watersample and a color scale comparison card. The color scale

comparison card for NH4+/NH3 rapid test kit reads 0.5 ppm,

1 ppm, 5 ppm and 10 ppm.. The limit of detection of the

test kit is 100 ppb for water samples .

Fabricated rapid test directions for use:The NH4

+/NH3 rapid test kit is a field technology for

determining total NH4+ content in water samples. An

instruction manual is included with each test kit.

1. In chosen well (A 1-12 to H 1-12), take one drop of

sample by Pasteur- plastic pipette.

2. Drop one drop from reagent (1), reagent (2) and (3);

respectively to each sample.

3. Shake the plate gently to avoid splash.

4. After seven minutes, put the plate on the colour scale

card to match sample color with colors correspond to

ammonium standards 0.5-10 ppm.

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5. Whenever you need to re-test the validity of reagents,

1 ppm ammonium standard provided with the test by

carrying steps 1-4 using another clean Pasteur pipette.

6. Rinse many times the used plate and Pasteur pipettes

after testing to avoid erroneous results.

7. Hit the plate many times while inverted on a tissue

paper to repel water droplets outside the wells, and then

leave it to dry in inverted position.

Validation Test Design:

All preparation, calibration, and analyses were

performed according to the ammonia NH4+/NH3 rapid test

directions for use stated above. The test kit was verified

in terms of its performance where results were recorded

manually. The results from the ammonia NH4+/NH3 test kits

were compared with those from the indophenols blue reference

method (American Public Health Association, 2005) toquantitatively assess precision, accuracy, linearity, and

detection limit.

Multiple aliquots of performance test samples

(fresh/brakish ponds in aquacultures and

surface/underground waters and were analyzed to assess

precision. Two operators used multiple the ammonia NH4+/NH3

rapid tests in order to analyze all the samples, so it wasassumed that kit-to-kit variability was similar for both

9

operators. Consequently, qualitative observations could be

made on operator bias.

Matrix interference effects were assessed by

challenging the ammonia NH4+/NH3 test kit with performance

test samples of known ammonium concentrations containing

both low-level and high-level interferences. False positives

and negatives were evaluated relative to the recently

established 5 ppm maximum contaminant level and 0.5 ppm as

minimum contaminant level (safe limit) for ammonium in

majority of aquaculture ponds waters

Test Samples:

Three types of samples were used in the

verification test, as shown in Table 1: quality control

(QC) samples, performance test (PT) samples, and

environmental water samples. The QC and PT samples were

prepared from certified standards. The QC sample

concentrations for ammonium were targeted at that 0.5

ppm level as stated above in the matrix interference

effect. The PT samples were targeted to range from 10%

to 1,000% of that level, i.e., from 0.05 to 5 ppm. The

environmental water samples were collected from various

sources [fresh (Kafr El-Shiekh) / brackish of maximum

salinity 40 parts per thouthands (Qarun lake) ponds in

aquacultures, surface (Burulus lake) / underground (El-

Natron valley) waters]. All samples were analyzed using

the ammonia NH4+/NH3 test kits and a reference method.

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Every tenth sample was analyzed twice by the reference

method to document the reference method’s precision. The

criterion in sampling rates is presented in table 1.

Results and discussion:

Effect of increasing phenate concentration on indophenols

formation:

The influence of phenate on the formation of

sensitized indophenols has been investigated using 1 ppm

ammonium, 0.6% sodium hypochlorite (Babko and Pilipinko,

1976) which represented short reaction time (20 min) and

0.0169% sodium nitroprusside at optimum pH (more than 12)

(Koroleff, 1969). The phenate concentration ranged

between 0.35-3.75% measured at arbitrary short 3 min.

time and room temperature 22-27°C. The results are shown

in fig.1 where maximum and constant absorbance of the

sensitized indophenols using 1.95- 3.75% thus, 1.95% of

phenate reagent was recommended for further

investigation. The result is in consistent with the fact

that the indophenols dye formed in presence of excess

phenol where ρ-aminophenol is formed first then it

further reacts with sodium hypochlorite to form

quinochloroimine in the second step. Finally

quinochloroimine reacts with the second molecule of

phenol to form indo phenol (Babko and Pilipinko, 1976).

11

Effect of increasing hypochlorite concentration onindophenols formation:

The effect of sodium hypochlorite on the formation of

sensitized indophenols has been investigated using 1 ppm

ammonium, 1.95% phenate and 0.0169% sodium nitroprusside

at optimum pH (more than 12). The sodium hypochlorite

concentration ranged between 0.01-2% measured at 3 min.

time and room temperature 22-27°C. The results are shown

in fig.2 where maximum and constant absorbance of the

sensitized indophenols using 1.25- 2 % thus, 1.25% of

hypochlorite reagent was recommended for the proposed

method. Ammonia determination was conducted according to

(Koroleff, 1969). Some modifications have been studied

such as adjustment of the reagent concentrations

suggested by Koroleff, the formation of indophenols blue

takes several hours at room temperature. The reaction can

be accelerated (i) by increasing the concentration of the

reagents as has been done in the majority of recent

procedures, (ii) by increasing the reaction pH to more than

12, (Scheiner, 1975), (iii) by increasing the reaction

temperature as in most automatic determinations and (iv) by

irradiation of samples with long-wave ultraviolet light

(Liddicoat et al., 1975). Babko and Pilipinko (1976)

previously concluded the maximum reaction conditions for

indophenols sensitization at room temperature 22-27°C and

20 min. reaction time which is coherent with our need for

12

ammonia rapid field test. Moreover, in previous studies

(Koroleff 1976; Baudinet and Galgani, 1991; Grasshoff,

1999; Hernandez-Lopez, 2003; Jonson 2011) 0.001- 0.068 %

sodium nitroprusside was used for color enhancement it

has no share in the indophenol formation reaction as

shown in (table 2). So a moderate concentration 0.0169%

sodium nitroprusside (Korokeff, 1969) will be

recommended; for the further investigations.

Calibration curves at different times:

Complete calibrations of ammonium concentration

ranges 0.5-10 ppm were carried out at different reaction

times to study the stability of indophenols formation at

the shortest time (fig.3). The calibration graph obtained

under optimum conditions given in the recommended

procedure adhered to Beer’s law up to 10 ppm of ammonium

after only 7 minutes. All these alternatives have been

studied it was found that a two-fold increase of the main

reagent concentrations (phenate and hypochlorite) is

advantageous as the reaction is completed in shortest

time ever. The reaction time proposed in this work (7

min.) is one-third shorter than the reaction time in

previous study (Babko and Pilipinko, 1976) without need

for either temperature control (at room temperature 22-

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27°C) or irradiation for shortening reaction time

(Liddicoat et al., 1975).

Fabrication and evaluation of rapid field test for total

ammonium/ammonia monitoring in waters:

Instruments used for measuring device colors

include colorimeters or micro-titer plate reader, auto-

analyzers and spectrophotometers in addition

alternatively less expensive digital cameras and image

scanners.  A simple experiment in which the principle of

absorbance may be demonstrated using digital color image

analysis of a photographed picture of solutions obtained

by a digital camera using a desktop scanner was published

before (Jansen et.al., 1998; Mathews et.al, 2004; Kohl et.al.,

2006). After scanning a standard 96-well micro-titer

plate, color gradations in the slots can be analyzed

quantitatively and be used for the estimation of enzyme

kinetics, binding assays or concentration determination.

Image analysis of the intensity of the complementary

color (blue) for each solution produced data that

conformed to the Beer–Lambert law.

Colorimetry is based on subjective visual

experiences - so called "color matching" (Sällström,

1998). Human trichromatic vision enables detection of

more than two million surface colors, wavelength

14

discrimination with precision <1 nm, and the unique

capacity to exploit color information for multiple

applications in myriad settings. A number of chemical

test kits can be bought that work according to the

principle of color matching. There are kits to test water

(e.g. in rivers, lakes, ponds and even swimming pools).

Gardeners use kits to test the quality of soil and some

kits are used in medical diagnosis, e.g. showing glucose

in urine which is a symptom of diabetes. The kits give

semi-quantitative results which mean that they give only

an approximate value. Although they give approximate

values but they fit to purpose. For example, universal

indicator solutions are mixtures of acid-base indicators

and their colour depends on pH. They can be used,

therefore, to find, very quickly, the approximate pH of a

solution (Sayour et. al., 2010). However, if a more exact

determination of the pH was needed, it should be

determined by using instruments.

Color matching sometimes gives only a “yes/no” answer

which can be entirely satisfactory in some instances, as

in the case of a pregnancy test or diagnosis of

infectious diseases. In addition these tests are cheap,

quick, easy to carry out and therefore more practical and

preferable, in certain cases, than other, more

sophisticated methods (Kenkel, 1994).

15

WHO recommends the screening infectious diseases or

food contaminants which are a leading cause of death in

developing countries by rapid test kits to avoid epidemic

crises even in the developed countries. The EPA’s

National Exposure Research Laboratory and its

verification organization partner, Battelle, operate the

Advanced Monitoring Systems (AMS) Center under

Environmental Technology Verification (ETV). The AMS

Center recently evaluated the performance of portable

analyzers based on sensors or biosensors, quick “smart”

test kits for pollutants in water purpose of meeting

legal requirements.

Small-scale farmers control their aquacultures the

cost of these laboratory-based monitoring techniques that

requiring micro-titer plate reader will be still high.

Moreover, in critical situations where decisions have to

be made in the shortest possible time frame, the

conventional way of doing laboratory analysis is often

unsatisfactory due to the fact that by the time the

analysis result is available, the problem might have

disappeared [ammonium/ammonia within an aquaculture pond

is continuously changing “Pond Dynamics” and must be

understood and monitored if a pond system is to be

controlled effectively (Boyed, 1998)]. In such cases

rapid screening field test kits can provide warning alarm

and immediate answers to the cause and corrective control

16

action can be made. Even in the laboratory the rapid

tests are suitable for checking plausibility, e.g. if a

mix- up of samples is suspected, they allow fast

clarification.

A commercial lab-fabricated rapid test colorimetric

micro-titer plate based, user-friendly, on-site, robust,

equipment-free, specific and very cheap is proposed for

determining concentrations of total ammonium/ammonia in

waters. The results of total ammonium/ammonia

concentrations are simply red visually by matching the

colors developed (according to above test directions for

use) on micro-titer plate by mounting it over color scale

card (image 1). For some field applications, visual

reading of the color of the reaction product in the wells

of the micro-titer plates is acceptable. Taking in

consideration that human trichromatic vision ability is

predicated on a normal complement of L, M and S cones

(Rabin, 2010). Hereditary color vision deficiency (CVD;

8% of males, 0.5% females) derives from a lack of L cones

(protanopia 1%) or M cones (deuteranopia 1%); or from a

cone spectral sensitivity shift (protanomaly: L shifted

toward M in 1%; deuteranomaly: M shifted toward L in 5%).

This why the choice calibration points on color scale

card should be sharp colors enough to be not confused and

misjudged. Two million surface colors and wavelength

discrimination (‹ 1 nm) in healthy persons can judge the

17

in between hues of colors corresponding to ammonium

concentrations even if they are not included in the color

scale card (Rabin, 2010; Ruitenberg, 1976).

This proposed rapid test is characterized by the

ease of multi-determination of 96 samples simultaneously

over other commercial available synonymous kits in the

markets which are used in sample by sample mode. The

applied concentration range (0.5-10 ppm of total

ammonium/ammonia) is covering most application fields for

water quality field monitoring (Fresh/marine ponds in

aquacultures and surface/underground waters). Moreover,

to the best of the author knowledge this proposed

equipment-free kit which is consuming the lowest both

sample and reagent volumes ever in addition to that the

fact that test kit needs only 7 minutes for the color

stability (shortest time ever) at room temperature 22-27

°C (no need for incubation) as shown in (table 2).

Validation of proposed rapid test kit for monitoring

waters of different sources:

According to EURACHEM Recommendations, United States

Environmental Verification Program and AOAC Performance

Tested Methods Program (EURACHEM, 1998; Unger-Heumann,

1996; Soler, 2006; U.S.EPA ETV, 2008; AOAC, 2009) the

proposed rapid test for total ammonium/ammonia will be

validated.

18

The validation will be performed according to criterion

of results of test samples stated before (table 1) for

both technical and non-technical operators and both QC,

PT and real samples. These results are presented in table

3 for both QC and PT while the real samples are presented

in table 4. The validation parameters are calculated

based on all data presented in both table 3, 4 and a

summary of qualitative accuracy results is presented in

table 5. Precision is evaluated in terms of relative

standard deviation (RSD) for both PT and QC samples where

RSD is found to equal 0%. The results for three of the

replicate sets were <0.5 ppm. The remaining replicate

sets for the non-technical operator had an RSD ranging

from 29 to 50%, and the remaining replicate set for the

technical operator had an RSD of 29%. For the freshwater

and brackish pond samples all results for two of the

replicate sets were <0.5 ppm. The remaining sets had an

RSD of 29 to 100% for the non-technical operator and 0 to

18% for the technical operator. The linearity of the

rapid test kit readings was assessed by means of a linear

regression of results against the reference method .

Rapid test kit for the non-technical operator, ppm = 0.90

(±0.086) x (reference, ppm) – 0.52 (±0.41) ppm, with r =

0.974, on the other hand for the technical operator, ppm

19

= 0.88 (±0.056) x (reference, ppm) - 0.45 (±0.27) ppm,

with r = 0.988.

The values in parentheses represent the 95% confidence

interval of the slope and intercept. Both regressions

show slopes that are significantly different from 1.0.

The rapid test results for both operators were all less

than the reference value, but in particular the technical

operator’s results were all identical (10 ppm), providing

no variation with which to quantitatively assess the

minimum detection limit (MDL). The non-technical operator

reported total ammonium/ammonia between 0.5 and 10 ppm.

Since the rapid test kit is only semi-quantitative, no

MDL was calculated from these data. Qualitative

indication of the rapid test kit (MDL) can be obtained

from the results of the PT2 and PT3 samples of

concentrations 0.5 and 1.0 ppm, respectively. With the

0.5-ppb samples, the non-technical operator reported

results of <5 ppb, whereas the technical operator

reported results of 0.5 ppm. With the 1.0-ppm samples,

all results were 1.0 ppm except for one result of 0.5 ppm

with the non-technical operator. The minimum change in

the reagent blank color can be observed by both operators

could be metaphorically equaled 0.1ppm.

20

Conclusion:

A rapid, field, equipment free and micro-

colorimetric assays that are based on a standard 96-well

micro-titer plate format and color matching that simplify

the quantification of total ammonium/ammonia with a

working linear range (0.5-10 ppm) was erased. Reaction

conditions of rapid test were optimized to give an ideal

rapid field test. The proposed rapid field test kit

showed fair validation parameters (accuracy, precision,

linearity and MLD) for different waters samples of

different sources upon using by both technical and non-

technical operators. This equipment-free technique offers

an alternative to standard commercial ammonium tests and

significantly decreases the cost (about 0.5 L.E per

sample) and time (7-min time test) of processing while

maintaining high precision and sensitivity.

In the future more rapid tests for other critical

parameters in environmental, food safety, health care

and industrial quality control applications era should

be investigated, fabricated and validated. These kinds

of tests may give approximate results (semi-

quantitative) but fit many purposes for smart decisions

21

to deal efficiently with environmental crises, save our

lives and money in production activities .

22

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Figure1: Effect of phenate conc. on indophenol formation upon

reaction with 1ppm ammonium with 0.6% hypochorite and 0.0169% sodium

nitroprusside, after 3min.

29

Figure 2: Effect of hypochlorite conc. on indophenol formation upon

reaction with 1ppm ammonium with 1.95% phenate and 0.0169% sodium

nitroprusside, after 3min.

Figure 3. Calibration curves 0.5, 1, 5, 10 ppm ammonium upon additionof 1.95% phenate and 1.25% hypochlorite and 0.0169% sodium nitroprusside at different times (1-9 min).

30

Image 1: Color card scale of total ammonium/ammonia.Table1: Criterion of Test Samples protocol for

Verification of the NH4+/NH3 test kit

Type of Sample

Sample Characteristics

Concentration

ppmNo. of Samples

Quality

control

Reagent blank 0 10% of total

number

Laboratory FortifiedMatrix

0.25 1 for each

site

Quality Control Sample

0.5 10% of total

number

St. ammonium

solution (1)

0. 5 3

31

Performance

test

St. ammonium

solution (2)

1.0 3

St. ammonium

solution (3)

5.0 3

St. ammonium

solution (4)

10 3

St. ammonium + low

spike

0.5+low 6

St. ammonium + high

spike

0.5+high 6

Real

samples

Fresh fish ponds

Unknown

3

Marine fish ponds

(sal. 40ppt)

3

Surface water

(Nile)

3

Underground water 3

Municipal

wastewater

3

Industrial

wastewater

3

32

Table2: Compromise of previous studies reaction conditions, volume measured and working range to those of the proposed

Ref

.

Final phenol conc. Hypochlor

ite

Nitroprus

side

Sample

volume

Vol.

measure

d

Wave

lengt

h

Temp Time Conc.

Range

µg

1969

0.33% in 95% ethyl 0.084% 0.0169% 50 ml 10 cm

1cm

640

nm

22-

27°C1 hr 5-500

0.5-50

197

6

1% in

0.296%acetone,

1.28% methyl,

0.43%NaOH

0.6% Not used 25 ml 2 cm

cuv.

610

nm

22-

27°C

20

minUp to 4

199 1.36% 0.136% 0.068% 260µL 440 µL 620

nm

60°C 2hr 0-0.05

33

1999

0.29% in30%ethyl 0.010 % 0.002% 50 ml 10 cm

1 cm

630

nm

37°C 1 hr 0.042-

127.5

34

200

3

0.625% 0.093% 0.031% 250 µL 320 µL 650

nm

60°C 1 hr 0.015-

0.425

201

1

0.165% 0.217% 0.0013% 200 µL 230 µL 630

nm

37°C 2 hr 0.004-

0.2

201

2This

metho

d

1.95% in 0.592%

acetone in 2.5%

methyl in 1.25%NaOH

1.25% 0.0169% 50 µL 200 µL 630

nm

22-

27°C

7

min

Up to2

µg

34

Table 3: Results of QC samples, performance test of both reagent blank and standardsSample Non-

technical

operator

Technica

l

operator

Reference

method

Sample Non-

technical

operator

Technical

operator

Reference

method

Reagent

blank1

‹ 0.5 ‹ 0.5 0.12 PT2 st3 5 5 5.7

Reagent

blank2

‹ 0.5 ‹ 0.5 0.11 PT3 st3 5 5 5.7

Reagent

blank3

NA ‹ 0.5 0.12 PT1 st4 10 ‹10 9.1

Reagent

blank4

NA ‹ 0.5 0.11 PT2 st4 10 ‹10 9.1

Reagent

blank5

NA ‹ 0.5 0.1 PT3 st4 10 ‹10 9.1

Reagent

blank6

‹ 0.5 ‹ 0.5 0.1 PT 1 st5+2

spike

5 ‹10 7.3

QC sample1 1 5 1.9 PT 2 st5+2

spike

5 ‹10 7.2

QC sample2 1 5 1.85 PT 3 st5+2

spike

5 ‹10 7.2

35

QC sample3 1 5 1.9 PT 4 st5+2

spike

5 ‹10 7.2

QC sample4 NA 5 1.9 PT 5 st5+2

spike

10 ‹10 7.2

QC sample5 NA 5 1.9 PT 6 st5+2

spike

5 ‹10 7.2

PT1 st1 0.5 ›0.5 0.55 PT 1 st5+5

spike

10 10 10.3

PT2 st1 0.5 ›0.5 0.6 PT 2 st5+5

spike

10 10 10.3

PT3 st1 0.5 ›0.5 0.55 PT 3 st5+5

spike

‹10 10 10.3

PT1 st2 1 ›1 1.6 PT 4 st5+5

spike

10 10 10.3

PT2 st2 1 ›1 1.6 PT 5 st5+5

spike

‹10 10 10.3

PT3 st2 1 ›1 1.6 PT 6 st5+5

spike

›5 10 10.3

PT1 st3 5 5 5.7

36

Table 4: Results of real samples from freshwater& brackish ponds, surface& underground

waters Sample Non-technical

operator

Technical

operator

Reference standard method

Reagent blank ‹0.5 ‹0.5 ‹0.1QC sample 1 1 1.1Fresh water pond1 (Kafr El-Shiekh) 1 ›0.5 0.72

Fresh water pond2(Kafr El-Shiekh) 1 1 1.25

Fresh water pond3(Kafr El-Shiekh) ‹0.5 ‹0.5 0.33

Reagent blank ‹0.5 ‹0.5 ‹0.5QC sample ›0.5 ›0.5 1Brackish water pond1 (Qarun lake) 1 1 1.2

Brackish water pond2(Qarun lake) 5 ‹5 2.1

Brackish water pond3(Qarun lake) ›5 ›5 5.1

Reagent blank ›0.5 ›0.5 0.1QC sample ›0.5 1 0.9Surface water1 (Buruls lake) 0.5 0.5 0.52

Surface water2(Buruls lake) ›0.5 1 1.1

37

Surface water3(Buruls lake) 1 ›1 2.1

Reagent blank ‹0.5 ‹0.5 0.1QC sample 1 1 1Under ground water1 (El-Natron

valley)

‹0.5 ‹0.5 ‹0.1

Under ground water2 (El-Natron

valley)

›0.5 ›0.5 0.68

Under ground water 3(El-Natron

valley)

›0.5 ›0.5 1.1

38

Table 5 Summary of qualitative accuracy results within 25%

Samples % accuracy non-technical

operator within 25%

% accuracy

technical operator

within 25%PT 71 55Fresh ponds (Kafr El-

Skiekh)

95 58

Brackish ponds (qarun lake) 72 93Surface water (Burulus

lake)

55 56

Underground water (El-

Natron valley)

92 97

39

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41