Laboratory PT #2

October 2023

2nd Round of Mercury Laboratory Proficiency Testing

Final Report

1 |Page1. Objective
The main purpose of this proficiency testing (PT) programme was to evaluate the performance of
mercury analyses conducted by the laboratories. It was expected to provide the individual
proficiency levels of participating laboratories and the collective mercury monitoring capacity in theregion.

2. Proficiency testing provider
This PT was organized by National Institute for Minamata Disease (NIMD) in collaboration with
United Nations Environmental Programme, Regional Office for Asia and the Pacific (UNEP-ROAP).
Asian Institute of Technology Regional Resource Centre for Asia and the Pacific (AIT RRC.AP)
distributed the test item and collected the analytical results. IDEA Consultants, Inc. prepared thetest item for this PT.

3. Implementation period
Call for participation:

September – December 2022

Test item distribution:

February – March 2023

Duration of test (analysis):

Until 8 June 2023

4. Participation fee
Free.

5. Test item (sample) and parameter
One (1) dried fish sample was used for analysing total mercury concentration, methylmercury
concentration or both (participating laboratories shoes the preferred option).

5.1. Test item preparation
The test item was made from the muscle tissue of a single yellowfin tuna. The tissue wascollected, freeze-dried, finely powdered, and sieved (106 μm) to ensure sufficient homogeneity. The
test item was also sterilized using gamma-ray.
Approximately 5 g of each test item was packaged in a brown glass bottle. The bottles of test
items were sealed in aluminium-lined laminate packs for distribution to the participants.

2 |Page5.2. Homogeneity testing
The following homogeneity testing of the test item was conducted to ensure that there were no
significant differences in the mercury concentrations in the test items between bottles that could
affect the result of the PT. Since the test item was collected from a single species and the ratio of
total mercury and methylmercury was assumed to be stable between test items, homogeneity
testing was conducted by analysing total mercury.
After the preparation of the test item (packed in bottles), ten bottles were selected, and the total
mercury analysis (acid digestion - aeration CVAAS measurement) was performed twice for eachtest item in a bottle.
The homogeneity of the test item was then analysed from the results of the total mercury
concentrations. Since the analytical results include the uncertainty due to the (chemical) analyticalprocedure, homogeneity was judged by the following criterion:

Criterion: Ss ≦ √𝐹1 × (0.3 × 𝜎𝑒𝑝 )2 + 𝐹2 × 𝑆𝑤 2
Ss: relative standard deviation of homogeneity testing
σep: (expected) relative standard deviation of the reported results from participants
wi2 =Σ (xgm2 - xg2) / (m-1)

xgm: result of m times analysis of the bottle

Sw2 =Σ wi2 / g

xg: average of the result of each bottle

F1 and F2 are values which are calculated from the probability distribution. In this homogeneity
testing (10 bottles testing), F1 and F2 were applied following numbers:
F1 = 1.88

F2 = 1.01(Even though these values are referred from the Annex B of ISO13528:2015, they are introduced
from the random variables of χ2 distribution and F distribution.)
Analysis results of this homogeneity testing are as follows:
Ss = 0.0090
Sw2 = 0.000934
Also, relative standard deviation of the results from the participants (used for evaluation) was asfollows:
σep = 0.0691
This standard deviation should be used the value which was used for the performance evaluation
for participants. As described in 8.2, the performance of participants was evaluated from themedian and normalized interquartile range (NIQR) of the results, thus relative NIQR was used for theconfirmation of the criterion.
Therefore, above criterion was judged as follows:
3 |Page√𝐹1 × (0.3 × 𝜎𝑒𝑝 )2 + 𝐹2 × 𝑆𝑤 2
= √1.88 × (0.3 × 0.0691)2 + 1.01 × 0.000934= 0.0418 > 0.0090 (Ss)
It was confirmed that the test item was sufficiently homogeneous to evaluate the performance ofparticipants’ results.

5.3. Stability testing
To ensure that the concentration of the target parameter (total mercury) was maintained without
significant changes during the PT, a following stability testing was conducted after the duration of
the analysis. Since the test item was collected from a single species and the ratio of total mercury
and methylmercury was assumed to be stable between test items, stability testing was conductedby total mercury analysis.
Ten test items were selected from the stored (not distributed to participants), and total mercury
analysis (acid digestion - aeration CVAAS measurement) was performed twice for each test item ina bottle.
The stability of the test item was then analysed by comparing the results before and after the
distribution of the test item. The stability of the test item was judged by the following criterion:

Criterion: | x - ȳ | ≦ 0.3 × σpt + 2 × √𝑢(𝑥) 2 + 𝑢(𝑦) 2
x: average of the item before distributionȳ: average of the item after proficiency testingu(x): uncertainty of the item before distribution
u(y): uncertainty of the item after proficiency testing
σpt: standard deviation for the proficiency evaluation. In this program, NIQR was applied toevaluation of performance of the participant.
Analysis results of test items before and after the PT are as follows:x = 4.126

u(x) = 0.037

ȳ = 4.139

u(y) = 0.047

Standard deviation of the result of all participants was as follows:
σpt = 0.279
This standard deviation should be used the value which was used for the performance evaluation
for participants. As described in 8.2, the performance of participants was evaluated from the
4 |Pagemedian and normalized interquartile range (NIQR) of the results, thus NIQR was used for theconfirmation of the criterion.
Therefore, above criterion was judged as follows:
0.3 × σpt + 2 × √𝑢(𝑥) 2 + 𝑢(𝑦) 2= 0.3 × 0.279 + 2 × √0.0372 + 0.0472= 0.203 > 0.013 (| x - ȳ |)It was confirmed that the concentration of total mercury in test item was not changed during thePT.

6. Target parameter
The target parameter of the PT was total mercury and/or methylmercury. Participants could
perform analysis and report either or both of total mercury/methylmercury. Participants conducted
three total mercury analyses and reported all results. Participants also conducted analysis ofmoisture in the test item. The result of moisture was used for the analysis of the reported data,
however, it was not the target of the PT, and the result of total mercury was not calculated bymoisture.
The moisture analysis procedure was instructed to the participants as follows:
1.

Take a test item of 100 mg or more and weigh it precisely.

2.

Dry the taken test item (100 °C, 2 hours).

3.

Weigh the dried test item again and calculate the moisture of the sample from the reducedmass.
It has also been instructed that the sample used for moisture analysis should not be used for total
mercury analysis.

7. Participating institutions
This PT was intended for public or university laboratories that perform mercury analysis. It was
requested to perform the analysis with a lower detection limit than 0.1 mg/kg on 0.5 g test item.
55 institutions registered in the PT and 48 institutions (total mercury) and 17 institutions(methylmercury) respectively reported the analysis results.
The number of participants for each parameter (total mercury/methylmercury) and status areshown in Table 1.

5 |PageTable 1 Number of participating laboratories for parameter and step
Number of laboratories
Category

Total mercury

Methylmercury

Registered55

19

Sample received

52

18

Result delivered

48

17

Laboratories are participated from all of UN Regions. Number of laboratories (who reported theresults) participated from each region is shown in Table 2.
Table 2 Number of participating laboratories per region
Number of laboratories
UN Region

6 |Page

Total mercury

Methylmercury

Africa62

Asia and the Pacific26

9Eastern Europe

11

Latin America and the Caribbean

72Western Europe and Other Group

83

8. Total Mercury Analysis Result
8.1. Basic statistic data of the PT result (total mercury)
The basic statistics of the result of PT are shown in Table 3.
Table 3 Summary of the results of the PT (total mercury)
Statistic data of the results (unit: mg/kg)
Average:

3.875

Median:4.046

Standard deviation:

0.791

Minimum

0.003

Maximum

4.707

25 percentiles

3.796

75 percentiles

4.173

Interquartile range (IQR)

0.377

Normalized IQR (NIQR)

0.279

Parameter related to distribution
Skewness of distribution-3.547

Kurtosis of distribution

14.88

The distribution of the results from the participants is shown in Fig. 1.

Fig. 1 Histogram of the report data (total mercury)
7 |PageSome registered participants were unable to receive the test item due to various issues such as
customs clearance, etc. There were also participants who were unable to report the analysis results
due to problems with the measuring instrument.
These statistical data were calculated from the average of each participant. There was one
participant who did not report 3 results (2 results were reported), but all data were used forstatistical analysis.
The distribution had a large kurtosis, indicating that a large number of reported values wereconcentrated around the median, while some other values deviated from it. This means theinterquartile range was relatively small compared to the standard deviation.
There were reported values that were much lower than the median. Thus, the skewness values
were negative. However, these data did not have a major impact on the performance evaluation
because the evaluation was estimated from the median and normalized interquartile range (NIQR).
Thus, performance was evaluated based on the data obtained from all reported values without
processing outliers.

8.2. Performance evaluation for participants
Median data of all laboratories was applied as agreement value. Performance of the results wasevaluated by the robust z score, which was calculated from the median and normalized interquartile
range (NIQR).
z score of each participant was calculated from the following equation.
z = [(average of reported result) – (median of all participants)] / NIQR
Performance of the result is classified by z score as follows:|z| ≦2: Performance is satisfactory (satisfactory)
2 < |z| < 3: Performance is questionable (caution)|z| ≧ 3: Performance is unsatisfactory (action)

8 |PageThe results and performances of laboratories are shown in Fig. 2.

Fig. 2 Result of each participant (total mercury)
The numbers of laboratories disaggregated for each z score range are shown in Table 4.
Table 4 Number of the laboratories in the range of z score (total mercury)z scorenz ≦ -33-3 <z< -22-2 ≦z≦ 2
40

2<z<3
3

z ≧3
0

As described in 8.1, IQR of reported results was relatively small. Therefore, satisfactory range ofthe result (mercury concentration) was relatively close and results around 21 % difference from themedian was the classified range of unsatisfactory result (absolute value of z-score over 3). Several
results had z-scores slightly above 2 in absolute value, but this does not immediately indicate poor
analytical procedure or situation.

9 |Page8.3. Regions of participating laboratories and the performances
Number of laboratories per region where the laboratories are located and the performance of the
PT are shown in Fig. 3.

Fig. 3 Number of laboratories per regions and performances (total mercury)
Regional difference was not examined due to the limited number of laboratories participating fromregions other than Asia and the Pacific being insufficient for comparison.

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8.4. Types of laboratories and the performances
Number of laboratories per type (academic, government, or non-government) and the performanceof the PT are shown in Fig. 4.

Fig. 4 Number of laboratories per types and performance (total mercury)
Statistical differences among the types of laboratories were not detected from the reported
results (Kruskal-Wallis one-way ANOVA on ranks, p=0.77).

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8.5. Analysis methods and results
The method of analysis was not specified for the PT and the participants performed analysis by
the method that they usually used in their routine analysis, or they were planning to use in the future.
The participants performed analysis of total mercury by the following methods:-

Thermal Decomposition Cold Vapour Atomic Absorption Spectrometry (TDAAS)-

Acid digestion, aeration Cold Vapour Atomic Absorption Spectrometry (CVAAS)-

Acid digestion, Cold Vapour Atomic Fluorescence Spectrometry (CVAFS)-

Acid digestion, Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

The distribution of the results from participants by analysis method is shown in Fig. 5.

Fig. 5 Distribution of the report data by analysis method (total mercury)
No significant difference was found among the analysis methods employed (Kruskal-Wallis oneway ANOVA on ranks, p=0.14). However, several data measured by CVAAS, which were significantly
different from the median. Since it is possible that errors in the data calculation process caused
these outliters, which might not be related to the analysis method. On the other hand, TDAAS
produced results with no outliers despite having the highest number of results. This can be
attributed to the specific instrument designed for mercury analysis, which requires no chemical
preparation and straightforward analysis procedure. Therefore, only a few parameters need to beconsidered to minimise error.

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9. Methylmercury Analysis Result
9.1. Basic statistic data of the PT result (Methylmercury)
The basic statistics of the methylmercury result of PT are shown in Table 5.
Table 5 Summary of the results of the PT (methylmercury)
Statistic data of the results (unit: mg/kg)
Average

3.394

Median3.557

Standard deviation

0.941

Minimum

0.027

Maximum

4.223

25 percentiles

3.380

75 percentiles

3.829

Interquartile range (IQR)

0.449

Normalized IQR (NIQR)

0.333

Parameter related to distribution
Skewness of distribution-3.145

Kurtosis of distribution

11.37

The distribution of the results from the participants is shown in Fig. 6.

Fig. 6 Histogram of the report data (methylmercury)
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Some registered participants did not receive the test item due to customs clearance issue, while
one did not report the analysis results.
These statistical data were calculated from the average of each participant. There was aparticipant who did not report 3 results (2 results were reported); however, all data were used for thestatistical analysis.
It was also shown in a large kurtosis of the distribution that relatively many reported values wereconcentrated in a narrow range around the median, while some reported values deviated from themedian. Thus, the results are in a relatively small interquartile range than the standard deviation.
There were reported values that were much lower than the median. Thus, the skewness values
were negative. However, these data did not have a major impact on the performance evaluation
because the evaluation was estimated from the median and normalized interquartile range (NIQR).
Thus, performance was evaluated based on the data obtained from all reported values without
processing outliers.

9.2. Performance evaluation for participants
Median data of all laboratories was applied as agreement value. Performance of the results wasevaluated by the robust z score, which was calculated from the median and normalized interquartile
range (NIQR).
z score of each participant was calculated from the following equation.
z = [(average of reported result) – (median of all participants)] / NIQR
Performance of the result is classified by z score as follows:|z| ≦2: Performance is satisfactory (satisfactory)
2 < |z| < 3: Performance is questionable (caution)|z| ≧ 3: Performance is unsatisfactory (action)

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The results and performances of laboratories are shown in Fig. 7.

Fig. 7 Result of each participant (methylmercury)
The numbers of laboratories disaggregated for each z score range are shown in Table 6.
Table 6 Number of the laboratories in the range of z score (methylmercury)z scorenz ≦ -31-3 <z< -2
1-2 ≦z≦ 2
14

2<z<3
1

z ≧3
0

As described in 9.1, IQR of reported results were relatively small. Therefore, satisfactory range ofthe result (mercury concentration) was relatively close and results around 28% difference from themedian was the classified range of unsatisfactory of the result. (absolute value of z-score over 3).
There was a result that had z-scores slightly above 2 in absolute value, but this does not
immediately indicate poor analytical procedure or situation.

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9.3. Regions of participating laboratories and the performances
Number of laboratories per region where the laboratories are located and the performance of the
PT are shown in Fig. 8.

Fig. 8 Number of laboratories per regions and performances
Regional difference was not examined due to the limited numbers of laboratories participating fromeach region being insufficient for comparison.

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9.4. Types of laboratories and the performances
Number of laboratories per type (academic, government, or non-government) and the performanceof the PT are shown in Fig. 9.

Fig. 9 Number of laboratories per types and performance (methylmercury)
Statistical differences among the types of laboratories were not detected from the reported
results (Kruskal-Wallis one-way ANOVA on ranks, p=0.62).

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9.5. Analysis methods and results
The method of analysis was not specified for the PT and the participants performed analysis by
the method that they usually used in their routine analysis, or they were planning to use in the future.
The participants performed analysis of methylmercury by some type of measuring instruments
after the solvent extraction of the target analyte. The measuring instruments used by participants
are as follows:-

Gas Chromatograph and Cold Vapour Atomic Fluorescent Spectrometer (GC/CVAFS)-

Gas Chromatograph and Electron Capture Detector (GC/ECD)-

Liquid Chromatograph and Inductively Conducted Plasma Mass Spectrometer (LC/ICP-MS)-

Thermal Decomposition Cold Vapour Atomic Absorption Spectrometer (TDAAS)-

Cold Vapour Atomic Absorption Spectrometer (CVAAS)*-

Inductively Coupled Plasma Mass Spectrometry (ICP-MS)*: About the report of CVAAS (one report), the method of extraction was not reported andit was unknown.

The distribution of the results from participants by analysis method is shown in Fig. 10.

Fig. 10 Distribution of the report data by analysis method (methylmercury)
Noticeable differences were not detected among the analysis methods used. (Kruskal-Wallis oneway ANOVA on ranks, p=0.28).

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10. Percentage of methylmercury
The summary of the percentage of methylmercury concentration against total mercury obtainedby the same participant is shown in Table 7.
Table 7 Summary of the percentage of methylmercury
Methylmercury / Total mercury (%)
Average

86.8

Median90.7

Standard deviation

23.6

Minimum

0.8

Maximum

107.8

The distribution of the percentage of methylmercury concentration is shown in Fig. 11.

Fig. 11 Histogram of the percentage of methylmercury against total mercury
The plots of the relationship between total mercury and methylmercury concentration are shown in
Fig. 12.

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*: One outlier data (ID# 11) is not shown in this plot.

Fig. 12 Scatter plot between total mercury and methylmercury

A significant positive correlation between total mercury and methylmercury are detected (r=0.440,
one-sided testing, p=0.039). It should be noted that the methodology and measuring instrumentused for both analysis are different, so the reason for this correlation is unknown. It is possible thatdeviations in the commonly used instruments, such as balances may have contributed to thisrelationship. However, it is unlikely that these instruments would deviate to the same degree as thedifferences in the participants' results under typical laboratory conditions.

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11. Moisture
The basic statistics of the reported moisture of test item is shown in Table 8. There wereparticipants who reported difference moistures for each analytical parameter (total
mercury/methylmercury). Therefore, statistics and analysis of moisture were conducted based oneach result for the analytical parameter individually.
The basic statistics of moistures are shown in Table 8.
Table 8 Summary of the result of moisture
Moisture (%)
Total mercury

Methylmercury

Average

4.90

4.63

Median4.45

4.14

Standard deviation

3.76

4.13

Minimum

0.003

0.01

Maximum

19.3

19.0

The distribution of the results of moistures are shown in Fig. 13.

Fig. 13 Histogram of the result of moisture

The plots of the relations of moisture and total mercury concentration are shown in Fig. 14.

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Fig. 14 Total mercury and moisture

The plots of the relations of moisture and methylmercury concentration are shown in Fig. 15.

Fig. 15 Methylmercury and moisture

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The reported moisture deviation was larger than the mercury deviation of the target parameter
analysis (total mercury/methylmercury), but no noticeable relationship between moisture and bothparameters of mercury concentration was found. The method used to measure moisture was tosubtract the weight measured after drying from the weight measured before drying. Since themoisture was relatively small (median was about 4-5%), it was considered that the uncertainty ofthe moisture analysis was larger than the change in moisture during the PT analysis period.

12. Conclusion
In both of the results of total mercury and methylmercury analysis, many of the reported resultsfrom the participants were concentrated around the median value and IQRs of the results wererelatively close. Thus, it was considered that the difference among many mercury analysis
laboratories were relatively small. Both of total mercury and methylmercury analysis, some sort ofanalysis methods were performed among the participating laboratories. Significant differences(bias) among the analytical methods were not detected in both of the target parameters (total
mercury / methylmercury).
The range of moisture reported from the participants were larger than the deviation of the target
parameters even it was not the target of the PT. Also, the correlation between the mercury andmoisture was not detected. Although the result of stability testing indicates that there was not asignificant change of testing item during the PT, it can be considered that the difference of moistureaffects the results of the PT because the deviations of the analysis results were small.

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