# Validation of Total Mercury in Marine Sediment and Biological Samples, Using Cold Vapour Atomic Absorption Spectrometry

^{1}

^{2}

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## Abstract

**:**

_{3}, H

_{2}SO

_{4}, and HF) with potassium dichromate solution in a hot block digestion system. A calibration curve was constructed (R

^{2}> 0.999). Two CRMs (Marine Sediment Reference Material (PACS-3) and Trace Elements in Muscle Tissue (Trace Elements and Methylmercury in Mussel Tissue (NIST2976)) were utilised for quality assurance and control. The limit of quantification (LOQ) calculated as 0.04 µg/kg, and uncertainty (U) calculated as 2%. The obtained results showed the suitability of this method for direct mercury measurement in environmental samples. Additionally, the proficiency of this method was recognised by accreditation under the standard of International Organization for Standardization (ISO/IEC 17025:2017) for competence of testing and calibration laboratories.

## 1. Introduction

## 2. Material and Methods

#### 2.1. Apparatus, Chemicals, and Reagents

^{®}ACS), potassium permanganate (EMSURE

^{®}ACS, Reag), hydroxylamine hydrochloride (99.999% trace metals basis), and tin-II-chloride (≥99.99% trace metals basis) were all obtained from Merck (Boston, MA, USA). Certified reference material (CRM) NIST-2976 (mussel tissue (trace elements and methylmercury), certified value: 0.061 ± 0.0036 mg/kg) were sourced from the National Institute of Standards and Technology (Gaithersburg, MD, USA). CRM PACS-3 (Marine Sediment Reference Material for Trace Metals and other Constituents), certified value: 2.98 ± 0.36 (mg/kg) was purchased from the National Research Council Canada (Ottawa, ON, Canada). Mercuric nitrate standard was used as a stock calibration standard (1001 ± 2 pg/mL, 2%, nitric acid in low TOC water (<50 ppb)).

#### 2.2. Sample Preparation

_{3}, and 3 mL H

_{2}SO

_{4}(Suprapur, Merck) to decompose and release matrix-bound mercury. The sample was then digested using a hot block set at 125 °C for 12 h with the cap closed to reduce mercury loss by volatilisation. After complete digestion, indicated by a clear solution, the tube was left to cool to room temperature after which 1 mL of resulting mixture was transferred into a 50 mL measuring flask together with 2 mL potassium dichromate, and the final volume made up to the mark with reagent water. This constitutes the sample solution. Other protocols employ different sample preparation techniques that may require different sample weights, utilise different digestion acids and other digestion apparatus [6,7].

#### 2.3. Sample Analysis

## 3. Results and Discussion

#### 3.1. Method Validation

#### 3.1.1. Selectivity

#### 3.1.2. Trueness

_{lab}= Mean of Measurement

_{ref}= Mean of certified value

_{ref}= Standard certified uncertainty

#### 3.1.3. Recovery and Matrix Effects

^{0}[14].

#### 3.1.4. Precision

#### Repeatability

#### Intermediate Precision

#### 3.1.5. Limit of Detection and Limit of Quantitation

#### 3.1.6. Linearity and Working Range

_{Cal}-equation was used to ascertain the linearity; If the (F

_{Cal}) is higher than (F

_{Tab}), the model cannot be considered fit for the data. i.e.,

_{0}there is no lack of fit (linear).

_{A}there is a lack of fit (nonlinear). Tabulated F value (F

_{Tab}) and calculated F value (F

_{Cal}) were calculated as per Equation (5). The F

_{Cal}value was 0.42 and F

_{Tab}was 2.56. Lack of fit is not statistically significant, and H

_{0}hypothesis was accepted.

#### 3.1.7. Ruggedness

## 4. Estimation of the Measurement Uncertainty (Quantifying Uncertainty in Analytical Measurement)

_{R}= Relative standard uncertainty.

## 5. Validation Characteristics Evaluation

_{Cal}) and (F

_{Tab})) to examine the criteria acceptance.

_{Cal}= 0.42) is less than (F

_{Tab}= 2.56), we accept the null hypothesis and the calibration curve was linear.

## 6. Conclusions

_{3}, H

_{2}SO

_{4}, and HF in a closed Teflon tube, thus reducing analyte loss and also reducing the risk of contamination. The preparation process was followed by CV AAS analysis. The obtained results were accurate and precise (Recovery > 90%, RSD ≤ 5%) when compared to the US-EPA method 3052 and 7474 when employed in the same samples.

## Author Contributions

## Funding

## Acknowledgments

## Conflicts of Interest

## References

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**Figure 1.**Spectrum of total mercury (T-Hg) analysis in two matrix reference materials at two different levels (high (

**A**), low (

**B**)) and blank sample noise background (

**C**).

**Figure 3.**Mean concentrations of two different CRMs (NIST2976, PACS3) compared with its certified values in six experiments at different preparation conditions.

**Table 1.**The mean of eight replicate results of two different matrices (biota and marine sediment certified reference materials (CRMs)).

CRM | n | NIST-2976 | Ref. Vale | PACS-3 | Ref. Vale |
---|---|---|---|---|---|

Mean value (mg/Kg) | 8 | 0.058 ± 0.002 | 0.061 ± 0.004 | 2.87± 0.15 | 2.98 ± 0.36 |

CRM(NIST-2976) | CRM(PACS-3) | ||
---|---|---|---|

Bias = −0.002 | Bias = −0.28 | ||

Relative bias = −4% | Relative bias = −9% | ||

SD = 0.006 | u(lab) = 0.12 | ||

u(ref) = 0.002 | u(ref) = 0.25 | ||

|z| | 0.4 | |z| | 1.0 |

**Table 3.**Comparison between our proposed method and traditional methods for total mercury analysis using cold vapour atomic absorption spectrometry (CV AAS) and comparable matrices.

Method | Matrix | Certified Value (µg/kg) | Avg. Measured Value (µg/kg) | Avg. % Rec | No. of Samples | RSD |
---|---|---|---|---|---|---|

EPA-7474 | NIST 8406 SED | 60 | 62 | 103% | 70 | 15% |

NIST 1566 Oyster tissue | 84 | 81 | 97% | 72 | 15% | |

EPA-7473 | Estuarine Sediment NIST SRM 1646 | 63 | 75 | 119% | N/A | 3% |

Oyster Tissue NIST SRM 1566a | 64 | 68 | 106% | N/A | 3% |

Level 1 (0.2 µg/L) | Level 2 (2 µg/L) | Level 3 (8 µg/L) | |
---|---|---|---|

Mean | 0.165 | 2.001 | 8.027 |

SD | 0.008 | 0.030 | 0.077 |

RSD | 5% | 2% | 1% |

**Table 5.**Intermediate precision concentration in mg/kg of eight CRM replicates between 15 March and 17 April.

CRM | Mean/mg/kg | St. Dev. | RSD |
---|---|---|---|

PACS3 | 2.784 | 0.143 | 5.10% |

NIST2976 | 0.062 | 0.003 | 4.80% |

Slope | Intercept | n | p | Nominator | Denominator | MSS_{error} | MSS_{lof} |
---|---|---|---|---|---|---|---|

0.0316 | 0.0011 | 7 | 5 | 5 | 28 | 1.0 × 10^{−5} | 2.5 × 10^{−6} |

**Table 7.**Ruggedness studies of total mercury analyses in marine sediment samples (PACS3) according to Plackett–Burman test.

Factor | Low Value (−1) | High Value (+1) | Experiment No. | Positive Effect | Negative Effect | Total Effect | Total Effect % | Mean Measured Values (mg/kg) | Ref. Value (mg/kg) | Recovery % |

Temp/°C | 100 | 125 | E1 | 3.07 | −2.88 | 0.2 | 7% | |||

Digestion time/h | 3 | 12 | E2 | 3.17 | −2.78 | 0.39 | 13% | |||

Dilution volume/mL | 50 | 100 | E3 | 3.17 | −2.78 | 0.39 | 13% | |||

Sample wt./g | 0.1 | 0.2 | E4 | 2.69 | −3.26 | −0.57 | −19% | |||

Reagents (HNO_{3} + H_{2}SO_{4}) mL | 3 + 5 | 6 + 10 | E5 | 2.85 | −3.1 | −0.26 | −9% | 2.97 | 2.98 | 99 |

**Table 8.**Ruggedness studies of total mercury analysis in biota samples (NIST2976) according to Plackett–Burman test.

Factor | Low Value (−1) | High Value (+1) | Experiment No. | Positive Effect | Negative Effect | Total Effect | Total Effect % | Mean Measured Values (mg/kg) | Ref. Value (mg/kg) | Recovery % |

Temp/°C | 100 | 125 | E1 | 0.07 | −0.09 | −0.03 | −0.90% | |||

Digestion time/h | 3 | 12 | E2 | 0.08 | −0.08 | −0.01 | −0.30% | |||

Dilution volume/mL | 50 | 100 | E3 | 0.08 | −0.08 | 0 | 0.10% | |||

Sample wt./g | 0.1 | 0.2 | E4 | 0.08 | −0.08 | 0 | −0.20% | |||

Reagents (HNO_{3} + H_{2}SO_{4}) mL | 3 + 5 | 6 + 10 | E5 | 0.07 | −0.09 | −0.01 | −0.50% | 0.065 | 0.061 | 107 |

Sum of Squares | df | Mean Square | F | Sig. | |
---|---|---|---|---|---|

Between Groups | 0.006 | 5 | 0.001 | 2.442 | 0.095 |

Within Groups | 0.006 | 12 | 0.000 | ||

Total | 0.012 | 17 |

Sum of Squares | df | Mean Square | F | Sig. | |
---|---|---|---|---|---|

Between Groups | 0.370 | 5 | 0.074 | 2.213 | 0.121 |

Within Groups | 0.401 | 12 | 0.033 | ||

Total | 0.771 | 17 |

Source of Uncertainty | Type | Measured Value | Error ± | Unit | u_{Relative} | Probability Distribution | Divisor | Squared Standard Uncertainty (u²) |
---|---|---|---|---|---|---|---|---|

Repeatability of prepared 0.6 µg/L CRM | A | 6 × 10^{−4} | 5.5 × 10^{−6} | mg/L | 9.2 × 10^{−3} | Normal, 1s | 1 | 8.5 × 10^{−5} |

Chemical Reagents purity | B | 1.00 | 5.0 × 10^{−3} | 5.0 × 10^{−3} | Rectangular | 1.73 | 8.4 × 10^{−6} | |

Pipette 1 mL repeatability | A | 1.0 × 10^{−3} | 7.8 × 10^{−8} | L | 7.8 × 10^{−5} | Normal, 1s | 1 | 6.6 × 10^{−9} |

Temperature effect on Pipette 1 mL Volume | B | 1.0 × 10^{−3} | 4.1 × 10^{−7} | L | 4.1 × 10^{−4} | Rectangular | 1.73 | 5.6 × 10^{−8} |

Calibration st certificate uncertainty of pipette 1 mL | B | 1.0 × 10^{−3} | 3.4 × 10^{−6} | L | 3.4 × 10^{−3} | Triangular | 2.45 | 1.9 × 10^{−6} |

Measuring flask 1 mL repeatability | A | 5.0 × 10^{−2} | 4.3 × 10^{−6} | L | 8.6 × 10^{−5} | Normal, 1s | 1 | 7.4 × 10^{−9} |

Temperature effect on flask 50 mL Volume | B | 5.0 × 10^{−2} | 2.0 × 10^{−5} | L | 4.1 × 10^{−4} | Rectangular | 1.73 | 5.6 × 10^{−8} |

Uncertainty of flask 50 mL | B | 5.0 × 10^{−2} | 6.0 × 10^{−5} | L | 1.2 × 10^{−3} | Triangular | 2.45 | 2.4 × 10^{−7} |

Concentration of the calibration standard | B | 1001 | 2 | mg/L | 2.0 × 10^{−3} | Normal, 2s | 2 | 1.0 × 10^{−6} |

Tare weight repeatability | A | 100 | 3.7 × 10^{−3} | mg | 3.7 × 10^{−5} | Normal, 1s | 1 | 1.3 × 10^{−9} |

Gross weight repeatability | A | 100 | 3.7 × 10^{−3} | mg | 3.7 × 10^{−5} | Normal, 1s | 1 | 1.3 × 10^{−9} |

Balance linearity contribution (gross weight) | B | 100 | 0.1 | mg | 1.0 × 10^{−3} | Rectangular | 1.73 | 3.3 × 10^{−7} |

Balance linearity contribution (tare weight) | B | 100 | 0.1 | mg | 1.0 × 10^{−3} | Rectangular | 1.73 | 3.3 × 10^{−7} |

Balance readability (gross weight) | B | 100 | 0.01 | mg | 1.0 × 10^{−4} | Rectangular | 1.73 | 3.3 × 10^{−9} |

Balance readability (tare weight) | B | 100 | 0.01 | mg | 1.0 × 10^{−4} | Rectangular | 1.73 | 3.3 × 10^{−9} |

Combined standard u_{c} | 0.0098 | |||||||

Expanded U at 95% confidence interval (k = 2) | 2.0% |

Parameter | Methodology | Acceptance Criteria | Results | Fulfil the Acceptance Criteria! |
---|---|---|---|---|

Selectivity | A specific and sharp peak of Hg produced during CRM analysis with no interferences | No interferences with the analyte peak. | Complies | Yes |

Trueness | Relative bias was calculated for ten repeated CRMs | Relative bias shall not lie outside the limit of ±10%, and Zeta score shall less than or equal 2 to be satisfactory | Complies | Yes |

Recovery | Two CRMs were used to calculate Recovery% = Measured value/Reference value. | ±10% of the reference value | Complies | Yes |

Precision | Repeated CRMs through one day of analysis and intermediate resection over a long time. | Relative standard deviation (RSD) shall not exceed 10% | Complies | Yes |

Limit of Detection and Limit of Quantitation | Replicates of low-level concentration CRM matrix used to calculate SD (σ). Calculate S/N ratio | Limit of detection (3σ), limit of quantitation (10σ). S/N between 2.5 to 10 | Complies | Yes |

Linearity: | Independent calibration curves signal used to calculate (F_{Tab}) and (F_{Cal}). | Lack of fit test (Linear if F_{Cal} < F_{Tab}) | Complies | Yes |

Range | The concentration interval over which linearity and accuracy are obtained and yields a precision of ≤3% RSD. | N/A | Complies | N/A |

Robustness | Various conditions tested using Placket-Burman test for experimental design ruggedness. One-way ANOVA | One-way ANOVA was used as statistical acceptance criteria of robustness at 95% confidence interval | Complies | Yes |

Uncertainty of measurement | Expanded Uncertainties were considered for in-house Calibration curve (U_{Cal}) and sample measurement (U_{measu}) to estimate the uncertainty from the main resources affecting the method. | U = k*u% | N/A | - |

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## Share and Cite

**MDPI and ACS Style**

Elezz, A.A.; Mustafa Hassan, H.; Abdulla Alsaadi, H.; Easa, A.; Al-Meer, S.; Elsaid, K.; Ghouri, Z.K.; Abdala, A.
Validation of Total Mercury in Marine Sediment and Biological Samples, Using Cold Vapour Atomic Absorption Spectrometry. *Methods Protoc.* **2018**, *1*, 31.
https://doi.org/10.3390/mps1030031

**AMA Style**

Elezz AA, Mustafa Hassan H, Abdulla Alsaadi H, Easa A, Al-Meer S, Elsaid K, Ghouri ZK, Abdala A.
Validation of Total Mercury in Marine Sediment and Biological Samples, Using Cold Vapour Atomic Absorption Spectrometry. *Methods and Protocols*. 2018; 1(3):31.
https://doi.org/10.3390/mps1030031

**Chicago/Turabian Style**

Elezz, Ahmed Abou, Hassan Mustafa Hassan, Hamood Abdulla Alsaadi, Ahmed Easa, Saeed Al-Meer, Khaled Elsaid, Zafar Khan Ghouri, and Ahmed Abdala.
2018. "Validation of Total Mercury in Marine Sediment and Biological Samples, Using Cold Vapour Atomic Absorption Spectrometry" *Methods and Protocols* 1, no. 3: 31.
https://doi.org/10.3390/mps1030031