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海水养殖尾水总磷测定方法的比较与优化

刘雁 佟飞 陈丕茂 袁华荣 冯雪

刘雁, 佟飞, 陈丕茂, 袁华荣, 冯雪. 海水养殖尾水总磷测定方法的比较与优化[J]. 南方水产科学. doi: 10.12131/20220056
引用本文: 刘雁, 佟飞, 陈丕茂, 袁华荣, 冯雪. 海水养殖尾水总磷测定方法的比较与优化[J]. 南方水产科学. doi: 10.12131/20220056
LIU Yan, TONG Fei, CHEN Pimao, YUAN Huarong, FENG Xue. Comparison and optimization of total phosphorus determination method in mariculture tailwater[J]. South China Fisheries Science. doi: 10.12131/20220056
Citation: LIU Yan, TONG Fei, CHEN Pimao, YUAN Huarong, FENG Xue. Comparison and optimization of total phosphorus determination method in mariculture tailwater[J]. South China Fisheries Science. doi: 10.12131/20220056

海水养殖尾水总磷测定方法的比较与优化

doi: 10.12131/20220056
基金项目: 广东省重点领域研发计划项目(2020B1111030002-2);深圳市科技创新计划(JCYJ20160331141759795)
详细信息
    作者简介:

    刘雁:刘 雁 (1988—),男,硕士研究生,研究方向海洋生物学。E-mail: 287475306@qq.com

    通讯作者:

    陈丕茂 (1969—),男,研究员,从事渔业资源保护与利用研究。E-mail: chenpm@scsfri.ac.cn

  • 中图分类号: S 912

Comparison and optimization of total phosphorus determination method in mariculture tailwater

  • 摘要: 为提高海水养殖尾水中总磷 (Total phosphorus, TP) 的检测效率,使用酶标仪代替分光光度计进行海水养殖尾水TP的测定,并对TP的消解方法、酶标板类型与加样量等进行优化。结果表明,使用控温加热板消解60 min与高压灭菌器消解30 min的效果无显著差异 (P>0.05);使用48孔板加样1 mL,检测的灵敏度优于其他实验组 (96孔板200 µL,96孔石英板200 µL,48孔板200 µL和500 µL,24孔板500 µL和1 mL),48孔板加样1 mL与96孔板加样200 µL,磷酸盐浓度与吸光度的线性相关最高 (R2=0.999 9);对养殖尾水TP测定的精确度和准确度验证结果显示,48孔板加样1 mL的相对误差 (Relative error, RE) 和相对标准偏差 (Relative standard deviation, RSD)分别为−2.97%~1.59%和0.42%~4.06%,对甘油磷酸二钠 (β-GLP) 和六偏磷酸钠 (SHMP) 的回收率分别为98.2%~99.6%和93.4%~97.1%,TP的测定限 (Limit of quantification, LOQ) 为0.25 µmol·L−1;96孔板加样200 µL的RE和RSD分别为−14.03%~0.21%和2.63%~14.23%,对β-GLP和SHM的回收率分别为94.7%~99.0%和88.9%~97.3%,TP的LOQ为0.55 µmol·L−1。在TP浓度介于0.55 µmol·L−1~6.4 µmol·L−1内,两种优化法的准确度和精密度均符合测定要求,可用于海水养殖尾水TP的测定。
  • 图  1  消解百分比与消解时间关系

    注:不同字母之间表示同一样品在不同消解时间的消解百分比差异显著 (P<0.05)。

    Figure  1.  Relationship between digestion percentage and digestion time

    Note: Different letters indicate that the digestion percentage of the same sample at different digestion time is significantly different (P<0.05).

    图  2  酶标仪测定法和常规方法测定的TP标准曲线

    Figure  2.  TP standard curve determined by microplate reader and conventional method

    表  1  不同酶标板与加样量和常规方法测定的回归方程

    Table  1.   Regression equations of different types of microwell plates, amount of sample and conventional method

    测定方式
    Determination method
    回归方程
    Regression equation
    相关性
    Relevance (R2)
    常规方法 conventional method A=0.019 1c+0.001 2 0.999 9
    48孔板1 mL 48-well plate 1 mL A=0.019 1c+0.002 1 0.999 9
    96孔板 200 µL 96-well plate 200 µL A=0.010 8c+0.002 3 0.999 9
    96孔石英板 200 µL
    96-well quartz plate 200 µL
    A=0.009 7c+0.007 5 0.999 3
    24孔板1 mL 24-well plate 1 mL A=0.010 0c+0.001 4 0.999 7
    48孔板500 µL 48-well plate 500 µL A=0.009 4c+0.001 2 0.999 0
    24孔板500 µL 24-well plate 500 µL A=0.004 7c+0.000 6 0.999 1
    48孔板200 µL 48-well plate 200 µL A=0.003 5c+0.000 4 0.995 5
    下载: 导出CSV

    表  2  96孔板加样200 µL与常规方法测定水样TP的结果比较

    Table  2.   Comparison of results of measuring TP of samples by 96-well plate with 200 µL of sample and conventional method

    样品号
    Sample No.
    优化法
    Optimized method/(µmol·L−1)
    常规方法
    Conventional standard method/(µmol·L−1)
    相对误差
    RE%
    相对平均偏差
    RAD/%
    相对标准偏差
    RSD/%
    11.50±0.0911.60±0.106−6.255.336.04
    23.00±0.1373.10±0.079−3.283.414.57
    34.26±0.1574.28±0.078−0.393.153.69
    41.48±0.1281.43±0.1023.737.048.61
    54.74±0.1184.73±0.0670.212.322.49
    62.44±0.1152.49±0.097−1.943.754.73
    70.87±0.1151.01±0.116−14.0310.5613.19
    83.17±0.1173.32±0.082−4.573.423.70
    90.96±0.1371.05±0.098−8.4112.6514.23
    101.87±0.1161.96±0.086−4.594.284.84
    110.90±0.0961.04±0.093−13.4610.0011.55
    123.05±0.1393.11±0.074−2.092.633.15
    135.30±0.1255.33±0.065−0.592.352.63
    143.88±0.1563.82±0.0731.532.953.24
    152.18±0.0932.28±0.080−4.533.524.57
    162.03±0.1472.09±0.090−3.113.954.74
    175.85±0.2045.64±0.0563.723.423.49
    181.19±0.1001.26±0.118−5.426.98.37
    注:以常规方法的测定均值为真值,相对偏差 (%) 是优化法与常规方法的偏差,测定浓度是尾水稀释20倍后的浓度;表4同此。 Note: The measured mean value of the conventional method is considered to be the true value, and the relative deviation (%) is the deviation between the optimization method and the conventional method, and the measured concentration is the concentration after tail water dilution of 20 times. The same case in Table 4.
    下载: 导出CSV

    表  3  96孔板优化法的样品加标回收率

    Table  3.   Sample spiked recovery by 96-well plate optimization method

    样品浓度
    Sample concentration/
    (µmol·L−1)
    加入浓度 (β-GLP)
    Added concentration (β-GLP)/
    (
    µmol·L−1)
    加标后浓度
    Found concentration/
    (µmol·L−1)
    平均回收率
    Average recovery/%
    加入浓度 (SHMP)
    Added concentration (SHMP)/(µmol·L−1)
    加标后浓度
    Found concentration/
    (µmol·L−1)
    平均回收率
    Average recovery
    /%
    3.051.504.53±0.0498.41.504.51±0.0497.1
    3.006.01±0.0498.73.005.97±0.0497.3
    2.421.503.91±0.0698.01.503.84±0.0393.6
    3.005.41±0.0399.03.005.34±0.0496.6
    2.211.503.64±0.0597.31.503.58±0.0493.1
    3.005.14±0.0498.63.005.08±0.0496.8
    1.191.502.61±0.0794.71.502.52±0.0788.9
    3.004.10±0.0497.13.004.02±0.0794.2
    下载: 导出CSV

    表  4  48孔板加样1 mL与常规方法测定水样TP的结果比较

    Table  4.   Comparison of the results of measuring TP of samples by 48-well plate with 1 mL of sample and conventional method

    样品号
    Sample No.
    优化法
    Optimized method/(µmol·L−1)
    常规方法
    Conventional standard method/(µmol·L−1)
    相对误差
    RE/%
    相对平均偏差
    RAD/%
    相对标准偏差
    RSD/%
    11.55±0.0291.60±0.106−2.501.91.10
    23.12±0.0243.10±0.0790.650.80.77
    34.31±0.0254.28±0.0780.700.50.58
    41.39±0.0431.43±0.102−2.802.13.09
    54.75±0.0214.73±0.0670.420.30.44
    62.45±0.0322.49±0.097−1.611.01.31
    70.98±0.0371.01±0.116−2.973.33.78
    83.36±0.0243.32±0.0821.200.70.71
    91.04±0.0311.05±0.098−0.952.32.98
    101.95±0.0271.96±0.086−1.521.61.38
    111.01±0.0411.04±0.093−2.882.94.06
    123.12±0.0293.11±0.0740.320.80.93
    135.37±0.0345.33±0.0650.750.60.63
    143.79±0.0333.82±0.073−0.790.80.87
    152.24±0.0302.28±0.080−1.751.21.34
    162.11±0.0352.09±0.090−1.401.51.66
    175.68±0.0245.64±0.0560.710.40.42
    181.28±0.0331.26±0.1181.592.12.58
    下载: 导出CSV

    表  5  48孔板优化法的样品加标回收率

    Table  5.   Sample spiked recovery by 48-well plate optimization method

    样品浓度
    Sample concentration/
    (µmol·L−1)
    加入浓度 (β-GLP)
    Added concentration
    (β-GLP)/(µmol·L−1)
    加标后浓度
    Found concentration/
    (µmol·L−1)
    平均回收率
    Average
    recovery/%
    加入浓度(SHMP)
    Added concentration
    (SHMP)/(µmol·L−1)
    加标后浓度
    Found concentration/
    (µmol·L−1)
    平均回收率
    Average
    recovery/%
    3.05 1.50 4.59±0.04 98.2 1.50 4.56±0.04 95.8
    3.00 6.11±0.02 99.6 3.00 5.98±0.03 95.2
    2.42 1.50 3.93±0.04 98.7 1.50 3.91±0.03 97.1
    3.00 5.42±0.03 99.4 3.00 5.35±0.03 96.8
    2.21 1.50 3.73±0.04 99.1 1.50 3.66±0.02 94.7
    3.00 5.22±0.02 99.3 3.00 5.12±0.03 95.9
    1.19 1.50 2.76±0.06 98.4 1.50 2.69±0.04 94.2
    3.00 4.23±0.04 98.4 3.00 4.08±0.04 93.4
    下载: 导出CSV
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  • 收稿日期:  2022-03-08
  • 修回日期:  2022-07-12
  • 录用日期:  2022-08-01
  • 网络出版日期:  2022-08-09

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