海洋藻类的食用和药用价值极高,含有丰富的天然维生素(A、B1、B2、B9、 B12、C、D、E、K)、矿物质元素(钙、铁、碘、镁、磷、钾、锌、铜、锰、硒、氟)、游离氨基酸、不饱和脂肪酸、植物性蛋白质、膳食纤维和多酚[1⇓⇓⇓⇓-6],是药食同源物质的典型代表之一,历来为舟山居民所食用。海带(Laminaria japonica)、浒苔[本研究中的浒苔消费品种一般指条浒苔,Enteromorpha clathrata (Roth)Greville,当地俗称苔菜、苔条]、裙带菜(Undaria pinnatifida Suringar)是当地居民膳食消费的主要品种。根据《2021舟山统计年鉴》[7]数据显示,截至2020年末,舟山地区主要海藻养殖品种海带的养殖面积为81 hm2,养殖产量已达4 687 t。当地市场上浒苔、裙带菜产品则多为野生品种采收加工而来。
由于海藻细胞壁主要由肽聚糖、磷脂和蛋白质组成的网状结构,表面积较大且具有黏性,并可提供许多能与离子结合的带负电荷的官能团,因此海藻易从周围环境中通过主动和被动吸收方式蓄积海洋环境中的金属离子,如镉(Cadmium)、铅(Lead)、砷(Arsenic)等[2,8⇓⇓⇓⇓⇓-14]。近年来全国海洋生态环境基本保持稳定,但是各类直排海污染源污水仍不可避免。据《2021年中国海洋生态环境状况公报》[15],在四大海区中,东海受纳污水排放量最多,其中直排海污染源污水中镉、铅、汞受纳总量分别达899.1、1 215.5、111.4 kg。陆源重金属污染物入海,加之海藻本身对海洋环境中的重金属表现出很高的亲和性,使海藻不可避免地富集以镉为代表的有毒有害元素,并且最终通过生物累积行为,对人类健康造成威胁[16]。
镉是一种人体非必需的有害微量元素之一,在联合国环境规划署列出的12种具有全球性意义的危险化学物质清单中,被列为首位[17-18]。美国毒物与疾病登记署(The Agency for Toxic Substances and Disease Registry,ATSDR)将其列为第6位危及人体健康的有毒物质[19-20]。近年来,国内外专家学者分析评估了食用海藻中以镉为代表的重金属危害隐患[13,21⇓⇓⇓⇓⇓⇓⇓⇓⇓-31],但是关于舟山地域性食用海藻的分析研究相对少见,基本被涵盖在水产品大类的宏观报导里[32-33]。综上,本研究针对舟山地区市场中常见的3种典型食用海藻产品(海带、浒苔、裙带菜)进行镉含量调查监测,并应用国家食品安全风险评估中心(China National Center for Food Safety Risk Assessment,CFSA)《食品中化学物健康风险分级技术指南》[34]的风险矩阵方法,对舟山典型食用海藻中镉膳食暴露健康风险进行等级评价,以期丰富食用海藻的质量安全评估体系的基础数据,拓展和深化水产品风险评估的层次。
1 材料与方法
1.1 样品来源
2020年在舟山市主城区及定海、普陀、岱山、嵊泗等县区农贸市场、超市、专业批发市场等市场流通环节中,定期随机采集包装、散装海带41份(干样)、浒苔19份(干样)、裙带菜21份(干样20份,湿样1份)等3种食用海藻样品。另从养殖环节采集海带12份(干样5份、湿样7份)作为补充。抽样依据GB/T 30891—2014 《水产品抽样规范》[35]的规定执行。样品带回实验室后,干样直接粉碎均匀,湿样经冷冻干燥后再粉碎均匀,两者均经100目过筛后再放置于干燥皿中待检。
1.2 仪器与试剂
Agilent 7900 电感耦合等离子体质谱仪,美国Agilent公司;ETHOS UP型微波消解仪,意大利Milestone公司;Milli-Q Reference超纯水系统,美国Millipore公司;ME204E/02型电子天平,梅特勒-托利多仪器(上海)有限公司;SCIENTZ-10N 冷冻干燥机,宁波新芝生物科技股份有限公司;氩气、氦气(气体纯度≥99.999%)。
硝酸(Trace metal grade,Assay:67%~70%),美国Fisher Chemical;30%过氧化氢(优级纯),国药集团化学试剂有限公司;镉单元素标准溶液(1 000 μg·mL-1 ),国家有色金属及电子材料分析测试中心;混合内标溶液(100 μg·mL-1 ),美国Agilent公司;国家标准物质——紫菜中砷、铅、镉成分分析标准物质(GBW08521,18 g/瓶),中国计量科学研究院。
本研究检测过程中所用玻璃器皿,均需在每次使用前先用10%~20%浓度的硝酸溶液浸泡24 h以上,再经超纯水多遍冲洗,自然阴干后方可使用,用于定容的50 mL容量瓶均已预先通过计量检定。
1.3 检测方法
取待检干样在天平上称重0.2~0.3 g(精确至0.1 mg)左右,经微波消解后,依据GB 5009.268—2016《食品安全国家标准食品中多元素的测定》[36] “第一法 电感耦合等离子体质谱法(ICP-MS)”进行测定。该方法中,镉的方法检出限为0.002 mg·kg-1,若未检出的数据比例<60%时,所有未检出数据用 1/2 LOD替代,若未检出的数据比例>60%时,所有未检出数据用LOD替代[37-38]。采用国家标准物质——紫菜中砷、铅、镉成分分析标准物质(GBW08521),与采集样品同步测定,实施质量控制。目前,我国《食品安全国家标准 食品中污染物限量》(GB 2762—2017)[39]未对藻类及其制品中镉提出明确限量要求,因此本研究中相关监测结果不作相关超标评价。
1.4 镉暴露健康风险等级评估方法
根据国家食品安全风险评估中心制定的《食品中化学物健康风险分级技术指南》[34],按照以下3个步骤进行3种食用海藻镉膳食暴露健康风险等级评估:1)确定潜在健康损害的程度或等级;2)确定健康损害发生的可能性等级;3)确定健康风险等级。
1.4.1 评估潜在健康损害等级
根据《食品中化学物健康风险分级技术指南》,评估潜在健康损害等级的步骤:1)确定Ha分级赋值;2)确定Hb分级赋值;3)计算潜在健康损害的程度或等级分值。其计算公式如下:
式(1)中:Ha赋值——急性毒性健康危害效应指标[40],依据《食品安全国家标准 急性经口毒性试验》(GB 15193.3—2014)[41],通过美国国家医学图书馆的化学品标识数据库(ChemIDplus)等权威门户网站查询镉的大鼠经口LD50数值,确定镉的急性毒性分级,对应《食品中化学物健康风险分级技术指南》中“急性毒性分级赋值标准”进行Ha分级及赋值,急性毒性等级依据实际无毒(大鼠经口LD50>5 000 mg·kg-1 BW)、低毒(大鼠经口LD50:501~5 000 mg·kg-1 BW)、中等毒(大鼠经口LD50:51~500 mg·kg-1 BW)、剧毒(大鼠经口LD50:1~50 mg·kg-1 BW)、极毒(大鼠经口LD50<1 mg·kg-1 BW),从低到高计分为1~5。Hb赋值——慢性毒性健康危害效应指标[41],参照国际癌症研究机构(IARC)致癌物分级、欧盟(EU)致突变物分级、EU生殖毒性(包含发育毒性)物质分级、美国环境保护署(EPA)神经毒性物质分级、世界卫生组织(WHO)化学品全球统一分类与标签制度(GHS)[34]等权威机构“证据权重法”[42]分级标准,在化学物致癌、致突变、生殖/发育毒性、神经毒性以及亚慢性/慢性毒性等食物中的化合物可能含有的多种毒性表征中选择最高等级,确定Hb 指标的分级及赋值,依据“是否对人类产生显著亚慢性/慢性毒性的物质”分别计分为4分(是)、3分(否)两档[34]。潜在健康损害等级分值——将Ha、Hb的赋值代入公式(1),得出潜在健康损害等级分值及对应等级标识(表1)。
表1 潜在健康损害等级分值对应等级标识[34]
Tab.1
| 潜在健康损害等级分值 Potential health risk rating score | 对应等级标识 Grade identification |
|---|---|
| 1 | - |
| 2 | + |
| 3 | ++ |
| 4 | +++ |
| 5 | ++++ |
1.4.2 评估健康损害发生的可能性等级
3种食用海藻中镉膳食暴露健康损害发生的可能性等级评估主要依据人群暴露风险因素的相关性指标:目标人群平均暴露量与健康指导值的比值(Pa)、个体暴露水平超过健康指导值的人群百分比(Pb)[34,43]。健康指导值(HBGV)是对食品中的化学品进行危害定性的一个重要的定量指标。HBGV是粮农组织/世卫组织食品添加剂联合专家委员会(JECFA)和农药残留联席会议(JMPR)等机构针对食品以及饮用水中的物质所提出的经口(急性或慢性)暴露安全范围的定量描述值,在一定时期内(24 h或终生)摄入低于该值的有害物质不会引起可觉察的健康风险,包括暂定每月可耐受摄入量(PTMI)、每日耐受/允许摄入量(TDI /ADI)等[44]。一般认为,膳食暴露量大于HBGV可能导致更大的健康风险[43,45]。因此,“目标人群平均暴露量与健康指导值的比值(Pa)” “个体暴露水平超过健康指导值的人群百分比(Pb)”被视作关键性指标。本研究中的HBGV来源于JECFA于2011年第73次会议提出的镉的PTMI(25 μg·kg-1b.w.),表达镉的可耐受摄入水平[46]。可能性等级的计算公式如下:
表2 健康损害发生的可能性分值对应等级标识[34]
Tab.2
| 健康损害发生的可能性分值 Rating score for probability of health risk | 对应等级标识 Grade identification |
|---|---|
| 1 | - |
| 2 | + |
| 3 | ++ |
| 4 | +++ |
| 5 | ++++ |
1.4.3 判断健康风险等级
表3 风险矩阵
Tab.3
| 可能性等级(分值) Rating score for probability | 潜在健康损害的等级(分值) Potential health risk rating score | ||||
|---|---|---|---|---|---|
| -(1) | +(2) | ++(3) | +++(4) | ++++(5) | |
| ++++(5) | 中(5) | 中(10) | 高(15) | 高(20) | 高(25) |
| +++(4) | 低(4) | 中(8) | 中(12) | 高(16) | 高(20) |
| ++(3) | 低(3) | 中(6) | 中(9) | 中(12) | 高(15) |
| +(2) | 低(2) | 低(4) | 中(6) | 中(8) | 中(10) |
| -(1) | 低(1) | 低(2) | 低(3) | 低(4) | 中(5) |
表4 健康风险等级划分[34]
Tab.4
| 健康风险等级 Health risk levels | 健康风险分值 Health risk scores |
|---|---|
| 高High | 15~25 |
| 中Medium | 5~12 |
| 低Low | 1~4 |
2 结果与分析
2.1 三种食用海藻中镉的含量水平
舟山地区市场上常见的3种典型食用海藻产品(海带、浒苔、裙带菜)的镉含量调查监测结果如表5所示。
表5 三种食用海藻中镉的含量水平(以干重计,n=2)
Tab.5
| 样品名称 Samples | 海带 Laminaria japonica | 浒苔 Enteromor-pha clathrata (Roth)Greville | 裙带菜 Undaria pinnatifida Suringar |
|---|---|---|---|
| 样品分类Systematic | 褐藻 | 绿藻 | 褐藻 |
| 样品数量Samples no. | 53 | 19 | 21 |
| 含量范围/(mg·kg-1) Content range | 0.207~1.32 | 0.176~2.13 | 0.452~2.02 |
| 平均值±SD/(mg·kg-1) Mean±SD | 0.624±0.249 | 0.903±0.651 | 1.18±0.441 |
| 中位值/(mg·kg-1) Median value | 0.610 | 0.601 | 1.25 |
对3组数据进行Jarque-Bera检验,显示海带、浒苔、裙带菜均没有呈现显著性(P>0.05)。3种食用海藻产品镉含量范围为0.176~2.13 mg·kg-1b.w.,比较3种海藻产品的含量平均值±SD:裙带菜﹥浒苔﹥海带。其中海带、裙带菜的平均值分别与其中位值比较接近,表明海带、裙带菜的含量水平分布基本呈对称分布。但即便同属褐藻类,海带的含量范围低于裙带菜,其最小值、平均值、中位值基本上在裙带菜对应数值的50%左右浮动。比较藻类种间差异时,综合海带、裙带菜的数据,褐藻的含量范围在0.207~2.02 mg·kg-1b.w.之间,与隶属绿藻的浒苔相近。
2.2 三种食用海藻镉暴露健康风险等级评估
2.2.1 潜在健康损害等级判定
首先,检索ChemIDplus数据库查询镉的大鼠经口LD50数值为2 330 mg·kg-1,依据《食品中化学物健康风险分级技术指南》中“急性毒性分级赋值标准”,确定3种食用海藻中镉膳食暴露急性毒性等级为低毒,为 Ha赋值2。其次,对照上述指南中“亚慢性/慢性毒性分级赋值标准”,镉的慢性毒性分类归类为第1类,为Hb赋值4。以上数值代入公式(1),得出潜在健康损害等级分值为3,参考表1“潜在健康损害等级分值对应等级标识”,归属“++”等级。
2.2.2 健康损害发生的可能性等级评价
式(4)中:c——三种食用海藻中的镉含量(mg·kg-1)。FIR——食物摄取率,本文中指各类海藻的人均日消费量,其中海带的消费量数据来源于《第五次中国总膳食研究》[49]中的“浙江省12类食品样品聚类后食物品种及消费量(按成年男子计算的每日实际食物消费量)”,为每人4.45 g·d-1,由于缺少女性消费量数据,依据风险最大化原则,本研究假设成年女子海带的人均日消费量数据等同男子;鉴于浙江省膳食消费调查中没有浒苔、裙带菜的相关数据,本研究借鉴了韩国保健产业振兴院的“2020年度国民健康营养调查”[50]中的男性日均摄入量(分别为0.18、0.70 g·d-1)以及女性日均摄入量(分别为0.48、0.53 g·d-1),其中浒苔在该营养调查数据中没有单独的食物分类,但因浒苔属石莼科藻类植物,其男、女性日均摄入量依据风险最大化原则,以该营养调查数据中石莼的对应日均摄入量计算。WAB——人群的平均体重(kg),据《第五次国民体质监测公报》[51]数据,中国成年男性平均体重约为72.5 kg,成年女性约为58.8 kg。计算结果保留3位有效数字。
表6 成年人通过三种食用海藻对镉的暴露评估及镉膳食暴露健康损害发生的可能性等级评价
Tab.6
| 海藻品种 Sampls | 性别 Gender | EMI平均值/ (μg·kg-1b.w.) EMI mean | 占PTMI 的比例/% Proportion of PTMI | Pa赋值 Pa assignment | 超PTMI的 人群比例/% Proportion of people with ultra-PTMI | Pb赋值 Pb assignment | 健康损害发生 的可能性分值 Rating score for probability of health risk | 健康损害发生 的可能性等级 Grade identification corresponding to rating score for probability of health risk |
|---|---|---|---|---|---|---|---|---|
| 海带 Laminaria japonica | 男 | 1.15 | 4.60 | 1 | 0 | 1 | 1 | - |
| 女 | 1.42 | 5.68 | 1 | 0 | 1 | 1 | - | |
| 浒苔 Enteromorpha clathrata (Roth) Greville | 男 | 0.0673 | 0.269 | 1 | 0 | 1 | 1 | - |
| 女 | 0.221 | 0.884 | 1 | 0 | 1 | 1 | - | |
| 裙带菜 Undaria pinnatifida Suringar | 男 | 0.341 | 1.36 | 1 | 0 | 1 | 1 | - |
| 女 | 0.318 | 1.27 | 1 | 0 | 1 | 1 | - |
图1
图1
三种食用海藻对成年人膳食镉暴露的贡献率
Fig.1
Contribution of three types of edible seaweeds to dietary cadmium exposure in adults
2.2.3 健康风险等级评估
表7 成年人通过三种食用海藻对镉的膳食暴露健康风险等级评估结果
Tab.7
| 海藻品种 Species sampls | 性别 Gender | 潜在健康 损害等级分值 Potential health risk rating score | 潜在健康 损害等级 Grade identification corresponding to potential health risk rating score | 健康损害发生 的可能性分值 Rating score for probability of health risk | 健康损害发生 的可能性等级 Grade identification corresponding to rating score for probability of health risk | 健康风险 分值 Health risk scores | 健康风险 等级 Health risk levels |
|---|---|---|---|---|---|---|---|
| 海带 Laminaria japonica | 男 | 3 | ++ | 1 | - | 3 | 低 |
| 1 | - | 3 | 低 | ||||
| 女 | |||||||
| 1 | - | 3 | 低 | ||||
| 浒苔 Enteromor-pha clathrata (Roth)Greville | 男 | ||||||
| 1 | - | 3 | 低 | ||||
| 女 | |||||||
| 1 | - | 3 | 低 | ||||
| 裙带菜 Undaria pinnatifida Suringar | 男 | ||||||
| 1 | - | 3 | 低 | ||||
| 女 |
3 结论与讨论
本研究针对舟山地区市场中常见的3种典型食用海藻产品(海带、浒苔、裙带菜)进行镉含量调查监测,结果表明3种食用海藻产品镉含量范围为0.176~2.13 mg·kg-1b.w.,3种海藻产品的含量平均值±SD:裙带菜﹥浒苔﹥海带。本研究中海带的含量范围为0.207~1.32 mg·kg-1,平均含量水平±SD为(0.624±0.249)mg·kg-1,与刘永涛[13]测定的烟台地区样品(0.68±0.32)mg·kg-1、大连地区样品(0.65±0.40)mg·kg-1较为接近,低于Almela C等[52]报道的在西班牙瓦伦西亚市的商店里销售的原产地日本的海带干样0.908 mg·kg-1的含量水平,高于俞国珍等[53]检测的三门县市售干海带平均浓度0.34 mg·kg-1、杨玉凤等[54]报道的2018年日照市市售海带(淡干)平均含量0.441 mg·kg-1、Hwang Y O等[55]研究的韩国市售干海带中平均含量0.296 mg·kg-1、Miedico O等[56]报道的意大利市售的东南亚进口海带产品中平均含量(0.539±0.300)mg·kg-1。
当前国内外对于藻类及其制品中镉污染判定标准各异,并未有统一的限量标准,如欧盟委员会法规(EU)2021/1323[57]规定“完全或主要由干海藻、海藻衍生产品或干双壳类软体动物组成的食品补充剂”上限为3.0 mg·kg-1;法国食品、环境及劳动卫生署[58]规定“食用海藻(拟直接供人食用、用作食品中某一成分、用于食品补充剂)”限量≤0.5 mg·kg-1(干重计);新加坡食品局发布的《食品销售法》[59]明确指出“任何海藻”镉限量≤2 mg·kg-1;泰国大众卫生部(MOPH)第(G/SPS/N/THA/263)号通报草案[60]规定“可供食用的干海藻”上限为2 mg·kg-1(干重计);印度尼西亚食品药品监督管理局《食品和药品管理条例》[61]规定“海藻(加工食品)” 镉限量≤0.05 mg·kg-1;而我国《食品安全国家标准 食品中污染物限量》(GB 2762—2017)、国际食品法典委员会(CAC)、美国食品药品监督管理局、日本厚生劳动省、韩国食品医药品安全部未对藻类及其制品中镉的限量值提出明确要求,因此无法就本研究中食用海藻含量水平进行超标量评价。
本研究运用国家食品安全风险评估中心《食品中化学物健康风险分级技术指南》的风险矩阵方法对舟山典型食用海藻中镉膳食暴露健康风险进行等级评价,显示海带、浒苔、裙带菜经成年人群摄入对镉的膳食暴露健康风险等级均属于“低风险”。海带在3种食用海藻中,对成年人膳食镉暴露的贡献率最高,占经3种海藻暴露镉总量的73%~74%。但本研究仍存在一定的局限性,譬如按照现行国标方法用硝酸和过氧化氢等强氧化剂对食用海藻样品进行前处理消解,实质是将所有形态的镉转化成无机镉[8],实测的是被检海藻样品中总镉含量。然而,镉的毒性程度和生物利用率取决于其化学形态[62-63],离子态镉的毒性较高,结合态和有机态镉无毒或毒性非常微弱[8,64-65]。已有研究表明,当海藻等植物受到有毒元素胁迫时,体内会生成植物螯合肽(Phytochelatins,PCs),对镉离子的螯合能力很强,因而海藻各提取态镉中蛋白质结合态或吸附态镉含量最高[8,66]。海藻细胞壁也可以提供许多能与离子结合的官能团,包括磷酸根等,与镉结合形成磷酸盐,海藻中镉磷酸盐含量因此也较高[8]。镉与有机酸、蛋白质结合形成的螯合物,以及形成的镉磷酸盐、草酸盐等难溶于水的化合物可限制镉在体内的移动[8,67-68],使镉毒性呈无毒或极微量毒性。而无机和有机水溶性镉,相较难溶于水的镉磷酸盐、草酸盐等化合物,迁移能力更强,毒性更高[63]。因此,下一步要在研究总镉含量的基础上,进一步深入分析主要食用海藻品种中各种镉形态的分布及含量水平。此外,未来还需考量海藻经烹调等热加工方式前、后镉元素的生物可给性存在的差异可能性,进一步补充和完善儿童及高端消费人群的消费量数据,以期为食用海藻镉含量调查与膳食暴露评估靶向化、多角度全方位升级化提供基础数据支撑。
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表1
图1
